Biochemical and Functional Characterisation of Phospholipase C-Η2

Biochemical and functional characterisation of Phospholipase C-η2

Petra Popovics, M.Sc.

This thesis is submitted in partial fulfilment for the degree of Doctor of Philosophy at the University of St Andrews

December 2012

Abstract

Phospholipase C enzymes are important cell signalling enzymes that catalyse the cleavage of phosphatidylinositol 4,5-bisphophate PI(4,5)P2 into two biologically active second messenger molecules. These are the inositol 1,4,5-trisphosphate which initiates Ca2+ release from the endoplasmic reticulum and the diacylglycerol that activates protein kinase C. Although this basic function is shared between the different isoforms, the PLC family encompasses a diverse collection of proteins with various domain structures in addition to the PLC-specific domains.

The neuron-specific “6th family” of these enzymes, PLCηs have most recently been identified with two members, PLCη1 and PLCη2. The aim of the thesis is to characterise the PLCη2 variant from several aspects. Firstly, it describes that PLCη2 possesses a high sensitivity towards Ca2+. Secondly, it investigates how the Ca2+- induced enzymatic activity of PLCη2 is controlled by its different domains. Also it provides evidence that the pleckstrin homology domain targets PLCη2 to membranes by recognising PI(3,4,5)P3. Moreover, the uniquely structured EF-hand is responsible for the Ca2+-sensitivity of the enzyme. Finally, it is demonstrated that the C2 domain is important for activity.

The initial biochemical characterisation is followed by the description of a physiological role for PLCη2. It is shown using a neuroblast model that PLCη2 is crucial for neuronal differentiation and neurite growth. Further efforts were made to assess how PLCη2 is responsible for this effect. It was revealed that it might be involved in regulating intracellular Ca2+ dynamics, transcriptional activity and actin reorganisation in differentiating neurons.

As the functions of PLCη2 are just beginning to come to light, more aspects for future research are also suggested in the thesis. Hopefully, this and the data presented within the thesis will stimulate even greater interest in this fascinating new field of research.

Declaration

I, Petra Popovics, hereby certify that this thesis, which is approximately 50,000 words in length, has been written by me, that it is the record of work carried out by me and that it has not been submitted in any previous application for a higher degree.

I was admitted as a research student in September, 2009 and as a candidate for the degree of Ph.D. in August, 2010; the higher study for which this is a record was carried out in the University of St Andrews between 2009 and 2012.

Date: November 30, 2012 Signature of candidate …………………..

Supervisor's declarations

I hereby certify that the candidate has fulfilled the conditions of the Resolution and Regulations appropriate for the degree of Ph.D. in the University of St Andrews and that the candidate is qualified to submit this thesis in application for that degree.

Date: November 30, 2012 Signature of supervisor …………………..

In submitting this thesis to the University of St Andrews I understand that I am giving permission for it to be made available for use in accordance with the regulations of the University Library for the time being in force, subject to any copyright vested in the work not being affected thereby. I also understand that the title and the abstract will be published, and that a copy of the work may be made and supplied to any bona fide library or research worker, that my thesis will be electronically accessible for personal or research use unless exempt by award of an embargo as requested below, and that the library has the right to migrate my thesis into new electronic forms as required to ensure continued access to the thesis. I have obtained any third-party copyright permissions that may be required in order to allow such access and migration. The following is an agreed request by candidate and supervisor regarding the electronic publication of this thesis: Embargo on both of printed copy and electronic copy for the same fixed period of two years on the following grounds: publication would be commercially damaging to the researcher and to the supervisor and the University; publication would preclude future publication.

Date: November 30, 2012 Signature of Candidate:......

Signature of Supervisor:......

iii

Acknowledgements

The completion of the thesis would not have been possible without the help and support of several individuals who, in one way or another, contributed to the completion of this study.

First and foremost I offer my sincerest gratitude to my supervisor, Dr. Alan Stewart, who has been supporting me throughout my Ph.D. studies and provided me with his knowledge and guidance whilst allowing me the room to work independently. I would like to thank him for giving me the opportunity to be his first Ph.D. student. I hope that I fulfilled his expectations.

I am also grateful to Dr. Gordon Cramb who has kindly shared his lab, materials and brilliant mind with me. I would be nowhere without his help. I will always be thankful to Dr. Simon Guild for teaching the Ca2+-permeabilising method which eventually became a key point of the thesis. I also thank to Dr. John Lucocq, who showed me how to measure neurites in a “smart way”.

I am thankful for Prof. Magdolna Kovács, who was supportive in my decision to come to Scotland and never forgot about me ever since. I am lucky to have her friendship.

I had the opportunity to work with fantastic and knowledgeable people in the lab who were always helpful and here I would like to owe my gratitude to them. I can`t thank them enough all their practical help, thoughtful discussions and the friendship. In particular, Dr. Eva Borger, Dr. Katherine Feeney, Dr. Taciana Kasciukovic and Dr. Elaine Campbell. I am also grateful for Laura McFarlane, Dr. Katarina Oravcova, Susana Moleirinho, Helen Popple, Steven Gellatly, Omar Kassaar and Ying Zheng for being my friends and making the 3 years pleasant for me. I would like to thank Mary Wilson for being a fantastic organiser of the lab.

Words fail me to express my appreciation to my husband Gábor who has dedicated his life to my carrier. Writing a Ph.D. would not have been possible without his love and support. I am also thankful to his family for accepting me as his wife and treating me as a family member already. I would like to thank my family for showing support and patience when I needed it.

And last but foremost, I would like to dedicate this thesis to the memory of my grandmother who has devoted her whole life to the family. I am lucky to inherit her tenacious personality which made it possible for me to get where I am at present. I will always keep you in my heart.

“Do not fear to be eccentric in opinion, for every opinion now accepted was once eccentric.”

“Science may set limits to knowledge, but should not set limits to imagination.”

Bertrand Russell

Contents

Abstract ...... i Declaration ...... ii Copyright declaration ...... iii Acknowledgements ...... iv Contents ...... vi List of figures ...... xi List of tables ...... xvii Abbreviations ...... xix Chapter 1.: Introduction 1 1.1. General overview of calcium signalling ...... 3 1.1.1. The versatility of calcium signalling ...... 3 1.1.2. Further regulation of intracellular calcium levels: ON and OFF reactions ...... 5 1.1.3. Regulators of calcium transport: pumps, exchangers and channels .. 6 1.1.4. Calcium signalling at the plasma membrane ...... 7 1.1.4.1. Basics of cell communication ...... 7 1.1.4.2. Receptor-operated calcium channels (ROCCs) ...... 8 1.1.4.3. Voltage-operated calcium channels (VOCCs) ...... 9 1.1.4.4. Capacitive calcium entry ...... 10 1.1.4.5. Second messenger-operated channels (SMOCs) ...... 11 1.1.4.6. Calcium pumps and -exchangers of the plasma membrane 12 1.1.4.7. Second messenger-coupled receptors involved in Ca2+ signalling ...... 12 1.1.5. Calcium stores and their molecular machinery ...... 15 1.1.5.1. What is the importance of accumulating calcium in the cell? ...... 15 1.1.5.2. Calcium homeostasis of the ER/SR ...... 15 1.1.5.3. Mitochondrial Ca2+ transporters ...... 16 1.1.5.4. Endosomes, lysosomes and secretory vesicles as calcium stores ...... 19 1.1.5.5. Nuclear calcium signalling ...... 19 1.1.5.6. Is the Golgi apparatus a calcium store? ...... 20 1.1.5 Calcium dynamics of the cytosol ...... 21 1.1.5.1. Cytosolic calcium buffers ...... 21 1.1.5.1. Calcium sensors of the cytosol ...... 21 1.1.5.1. Calcium sensitive enzymes and processes - the effectors ... 22

1.2. The classification and comparative description of Phospholipase C enzymes ...... 23 1.2.1. Phospholipids: their regulation and their importance in cellular signalling ...... 23 1.2.2. Phospholipase enzyme superfamily ...... 26 1.2.3. Phospholipase C enzymes ...... 28 1.2.3.1. A short history of the discovery of PLCs...... 28 1.2.3.2. Domain structure of PLCs ...... 32 1.2.3.2.1. General overview...... 32 1.2.3.2.2. Pleckstrin homology domain ...... 32 1.2.3.2.3. The EF-hands...... 34 1.2.3.2.4. The catalytic domains ...... 36 1.2.3.2.5. The C2 domain ...... 37 1.2.3.2.6. PLC isozyme-specific domains ...... 38 1.2.3.3. Regulation of PLCs ...... 39 1.2.3.4. The role of PLCs in neuronal physiology ...... 43 1.2.3.4.1. Neuronal pattern of PLC expression ...... 43 1.2.3.4.2. PLC activity-linked neuronal functions ...... 45 1.2.3.5. The latest PLC family, PLCηs ...... 47 1.2.3.5.1. First characterisation of PLCη enzymes ...... 47 1.2.3.5.2. Biochemical properties ...... 49 1.4. Thesis outline ...... 50 Chapter 2.: Materials and Methods 53 2.1. Cell Culture Methods ...... 55 2.1.1. Cell lines and their maintenance ...... 55 2.1.2. Cryopreservation of cell lines...... 56 2.1.3. Rescue of frozen cells...... 56 2.1.4. Transfection by electroporation...... 57 2.1.5. Lipofection of Neuro-2a cells ...... 57 2.1.6. Generation of stable PLCη2 knock-down cell lines ...... 57 2.2. Cell assays and treatments ...... 58 2.2.1. Immunocytochemistry and fluorescent microscopy ...... 58 2.2.2. Differentiation of Neuro-2a cells ...... 60 2.2.3. [3H]Inositol-phosphate release assays (IP-assay) ...... 61 2.2.4. Permeabilisation of COS-7 cells to Ca2+ ...... 62 2.2.5. Dual-Luciferase reporter assay ...... 63 2.3. Molecular biology methods ...... 65 2.3.1. RNA and DNA isolation, analysis and modification ...... 65 2.3.1.1. RNA isolation ...... 65

vii

2.3.1.2. DNA digestion and Reverse Transcription (RT) ...... 65 2.3.1.3. Conventional (semi-quantitative) Polymerase Chain Reaction (PCR) ...... 65 2.3.1.4. Quantitative PCR (qPCR) ...... 66 2.3.1.4. Agarose Gel Electrophoresis ...... 67 2.3.1.5. Purification of DNA-bands from agarose gels ...... 67 2.3.1.6. Digestion with restriction endonucleases ...... 67 2.3.1.7. Molecular cloning ...... 68 2.3.1.8. Site-directed mutagenesis ...... 69 2.3.1.9. Transformation of vectors into E. coli ...... 69 2.3.1.10. Isolation of plasmid DNA ...... 70 2.3.2. Protein analysis ...... 70 2.3.2.1. Western-blot analysis ...... 70 2.3.2.2. Purification of GST-tagged PH domains ...... 72 2.3.2.3. Förster resonance energy transfer (FRET)-based detection of protein-lipid interactions ...... 73 2.3.2.4. Detection of protein-lipid interactions by lipid-adsorbed membranes (PIP strips) ...... 73 2.3.2.5. Identification of protein-protein interactions by bacterial two-hybrid screen ...... 74 2.3.2.6. Myc-tag co-immunoprecipitation (pull-down) ...... 75 2.4. Statistical analysis ...... 75 Chapter 3.: Biochemical characterisation of PLCη2 77 3.1. Introduction ...... 79 3.2 Characterisation of PLCη2 Ca2+-activation ...... 80 3.2.1. PLCη2 is activated by monensin ...... 80 3.2.2. Monensin-induced activation of PLCη2 is directed by Ca2+ ...... 82 3.2.3. PLCη2 is activated by physiological levels of Ca2+ in permeabilised COS-7 cells ...... 84 3.2.4.: Localisation of PLCη2 in COS-7 cells ...... 85 3.3. Investigation of the PLCη2 PH domain ...... 89 3.3.1. PLCη2 PH domain is required for membrane-binding ...... 89 3.3.2. Membrane association of PLCη2 is essential for its activity ...... 91 3.3.3. Purification of GST-tagged PH domains of PLCη1 and PLCη2 ..... 92 3.3.4. Determination of the specificity of PLCη PH domains towards phosphoinositides by a FRET-based assay ...... 94 3.3.5.: Detection of PLCη PH domain-phospholipid interactions by lipid- absorbed membranes ...... 98 3.3.6.: Localisation of PH-PLCη2 in Neuro-2a cells in relation to the

viiivii

distribution of PI(3,4,5)P3 and PI(4,5)P2 ...... 100 3.4. An EF-hand directs the Ca2+-sensitivity of the enzymePLCη2 – the description of a novel EF-hand structure ...... 101 3.5.: Ca2+-regulated membrane association of the C2 domain is essential for PLCη2 enzyme activity ...... 107 3.6.: Discussion ...... 113 3.6.1. The initial characterisation ...... 113 3.6.2. Targeting mechanism of the PH domain ...... 115 3.6.3. Identification of a novel EF-hand in PLCη2 ...... 117 3.6.4. C2 domain supports the secondary membrane-docking of PLCη2 118 3.6.5. Conclusion ...... 120 Chapter 4.: The role of PLCη2 in neuronal physiology 123 4.1. Introduction ...... 125 4.2. Results ...... 126 4.2.1. The distribution of PLCη2 in the mouse brain ...... 126 4.2.2. Different splice variants of PLCη2 are expressed in Neuro-2a cells and in mouse brain ...... 131 4.2.3.: PLCη2 level gradually elevates during retinoic acid induced differentiation of Neuro-2a cells ...... 135 4.2.4. The physiological level of PLCη2 is essential for the retinoic acid- induced differentiation of Neuro-2a cells ...... 141 4.2.5. PLCη2 is involved in the regulation of retinoic acid receptor signalling ...... 146 4.2.6. The interaction of PLCη2 and Lim domain kinase-1 and its consequences for neuronal outgrowth ...... 152 4.2.7. The localisation of PLCη2 in differentiated Neuro-2a cells is

controlled by PI(3,4,5)P3 levels ...... 163 4.3. Discussion ...... 166 4.3.1. Implications of the specific pattern of PLCη2 in the brain ...... 166 4.3.2. The gradual elevation in PLCη2 protein levels is essential for neuronal differentiation of Neuro-2a cells ...... 168 4.3.4. The role of PLCη2 in nuclear signalling ...... 170 4.3.5. The role of PLCη2-LIMK1 interaction for neuronal outgrowth of Neuro-2a cells ...... 171

4.3.6. PLCη2 localisation is regulated by PI(3,4,5)P3 ...... 172 4.3.7. Final conclusion ...... 172 Chapter 5.: Future experiments 175 5.1.: How does Ca2+ activate PLCη2? ...... 177 5.2.: What are the characteristics of PLCη2 C2 domain phospholipid-binding 177

5.3. Cellular localisation – still in doubts ...... 178 5.4. PLCη2 levels throughout life and in disease ...... 179 5.5. Splice variants of PLCη2 ...... 180 5.6. LIMK1-PLCη2 interaction: more evidence is required ...... 181 5.7. What is the role of PLCη2 in nuclear signalling? ...... 181 Chapter 6.: Bibliography 185 Chapter 7.: Appendices 223 A Sequences ...... 225 B Plasmid maps ...... 228 C Abbreviations ...... 230 D Publications ...... 230

ixx

List of figures

1.1: The mechanism of Ca2+ release from the ER/SR ...... 4 1.2: Chemical communication of cells ...... 7 1.3.: Biosynthesis of phosphatidylinositol ...... 24 1.4.: Phosphatidylinositol metabolism ...... 25 1.5.: Inositol-phosphate metabolism...... 26 1.6.: The site of hydrolysis catalysed by different phospholipases...... 27 1.7.: Generation of second messengers by PLCs...... 28 1.8.: Domain structure of PLCs...... 32 1.9.: Structure of the EF-hand...... 35 1.10.: Structure of the rat PLCδ1 C2 domain...... 38 1.11.: Various activation of PLC isoforms...... 42 1.12.: Domain structure of PLCη enzymes...... 47 1.13.: Amino acid motifs of the PLCη2 C-terminal that might be targeted by kinases/phosphatases...... 48 2+ 3.1.: The effects of various Ca -mobilising agents on PI(4,5)P2 hydrolysis in COS-7 cells transfected with PLCη2...... 81

3.2.: Inhibition of monensin-induced PI(4,5)P2 hydrolysis in PLCη2-transfected COS-7 cells by various inhibitors...... 83 3.3.: Measurement of [3H]inositol phosphate release in permeabilised COS-7 cells transfected by empty vector or PLCη2 in the presence of various concentrations of free Ca2+...... 85 3.4.: GFP-PLCη2 possesses a low co-localisation rate with the mitochondria in COS-7 cells...... 86 3.5.: Measurement of monensin-induced accumulation of [3H]inositol phosphate in COS-7 cells transfected with constructs encoding wild-type and GFP-PLCη2...... 87 3.6.: Exogenously introduced PLCη2 exhibits a high co-localisation rate with mitochondria and induce morphological changes in COS-7 cells...... 88 3.7.: Localisation of GFP-PLCη2 and its PH-domain lacking deletion mutant in COS-7 cells...... 89 3.8.: Membrane-binding ability of PLCη2 is reduced in its PH-lacking mutant as it was shown by western blot from membrane enriched fractions...... 90 3.9.: Measurement of monensin-induced accumulation of [3H] inositol- phosphate COS-7 cells transfected with constructs encoding wild-type and ΔPH PLCη2...... 91

3.10.: Measurement of PI(4,5)P2 hydrolysis in wild-type and ΔPH PLCη2-

expressing permeabilised COS-7 cells in the presence of various free Ca2+ concentrations...... 92 3.11.: ClustalW alignment of protein sequences corresponding to the PH domains of PLCη1 and PLCη2...... 91 3.12.: Polyacrylamide gel demonstrating the purity of GST-PH-PLCη in the elution buffer...... 93 3.13.: Principles of detecting PH domain-phospholipid interactions by FRET...... 95 3.14.: Measurement of the association of PLCη1 and PLCη2 PH domains with biotinylated phosphoinositides by using TR-FRET detection...... 95

3.15.: FRET measurement of the dissociation of biotinylated PI(4,5)P2-PLCη PH domain sensor complexes by unbiotinylated phosphoinositides...... 97 3.16.: Unique structure of PLCη PH-domains suggested by their alignment with PLCδ1...... 98 3.17.: Investigation of PLCη PH-domain phospholipid selectivity by lipid- protein overlay assay...... 99 3.18.: Localisation of GFP-PH-PLCη2 in Neuro-2a cells in relation to

PI(4,5)P2 and PI(3,4,5)P3...... 101 3.19.: Coordination of Ca2+ in EF-hands and the alignment of these domains from PLCη enzymes and other proteins...... 102 3.20.: Western-blot showing that wild-type and mutants of PLCη2 are expressed in transfected COS-7 cells...... 103 3.21.: Measurement of monensin-induced accumulation of [3H]-labelled inositol phosphate in COS-7 cells transfected with constructs encoding wild-type or mutant PLCη2 (D256A and D292A)...... 104 3.22.: Measurement of monensin-induced accumulation of [3H]-labelled inositol phosphate in COS-7 cells transfected with constructs encoding wild-type or mutant PLCη2 (H296A, Q297A and E304A)...... 105 3.23.: Measurement of the accumulation of [3H]-labelled inositol phosphates in permeabilised COS-7 cells transfected with constructs encoding wild-type or mutant PLCη2 (D256A and D292A) in the presence of various free Ca2+- concentrations...... 105 3.24.: Measurement of the accumulation of [3H]-labelled inositol phosphates in permeabilised COS-7 cells transfected with constructs encoding wild-type or mutant PLCη2 (H296A, Q297A and E304A) in the presence of various free Ca2+-concentrations...... 106 3.25.: Alignment of murine sequences corresponding to the C2 domains of PLCη2, PLCη1, PLCδ1 and synaptotagmin-I (Syt-1)...... 108 3.26.: Western blot showing equal protein levels of wild-type and mutant PLCη2 (D861A, D861N and D920A) in whole cell lysates from transfected COS-7

xii

cells...... 109 3.27.: Monensin-induced accumulation of [3H] inositol phosphate in COS-7 cells transfected with wild-type PLCη2 or D861A, D861N and D920A mutants...... 109 3.28.: Measurement of the accumulation of [3H]-labelled inositol phosphates in permeabilised COS-7 cells transfected with constructs encoding wild-type or mutant PLCη2 (D861A, D861N and D920) in the presence of various free Ca2+-concentrations...... 110 3.29.: Mutations in the C2 domain did not alter the membrane-binding properties of PLCη2 in COS-7 cells...... 111 3.30.: Western blot showing the protein levels of wild-type and mutant PLCη2 (D861A, D861N and D920A) in membrane-enriched fractions of transfected COS-7 cells...... 112 3.31.: Illustration of the role of PLCη2 domains in supporting enzymatic cleavage...... 121 4.1: The expression of PLCη2 mRNA in the mouse brain detected by in situ hybridisation...... 127 4.2: PLCη2 is highly abundant in the cerebellum...... 128 4.3.: High expression levels of PLCη2 were found in the hippocampus, medial habenula, suprachiasmatic nucleus and pineal gland...... 130 4.4.: Increased PLCη2 mRNA levels are found in the main olfactory bulb, pyriform cortex and the olfactory tubercule...... 131 4.5: Exon composition of the C-terminus in different splice variants of PLCη2...... 132 4.6.: Detection of PLCη2 splice variants in Neuro-2a cells and in mouse brain by RT-PCR...... 133 4.7.: Immunoblot of Neuro-2a cell lysates demonstrating the expression of the 21a/23 and 21a/22/23 splice variants of PLCη2...... 134 4.8.: PLCη2 protein levels increase during retinoic acid-stimulated differentiation of Neuro-2a cells...... 136 4.9.: Cellular localisation of endogenous PLCη2 in undifferentiated and differentiated Neuro-2a cells counterstained with the nuclear marker, DAPI...... 137 4.10.: Cellular localisation of GFP-tagged PLCη2 in undifferentiated and differentiated Neuro-2a cells counterstained with the nuclear marker, DAPI...... 139 4.11.: Endogenous PLCη2 is partially colocalised with mitochondria in non- differentiated and differentiated Neuro-2a cells...... 140 4.12.: PLCη2 protein level was downregulated by the stable transfection of shRNA in Neuro-2a cells...... 142 4.13: Calcium induced phospholipid turnover is significantly reduced in

xiii

PLCη2 knock-down cells...... 144 4.14.: The retinoic acid-induced differentiation of Neuro-2a cells is reduced in PLCη2 knock-down cells...... 145 4.15.: Retinoic acid-induced neuronal outgrowth is significantly reduced in PLCη2 knock-down cells...... 146 4.16.: RT-PCR showing the expression of retinoid receptors in Neuro-2a cells and in mouse brain...... 148 4.17.: Quantitative PCR showing the relative levels of RARα mRNA in control and knock-down Neuro-2a cell lines...... 149 4.18.: Simple representation of luciferase reporter constructs designed for the detection of retinoic acid induced gene transcription...... 149 4.19.: Basal retinoid receptor activity in control and PLCη2 knock-down cell lines as determined by a dual-luciferase assay...... 151 4.20.: Retinoic acid-stimulated signalling is suppressed in PLCη2 knock- down cell lines as detected by dual-luciferase assay...... 151 4.21.: Simplified description of the 2-hybrid system...... 153 4.22.: Domain structure of the human LIMK1...... 154 4.23.: E. Coli reporter strain encompassing both PLCη2-pBT and LIMK1- pTRG plasmids grow on selective plate containing streptomycin, 3-AT, tetracyclin and chloramphenicol...... 155 4.24.: Co-immunoprecipitation experiment failed to show the interaction between LIM kinase 1 and PLCη2. C-myc-LIMK1, PLCη2 or both constructs were transfected into COS-7 cells...... 156 4.25.: Structure of the kinase domain of human LIMK1...... 157 4.26.: Endogenous PLCη2 and LIMK1 co-localise in the nucleus of undifferentiated and differentiated Neuro-2a cells...... 158 4.27.: GFP-tagged PLCη2 and LIMK1 co-localise in the nucleus of undiffer entiated and differentiated Neuro-2a cells...... 159 4.28.: Retinoic acid stimulated LIMK1 and CREB phosphorylation levels are reduced in PLCη2 knock-down cells...... 160 4.29.: The actin dynamics is disturbed in PLCη2 knock-down cells...... 162

4.30.: PI(3,4,5)P3 is co-localised with endogenous PLCη2 and transfected GFP-PLCη2 in differentiated Neuro-2a cells...... 164 4.31.: Localisation of endogenous PLCη2 and GFP-PLCη2 relative to the

PI(4,5)P2 marker, mCherry-PH-PLCδ1 in differentiated Neuro-2a cells...... 165 4.32.: PLCη2 activity contributes to neuronal differentiation on multiple levels...... 173 B.1.: Plasmid map of pcDNA3.1D/V5-His-TOPO...... 228 B.2.: Plasmid map of pcDNA3.1/NT-GFP-TOPO...... 229

xiiixiv

B.3.: Plasmid map of psi-H1 encoding the control shRNA...... 229

List of tables

Table 1.1.:Tissue specific distribution of the different PLC isoforms collected from the UniGene Database...... 31 Table 1.2.: Summary of known information relating to the distribution of PLC- isoforms in the brain...... 44 Table 2.1.: Cell-lines and there maintenance...... 55 Table 2.2.: PLCη2-specific sequences in shRNA constructs...... 58 Table 2.3.: Agents used in combination with [3H]inositol phosphate release assays...... 61

Table 2.4.: Concentrations of CaCl2 and MgCl2 required to obtain various concentrations of free Ca2+ in equilibrium with 5 mM EGTA...... 63 Table 2.5.: Primer sequences used for RT-PCR...... 66 Table 3.1.: Half-maximal inhibitory values (IC50) calculated from the FRET- assay measuring the ...... 97 Table 3.2.: Summary of mutations generated in PLCη2 and their effects on enzyme activity...... 120 Table 4.1.: Detection of PLCη2 spliceforms by using specific primers ...... 133 Table 4.2.: Splice variant-specific PCR products detected in Neuro-2a cells and in mouse brain...... 134 Table C.1.: Amino acid codes ...... 230

xvii

Abbreviations

3-AT 3-aminotriazole 5-HT3 type 3 serotonin receptor AA arachidonic acid AC adenlylate cyclase enzyme ACB nucleus accumbens AMPA α-amino-3-hydroxyl-5-methyl-4-isoxazole propionate ANT adenine nucleotide translocase AOB accessory olfactory bulb APC allophycocyanin ARC arachidonic acid (AA) regulated Ca2+ channels ATP adenosine 5'-triphosphate BDNF brain-derived neurotrophic factor C2 domain constant 2 domain CA Ammon`s horn/ Cornu ammonis CAMKs Ca2+/calmodulin-dependent protein kinases cAMP cyclic adenosine monophosphate CBX cerebellar cortex cGMP cyclic guanosine monophosphate CLRs cysteine-loop receptors CP caudoputamen CREB cAMP responsive element binding protein CTD cytidine diphosphate CTX cerebral cortex CXCR4 CXC chemokine receptor 4 CypD cyclophilin D DAG diacylglycerol DAPI 4',6-diamidino-2-phenylindole DG dentate gyrus DHP dihydropyridine receptor DLRA Dual-Luciferase Reporter Assay DMEM Dulbecco`s modified Eagle`s medium ECACC The European Collection of Cell Cultures ECL enhanced chemiluminescent substrate EMEM Eagle's minimal essential medium ER/SR endo/sarcoplasmic reticulum FRET Förster resonance energy transfer GA Golgi apparatus GFP green fluorescent protein GluRs glutamate receptors GPCR G-protein-coupled receptor Grp1 general receptor for phosphoinositides-1 HCA H+/Ca2+ antiporter

xix

HPF hippocampal formation HsCalD9K human calbindin D9K HsCaMEF2 human calmodulin EF-hand domain 2 HsNCS-1 human neuronal calcium sensor-1 HsTropCII human troponin C type 2 HY hypothalamus I(1,3,4)P3 inositol 1,3,4-trisphosphate I(1,3,4,5)P4 inositol 1,3,4,5-tetrakisphosphate I(1,4,5)P3 inositol 1,4,5-trisphosphate IC inferior colliculus IMM inner mitochondrial membrane IP3R inositol 1,4,5-trisphosphate receptor IP-assay [3H]Inositol-phosphate release assays IPTG isopropyl β-D-1-thiogalactopyranoside ITC isothermal titration calorimetry LH lateral habenula LIMK1 lim domain kinase-1 LPA lysophosphatidic acid LPC lysophosphocholine LTD long-term depression LTP long-term potentiation MB midbrain MCU mitochondrial Ca2+ uniporter MH medial habenula MOB main olfactory bulb mPTP mitochondrial permeability transition pore MRN midbrain reticular nucleus MY medulla NAADP nicotinic acid adenine dinucleotide phosphate nAChR nicotinic variant of acetylcholine receptors NADP nicotinamide adenine dinucleotide phosphate NCX Na+/Ca2+ exchanger + 2+ NCXmito mitochondrial Na /Ca exchanger NE nuclear envelope NLS nuclear localisation signal NMDA N-methyl-D-aspartic acid NOS nitric oxide synthase och optic chiasm OMM outer mitochondrial membrane OT olfactory tubercule P pons PA phosphatidic acid PC phosphatidylcholine PDK1 3-phosphoinositide dependent protein kinase-1 PE phosphatidylethanolamine

PG phosphatidylglycerol PH domain pleckstrin homology domain PI phosphatidylinositol PI(3)P phosphatidylinositol 3-phosphate PI(3,4)P2 phosphatidylinositol 3,4-bisphosphate PI(3,4,5)P3 phosphatidylinositol 3,4,5-trisphosphate PI(4)P phosphatidylinositol 4-phosphate PI(4,5)P2 phosphatidylinositol 4,5-bisphosphate PI3K phosphatidylinositol 3-kinase PIN pineal gland PKA protein kinase A PKC protein kinase C PLC phospholipase C PMCA plasma membrane Ca2+-ATPase po polymorph layer PS phosphatidylserine PTB phosphotyrosine-binding domain RA ras association RARE retinoic acid responsive element RasGAP Ras GTPase activating protein RasGEF Rasvguanine nucleotide exchanger RhoGEF Rho guanine nucleotide exchange factor RNAP RNA polymerase ROC receptor-operated channel ROCK rho-associated protein kinase RTK receptor tyrosine kinase RyR ryanodine receptor S(1)P sphingosine 1-phosphate SAM sterile α motif SC superior colliculus SCH suprachiasmatic nucleus SDF-1 stromal derived factor 1 SEM standard error of the mean SERCA sarco/endoplasmic reticulum ATPase sg granule cell layer SH2 SRC homology 2 SMOC second messenger-operated channels SPCA secretory pathway Ca2+ ATPase STIM1 stromal interaction protein 1 Syt-1 synaptotagmin-1 TH thalamus TIM-barrel triose phosphate isomerase-barrel TPC two-pore channels TRITC tetramethylrhodamine B isothiocyanate TRPC transient receptor potential channel

xxi

VDAC voltage dependent anion channel VOC voltage-operated channels VOCC voltage-operated calcium channels VTA ventral tegmental area Wt wild type

xxii

Chapter 1.

Introduction

CHAPTER 1. INTRODUCTION

Chapter 1 focuses on two main topics that are essential to understand the results of Chapter 3 and 4 and their outcome. The first part of this Chapter provides an overall view of calcium signalling and how it is regulated by introducing the main groups of molecules that are involved in this process. The second part concentrates on the phospholipase C (PLC) family, an effector of the calcium signalling toolkit, and its newly identified member PLCη2. This latter enzyme is the subject of the analysis described within Chapters 3 and 4.

1.1. General overview of calcium signalling

1.1.1. The versatility of calcium signalling

A central role for Ca2+ has been described in various cellular processes such as fertilisation, muscle contraction and neurotransmission (Miledi 1973; Steinhardt et al. 1977; Ringer 1882). The most intriguing aspect about Ca2+ signalling is provided by its diversity. In principle, its action is simple: when a calcium signal is generated, the concentration of Ca2+ rises from 100 nM to 500-1000 nM in the cytosol (Berridge et al. 2000). The versatility of actions is likely to reside in the complex temporal and spatial nature of Ca2+ signalling (Thomas et al. 1996). Also, the wide range of effector molecules broadens the diversity of activated processes.

A possible classification of the various Ca2+ signals was provided by Berridge and colleagues (Figure 1.1) (Berridge et al. 2000), who organised the existing terms and built a comprehensive system for this purpose. Ca2+ is derived from extra- or intracellular sources. The most characterised intracellular source of Ca2+, the endo/sarcoplasmic reticulum (ER/SR), generates Ca2+ signals mainly via ryanodine or inositol 1,4,5-trisphosphate receptors (RyRs or IP3Rs) (Imagawa et al. 1987; Miyawaki et al. 1990). These receptors are focused into clusters on the ER/SR membrane. When a stimulus is generated, it activates individual channels producing 2+ small discharges of Ca termed as a `blips` (from IP3R) or `quarks` (from RyR). A stronger stimulus may activate a whole cluster of receptors that provides Ca2+ ’puffs’

CHAPTER 1. INTRODUCTION

2+ (from IP3R) or `sparks` (from RyRs). The resulting elevation in Ca concentration is high, but transient and localised to a small cytoplasmic region. Transient Ca2+ spikes are able to evoke the opening of neighbouring receptor clusters. In some cases, this activation spreads in a saltatory manner triggering the generation of a Ca2+ wave throughout the cell. In cells which are connected by gap junctions assembled from connexins, the Ca2+ wave may be transmitted from cell to cell (Berridge et al. 2000).

Figure 1.1: The mechanism of Ca2+ release from the ER/SR. A: The ER/SR membrane is enriched in IP3Rs and RyRs. B: Weak stimulation results in the opening of individual receptors. The generated Ca2+ signals are termed blips and quarks, respectively. C: Clusters of these receptors are activated by a stronger stimulus 2+ providing a larger Ca release called ’puff’ (from IP3R) or `spark` (from RyRs). D: Ca2+ transients provided by a specific receptor cluster may activate neighbouring receptors that may result in a Ca2+ wave along the ER/SR membrane. E: Ca2+ signal may be transmitted to another cell via gap junctions. Figure was produced by the author of this thesis.

CHAPTER 1. INTRODUCTION

The existence of Ca2+ waves was first implicated in the process of muscle contraction and fertilisation (Fatt and Ginsborg 1958; Endo et al. 1970; Steinhardt et al. 1977; Gilkey et al. 1978; Kort et al. 1985). It was followed by the recognition that the local rises in Ca2+, also known as Ca2+ microdomains, possess similar importance in cell signalling (Rizzuto and Pozzan 2006). The transient surges in Ca2+ concentration are restricted to defined regions of the cell due to the limited range of diffusion of this ion within the cytosol directed by different Ca2+-binding mechanisms (Allbritton and Meyer 1993).

1.1.2. Further regulation of intracellular calcium levels: ON and OFF reactions.

A wide range of molecules participate in the regulation of Ca2+ signalling. These are collectively known as the Ca2+ signalling toolkit. The toolkit consists of receptors, transducers, ion pumps, ion exchangers, ion channels, Ca2+ buffers, Ca2+ sensors and Ca2+ sensitive enzymes (Berridge et al. 1998). Each cell type expresses a unique set of components from the Ca2+ signalling toolkit to create Ca2+ signalling dynamics with different spatial and temporal properties.

Ca2+ signals are generated from both external and internal sources of Ca2+ primarily by the activation of various ion channels, ion exchangers and ion pumps (Berridge et al. 2003; Bootman et al. 2001). These events, which increase cytoplasmic Ca2+ levels, are the ON reactions. To maintain the ability of the ON reactions to evoke efficient rises in Ca2+, the OFF reactions restore the resting level of Ca2+ after each signal. This occurs by the transport of cytoplasmic Ca2+ into internal stores or to the extracellular space. At any time the level of cytosolic free Ca2+ depends on the balance between the ON and OFF reactions (Bootman et al. 2001; Berridge et al. 2003).

In the following sections, the molecular machinery directing the temporal and spatial distribution of Ca2+ will be introduced based on where they regulate Ca2+ transport in

CHAPTER 1. INTRODUCTION the cell. According to this, Ca2+ inflow to the cell may arise from the extracellular space or from intracellular Ca2+ stores such as the ER/SR, mitochondria, golgi apparatus (GA), nuclear envelope or such unusual compartments as lysosomes or transmitter vesicles at the neuronal synapse (Verkhratsky and Petersen 1998; Mitchell et al. 2001; Churchill et al. 2002). The description of the molecular groups that direct the movement of Ca2+ across membranes is given in the following section.

1.1.3. Regulators of calcium transport: pumps, exchangers and channels

Molecular groups that regulate Ca2+ movements are explained based on G.R. Dubyak`s description (Dubyak 2004). Ca2+ transporters move ions against their electrochemical gradient and therefore they require external energy for the process. Traditionally, Ca2+ transporters are divided into two groups: Ca2+ pumps and Ca2+ exchangers (Dubyak 2004; Gadsby 2004). Ion pumps selectively recognise ions which bind and initiate a conformational change in the transporter protein to permit the movement of the ion across the membrane. The typical energy source which utilises the transfer of the ion pumps is adenosine 5'-triphosphate (ATP). Ion exchangers use the energy of a passively flowing ion to transport another ion against its electrochemical gradient. Pumps and exchangers are usually responsible for maintaining the resting level of ions and to preserve the releasable Ca2+ pool within intracellular stores.

Ion channels permit the movement of ions down their electrochemical gradient (passive flow) which allow a relatively low energetic interaction between the channel protein and the ion. Ca2+ channels structurally consist of a membrane-crossing pore and a highly regulated gate which only opens when it is activated. When a channel opens, the ion flow is much higher than in transporters, allowing the channel to generate a rapid intracellular response upon sensing the extracellular stimuli. These

CHAPTER 1. INTRODUCTION stimuli may be receptor ligands (receptor-operated channels, ROCs), changes in membrane potential (voltage operated channels, VOCs), or second messengers (second messenger-operated channels, SMOCs) (Dubyak 2004; Gadsby 2004).

1.1.4. Calcium signalling at the plasma membrane

1.1.4.1. Basics of cell communication

Figure 1.2: Chemical communication of cells. Molecules liberated from the cell surface or released from the inside of the cell by diffusion or secretion may act on different cells in the body. These messengers may enter the blood stream and reach a different organ than the original (endocrine) or they may diffuse in the tissue of origin and act on a neighbouring cell (paracrine). A cell may regulate its own activity by binding its previously released molecules (autocrine). Moreover, a specific type of chemical communication may occur between two cells when the ligand of a receptor is presented on the cell surface and its specific receptor appears on another cell, the transfer of information is linked to cell-to-cell interaction (juxtacrine). Figure was produced by the author of this thesis.

Cell communication in an organism is crucial and is maintained in a highly regulated manner. Two cells may interact directly by transmitting an electrical current (electrical communication) or otherwise, messenger molecules may be secreted to reach their target cell by diffusion (chemical communication). The transfer of the chemical information is classified based on the distance that the messenger molecule is required to travel from the donor to the recipient cell. According to this, the

CHAPTER 1. INTRODUCTION molecule may travel a long distance in the blood stream to act upon a different tissue (endocrine), it may target a neighbouring cell in the same tissue (paracrine), or may have a direct feedback on the donor cell (autocrine). A special form of communication occurs when the ligand of a receptor is membrane bound and the receptor on another cell is activated upon the direct contact of the donor cell (juxtacrine) (Berridge 2012b).

1.1.4.2. Receptor-operated calcium channels (ROCCs)

Chemical messengers bind to specific receptors, some of which bear ion-channel properties (ROCs) and gate calcium in response to ligand-binding. ROCs are present in the receptor groups cysteine-loop receptors (CLRs), glutamate receptors (GluRs) and the ATP-sensing P2X receptors (Berridge 2012c).

From the members of CLRs only the nicotinic variant of acetylcholine receptors 2+ (nAChR) and the type 3 serotonin receptor (5-HT3) gate Ca . The nAChR is differentiated from its metabotropic variant in that the stimulation of the latter is linked to the generation of second messengers (Liu et al. 1996; Unwin 1993). Neuronal variants of nAchRs are found on pre-, post- and extrasynaptic membrane surfaces (cholinergic synapses) (Horch and Sargent 1995). Presynaptic Ca2+ transients derived from nAchR activation are involved in the stimulation of transmitter release (Li et al. 1998) and their activation has been implicated in the physiology of learning (Matsuyama et al. 2000). Cholinergic projections are often mixed with non-cholinergic components which might be explained by the stimulatory effect of nAChR on the release of other transmitters (Rye et al. 1984). Apart from the brain, nAChR is dominant on neuromuscular junctions where it transmits the excitation of the nerve onto skeletal muscle cells (Tsuneki et al. 1997). 2+ As stated before, from all serotonin receptors, only the 5-HT3 is a ligand-gated Ca channel (Hargreaves et al. 1994). 5-HT3 directs neurotransmission in parasympathetic synapses of the gut and in the central nervous system in hippocampus, amygdala and hypothalamus (Akasu et al. 1987; Tecott et al. 1993).

CHAPTER 1. INTRODUCTION

Similarly to nAChR, serotoninergic stimulation of presynaptic Ca2+ release is implicated in the facilitation of neurotransmitter release (Turner et al. 2004).

Glutamate is a frequently appearing neurotransmitter in the brain activating both ionotropic (ion-gating) and metabotropic (second messenger coupled) receptors. Ionotropic GluRs have been classified on the basis of their activation properties in response to different chemical compounds (Nakanishi 1992). N-methyl-D-aspartic acid (NMDA) stimulated receptors are highly permeable to Ca2+ (Mayer and Westbrook 1987). Moreover, a subset of α-amino-3-hydroxyl-5-methyl-4-isoxazole- propionate (AMPA)- and kainate-stimulated GluRs are also able to gate Ca2+ ions in addition to Na+ (Murphy and Miller 1989; Gilbertson et al. 1991). Cooperation between AMPA and NMDA receptors has also been demonstrated. It is initiated by the AMPA receptor which triggers the depolarisation of the membrane leading to the removal of a Mg2+ ion from the NMDA receptor which serves to block the Ca2+ flow in its unstimulated state (Rao and Finkbeiner 2007).

The ionotropic P2X receptors are ATP-gated cation channels widely distributed in the central and peripheral nervous system (Collo et al. 1996). They are thought to play a role in nociception, through sensing ATP leakage from damaged cells (North 2004). In addition, P2X receptors have diverse functions in the developing and adult brain (Majumder et al. 2007).

1.1.4.3. Voltage-operated calcium channels (VOCCs)

Electrical communication of cells is directed by voltage-sensitive channels, some of which are Ca2+ channels. The classification of VOCCs is based on their structural and pharmacological properties, but also on their ability to induce different currents (Ertel et al. 2000; Catterall et al. 2005). Accordingly, 3 groups of VOCCs are distinguished: CaV1, CaV2 and CaV3. CaV1 channels are activated by a strong depolarisation evoking the prolonged opening of the channel. They are also called L- type channels referring to their long lasting activation. Cav1.1/dihidropyridine receptor (DHP) has a critical role in skeletal muscle contraction. DHP interacts with

CHAPTER 1. INTRODUCTION ryanodine receptors on the sarcoplasmic reticulum (SR), and the conformational change in the activated channel protein triggers Ca2+ release from the SR (Flucher et al. 1993). Additionally, the DHP induced Ca2+ influx also stimulates ryanodine receptors which are not DHP-coupled and together, these events are responsible for the Ca2+ rise that evokes the contraction of muscle fibers (Meissner and Lu 1995).

Members of the CaV2 channel family (N-, P-,Q- and R-type VOCCs) also require strong depolarisation (Ertel et al. 2000). They are classified by selective toxin antagonists used to detect their specific contributions to neurotransmission (Wright and Angus 1996). Their main role is to direct excitation-coupled secretion at synapses (Scheuber et al. 2004; Kaja et al. 2007). The currents induced by T-type

VOCCs (CaV3) are transient, and they possess a low threshold for activation (Perez- Reyes et al. 1998). Their presence was demonstrated in a variety of cell types with different functions (Perez-Reyes 2003).

1.1.4.4. Capacitive calcium entry

In addition to the mechanisms described above, Ca2+ may also enter to the cell when the intracellular stores are depleted and extracellular Ca2+ is required for the reconstitution of basal Ca2+ concentrations of cell compartments. A model for the so- called capacitive Ca2+ entry was first proposed by Ginsborg et al., who suggested that the hyperpolarizing responses to dopamine in cockroach salivary gland cells depend on a calcium influx from the extracellular space which is filled into an intracellular store (Ginsborg et al. 1980). The first proteins which were implicated in directing the ER/SR depletion-dependent Ca2+ uptake were identified by a siRNA screen against 2,304 proteins in HeLa cells. In this study, the stromal interaction protein 1 (STIM1) was identified to play an important role in maintaining Ca2+ stores as the downregulation of these proteins resulted in decreased Ca2+ uptake after store depletion by thapsigargin or histamine (Liou et al. 2005). Later the following mechanism for capacitive Ca2+ entry was proposed: the channel is formed by the interaction of STIM1 on the ER/SR and the ORAI1 pore forming protein on the plasma membrane (Prakriya et al. 2006). The luminal part of STIM1 contains a SAM

CHAPTER 1. INTRODUCTION

(sterile α motif) structure and an EF hand which undergoes dimerisation in the absence of Ca2+ initiating the opening of the channel (Stathopulos et al. 2006). Transient receptor potential channel (TRPC) family has been also implicated to interact with STIM1 and direct store operated Ca2+ uptake under certain conditions (Alicia et al. 2008).

1.1.4.5. Second messenger-operated channels (SMOCs)

Extracellular stimuli are able to induce Ca2+ uptake indirectly via the activation of receptors coupled to second messenger generation (metabotropic receptors). Although metabotropic receptors of the Ca2+-signalling toolkit will be mentioned later, this section describes Ca2+ channels which are sensitive to the molecules produced by these receptors. Ca2+ channels in this group are collectively known as SMOCs.

Cyclic nucleotide gated channels (CNGCs) are activated by cAMP (cyclic adenosine monophosphate) or cGMP (cyclic guanosine monophosphate) and are permeable not only to Ca2+ but also to Na+ and K+. CNGCs are well characterised in photoreceptors where cGMP as a second messenger was first established (Fesenko et al. 1985; Gerstner et al. 2000). CNGCs are active participants of phototransduction being permeable to cations in dark when the cGMP level is high and shutting the inward current when cGMP is hydrolysed in response to light (Koch et al. 2002).

Arachidonic acid (AA) regulated Ca2+ channels (ARCs) are novel members of SMOCs (Mignen and Shuttleworth 2000). AA is generated by the Phospholipase A2 enzyme in response to receptor stimulation (Murakami and Kudo 2002). ARCs are composed of ORAI1 (plasma membrane constituent of capacitive Ca2+ entry, section 1.2.2.4.) and Orai3 subunits (Mignen et al. 2009). Unfortunately, little is known about their function. They have been found to trigger Ca2+ oscillations in pancreatic acinar cells in response to cholecystokinin (Mignen et al. 2005).

CHAPTER 1. INTRODUCTION

A subgroup of the diverse family of TRPCs is activated by the second messenger diacylglycerol (DAG) (Hofmann et al. 1999). Their contribution is required for the transduction of sensory signals such as nociception (Woo et al. 2008). The mechanism which provides the production of DAG will be discussed later.

1.1.4.6. Calcium pumps and -exchangers of the plasma membrane

As described earlier, transporters and pumps maintain the resting levels of Ca2+ in the cytoplasm. The most critical partners in this process are the plasma membrane Ca2+- ATPase (PMCA) and the Na+/Ca2+ exchangers (NCXs). PMCA senses the levels of the intracellular Ca2 by being stimulated directly by Ca2+ or by the Ca2+-binding molecule calmodulin. It utilises the energy of ATP to move Ca2+ across the membrane (Wetzker et al. 1987). By possessing high affinity to Ca2+ and a slow rate of Ca2+-transport it has been implicated in the fine tuning of intracellular Ca2+ levels (Brini and Carafoli 2011). In contrast, NCXs utilise the energy of the passively flowing Na+ to transfer 2 Ca2+ against its electrochemical gradient. NCX has a low affinity towards Ca2+ whereas its transport rate is high indicating its role in rapid response to high Ca2+ levels. Consequently, PMCA and NCX possess a dynamic cooperation to extrude Ca2+ from the cells and restore basal levels (Juhaszova et al. 2000). In addition, NCX is capable of switching into a reverse mode (transporting Ca2+ to the cytosol) during depolarisation or when the intracellular Ca2+ or Na+ concentrations are low or high, respectively (Pieske et al. 2002; Weber et al. 2002). This further substantiates its role in restoring physiological basal Ca2+ levels.

1.1.4.7. Second messenger-coupled receptors involved in Ca2+ signalling

G-protein-coupled receptors (GPCRs) constitute the largest protein family in the human body comprising more than 1,000 members (Nemoto et al. 2011). The common feature of GPCRs is the ability to activate heterotrimeric G proteins which is accompanied by the separation of the trimer into the second messengers Gα and the

CHAPTER 1. INTRODUCTION

Gβγ dimer. In the heterotrimeric form, Gα is bound to GDP which is released upon GPCR activation. The subsequent binding of GTP triggers the dissociation of the subunits, however the intrinsic GTPase activity of the Gα leads back to the formation of the receptor-heterotrimer complex (Morris and Malbon 1999). All GPCRs have seven transmembrane segments connected by extra- and intracellular loops (Unger et al. 1997). GPCRs are divided into 3 subfamilies (Gether 2000): the rhodopsin/β2 adrenergic receptor like subfamily (classical structure, receptors for gonadotropins, neurotransmitter amines, oxytocin, vasopressin, melatonin and others), the glucagon/vasoactive intestinal polypeptide/calcitonin receptor like subfamily (long N terminus with disulfide bridges, receptors for growth hormone releasing hormone, glucagon, corticotropin-releasing factor, calcitonin, vasointestinal peptide and others) and the metabotropic neurotransmitter/calcium receptors (long N and C terminus, short third intracellular loop, receptors for metabotropic glutamate and GABA receptors, calcium receptors, pheromone and taste receptors and others).

The diversity of GPCR functions resides in their specific coupling to one of the numerous G-proteins. Classification of G proteins is based on the Gα subunit.

According to this, 4 subfamilies are distinguished: the Gs (Gs, Golf), the Gi/o (Gi, Gt,

Go, Ggust and Gz) the Gq/11 (Gq, G11, G14, G15, G16) and the G12/13 families (G12, G13) (Hamm and Gilchrist 1996; Neves et al. 2002). In each subfamily, G-protein activation is coupled to the regulation of different effectors. Members of the Gq family are linked to PLC activation (Smrcka et al. 1991). PLCs cleave phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and generate the second messengers, inositol 1,4,5-triphosphate (I(1,4,5)P3) and diacylglycerol (DAG). 2+ I(1,4,5)P3 triggers Ca release from I(1,4,5)P3 sensitive stores and DAG activates protein kinase C (PKC) (Wu et al. 1992a; Jhon et al. 1993). Activation of PLCs and the importance of their enzyme activity will be further discussed later in this Chapter.

GαS triggers the activation of the adenlylate cyclase enzyme (AC) resulting in an elevation in cAMP concentration that consequently activates protein kinase A (PKA) (Alousi et al. 1991). This cascade continues with the stimulation of Ca2+ channels

CHAPTER 1. INTRODUCTION such as L-type VOCs or dihydropyridine-sensitive channels by the activated PKA

(Rotman et al. 1995; Kavalali et al. 1997; Basavappa et al. 1999). Conversely, Gi reduces intracellular cAMP level, by inhibiting the activity of AC (Taussig et al.

1993). Transducin, a photoreceptor-specific Gi, directs the molecular process of photoreception by activating the cGMP phosphodiesterase to hydrolyse cGMP in response to light which leads to the closure of CNGCs (section 1.2.2.3) (Deterre et al. 1988). A unique type of Gi, gustducin, has also been found to participate in the signalling events of bitter taste sensation (Yan et al. 2001).

The G12 and G13 proteins are recently discovered members and they have been shown to interact with a range of proteins including Rho guanine nucleotide exchange factor (RhoGEF), Ras GTPase activating protein, phospholipase D, and PLCε (Kurose 2003).

The Gβγ dimer that remains after the dissociation of Gα from the heterotrimer also exerts stimulatory activity on a range of proteins. For instance, it activates several PLC isoforms (Boyer et al. 1992; Park et al. 1993; Wu et al. 1993; Wang et al. 2000; Wing et al. 2001; Zhou et al. 2008), stimulates phosphoinositide 3-kinase (Zaballos et al. 2008) and induce the phosphorylation of the transcription factor STAT3 (Yuen et al. 2010).

An additional receptor family whose activation is involved in the regulation of Ca2+ dynamics are the receptor tyrosine kinases (RTKs). RTKs are predominantly monomers that undergo dimerisation upon ligand-induced stimulation which event triggers the autophosphorylation of the C-terminus facing the cytoplasm. In certain cases they may possess a tetrameric structure which was described for insulin receptors (Van Obberghen et al. 1985; Hubbard et al. 1998). The activated RTK recruits and phosphorylates SRC homology 2 (SH2) or phosphotyrosine-binding domain (PTB)-containing proteins such as PLCγ1, small G-proteins or the GTPase activating protein (Anderson et al. 1990; Hubbard et al. 1998).

CHAPTER 1. INTRODUCTION

1.1.5. Calcium stores and their molecular machinery

1.1.5.1. What is the importance of accumulating calcium in the cell?

Releasable intracellular Ca2+ pools have a major role in regulating the physiological response to cellular processes. As described before, the resting intracellular Ca2+ concentration is maintained at a low level (~10-7 M) by ion pumps and exchangers of extra- and intracellular membranes (Usachev et al. 1995; Bygrave and Benedetti 1996). By the accumulation of Ca2+ into membrane enclosed structures nature has found an incredible way to broaden the versatility of Ca2+ dynamics in the cell. The most studied and perhaps most important Ca2+ store is the ER/SR. Although since its discovery many intracellular organelles have been shown to deposit and release Ca2+ in response to stimuli (Fatt and Ginsborg 1958; Endo et al. 1970; Koch 1990; Verkhratsky and Petersen 1998; Calcraft et al. 2009;). These stores differ in the concentration of Ca2+ they are capable of accumulating. The ER/SR is able to maintain a Ca2+ concentration of 10-3 M at rest. In contrast, the mitochondrial basal Ca2+ level is similar to that of the cytosol and its Ca2+ uptake is associated with cytosolic Ca2+ rises (Bygrave and Benedetti 1996; Moreau et al. 2006). In the following sections the molecular machinery that controls Ca2+ uptake and release in these stores is discussed.

1.1.5.2. Calcium homeostasis of the ER/SR

As mentioned above the ER/SR is a crucial Ca2+ store being able to maintain a large pool of Ca2+. Its Ca2+ uptake mechanism is controlled by the sarco/endoplasmic reticulum ATPase (SERCA). SERCA is similar to the PMCA, however, it is capable of transporting two Ca2+ ions from the energy generated by a single ATP molecule whereas its plasma membrane equivalent only carries one Ca2+/ATP (Filomatori and Rega 2003). A large proportion of the accumulated Ca2+ is not free but rather bound by local Ca2+ buffers such as calreticulin (luminal) and calnexin (integral to the membrane) (Tjoelker et al. 1994; Mery et al. 1996). This buffering capacity is crucial

CHAPTER 1. INTRODUCTION for the ability of the ER/SR to store large amounts of Ca2+ (Mery et al. 1996). Additionally, Ca2+ buffers of the ER/SR serve as chaperones and Ca2+ regulates the folding of newly synthesised proteins (Lodish et al. 1992; Saito et al. 1999).

The release of the ER/SR-based Ca2+ pool is dictated by two main receptor groups

RyRs and IP3Rs. RyRs have been identified in the SR of skeletal muscle cells where they are physically coupled to DHP receptors and are stimulated by depolarisation (Lai et al. 1988; Marty et al. 1994). However, extracellular Ca2+ inflow is able to induce Ca2+ release from the SR generating calcium-induced calcium release (CICR) from the uncoupled RYRs and this mechanism of activation is crucial in non-muscle cells where DHP is absent (Kim et al. 1983; McPherson et al. 1991). Recent evidence also suggests that intraluminal Ca2+ modulates the open probability of RyR (Gyorke and Gyorke 1998). Additionally, RyRs are activated by other factors or second messengers such as ATP, Mg2+ or cyclic adenosine diphosphate ribose (cADPR) (Meissner et al. 1986; Ogunbayo et al. 2011)

2+ Ca release by IP3Rs is stimulated by the PLC enzymatic product, I(1,4,5)P3. IP3Rs are also stimulated by intracellular Ca2+, although if the cytosolic [Ca2+] reaches 300 2+ nM it becomes inhibitory on IP3R activity (Iino 1990). Ca release by IP3R activation or by other sources is able to stimulate the activity of Ca2+-sensitive PLCs (PLCδ, PLCζ and PLC η) and this pathway has been proposed as a unique PLC- dependent CICR mechanism (Popovics and Stewart 2012b; Yamamoto et al. 2000).

IP3Rs and RyRs coexist in a variety of cells and crosstalk between the two has been described (Boittin et al. 1999; Haak et al. 2001; Gordienko and Bolton 2002).

1.1.5.3. Mitochondrial Ca2+ transporters

It has been long known that mitochondrial Ca2+ sensitive enzymes are finely tuned by small changes in matrix Ca2+ levels (Denton and McCormack 1980; Jouaville et al. 1999). Subsequently, it was revealed that mitochondria are capable of sequestering Ca2+ in a rapid manner (Rizzuto et al. 1992) which may lead to the matrix Ca2+ concentration reaching millimolar levels in certain cases (Montero et al.

CHAPTER 1. INTRODUCTION

2000). In contrast to the ER/SR, the mitochondrial Ca2+ uptake is activated only when the cytosolic Ca2+ concentration rises above 300 nM (Colegrove et al. 2000). In addition, the matrix Ca2+ concentration precisely follows cytosolic Ca2+ oscillations and therefore a role for mitochondria in buffering intracellular Ca2+ signals has been widely suggested by numerous groups (Werth and Thayer 1994; Robb-Gaspers et al. 1998).

The inner and outer mitochondrial membranes (OMM and IMM) largely differ in Ca2+ permeability. Transport across the OMM is regulated by the voltage dependent anion channel (VDAC) which lets small molecules including Ca2+ pass non- selectively (Hodge and Colombini 1997; Gincel et al. 2001). However, the depolarisation-induced closure of VDAC blocks the transport of small metabolites and facilitates the transport of Ca2+ to mitochondria (Tan and Colombini 2007). Although, a variety of mechanisms have been proposed to direct the transfer of Ca2+ through the IMM, only the role of the Ca2+ uniporter (MCU) has been verified (Jean- Quartier et al. 2012). MCU, contrary to its name, acts more like a channel as it does not require energy for the transport. Moreover, it is activated by Ca2+ itself and therefore directs Ca2+-induced Ca2+ uptake to the mitochondria (Moreau et al. 2006). Recent findings suggest that MCU is a multiprotein complex but only two of its components have been identified to date (Perocchi et al. 2010; Baughman et al. 2011).

Mitochondrial Ca2+ efflux is provided by the mitochondrial Na+/Ca2+ exchanger + 2+ (NCXmito) and the H /Ca antiporter. Although their existence and mechanism have + 2+ been known for some time, candidates for the genes encoding NCXmito and H /Ca antiporter (HCA) have only recently been suggested (Jiang et al. 2009; Palty et al.

2010). NCXmito operates similarly to its plasma membrane relative, however its main role is the opposite: it extrudes Ca2+ from the mitochondria to the cytosol (Palty et al. 2010). HCA is able to change the direction of Ca2+ transport depending on the extra- and intramitochondrial Ca2+ levels and pH suggesting a possible interplay with the Ca2+ uniporter in Ca2+ uptake (Jiang et al. 2009).

CHAPTER 1. INTRODUCTION

The mitochondrial permeability transition pore (mPTP) is a molecular complex that forms across the IMM and OMM when mitochondria are overloaded with Ca2+ at micromolar cytosolic [Ca2+] (Crompton 1999). It is composed of at least three proteins: the previously mentioned VDAC in the outer membrane, the adenine nucleotide translocase (ANT) in the inner membrane and the cytosolic protein, cyclophilin D (CypD) (Crompton 1999). The channel complex is suggested to exhibit two phases of activation. In its low conductance state, mPTP opens in a pH dependent manner and only extrudes small ions such as Ca2+ out of the mitochondria. The pore is switched to a high conductance state depending on the matrix Ca2+ concentration and leads to a nonspecific release of both metabolites and ions. The opening of the second type of mPTP is usually paired with apoptotic cell death (Ichas and Mazat 1998; He and Lemasters 2002). In conclusion, the mitochondrial Ca2+ transporting system has an important role in modifying cytoplasmic Ca2+ signals and regulating apoptosis.

A newly evolving field in the research of Ca2+ store dynamics is the investigation of 2+ the cross-talk between ER/SR and mitochondrial membranes. Ca released by IP3Rs is immediately taken up by the mitochondria. This mechanism is most pronounced at the mitochondria-associated ER-membranes where a direct contact of the two organelles has been shown (Rizzuto et al. 1998). Interestingly, IP3R and VDAC is physically coupled through GRP75/mortalin which further indicates the existence of a direct Ca2+ flow from the ER to the mitochondria (Szabadkai et al. 2006). In addition, ER/SR-depletion-controlled store-operated Ca2+ entry is inhibited by mitochondrial depolarisation (Glitsch et al. 2002). Mitochondrial Ca2+ uptake also 2+ inhibits the positive feedback of Ca on IP3Rs substantiating the existence of a strong interplay between these 2 organelles (Hajnoczky et al. 1999).

CHAPTER 1. INTRODUCTION

1.1.5.4. Endosomes, lysosomes and secretory vesicles as calcium stores

Secretory vesicles are also capable of behaving as releasable Ca2+ stores.

Interestingly, secretory vesicle membranes contain RyRs rather than IP3Rs (Mitchell et al. 2001). The Ca2+-induced Ca2+ release from this store has been proposed to modulate the exocytosis of already primed vesicles (Mitchell et al. 2001). Lysosomes, endosomes and secretory vesicles are functionally related in responding to nicotinic acid adenine dinucleotide phosphate (NAADP) by releasing Ca2+ (Churchill et al. 2002; Mitchell et al. 2003). NAADP is converted from nicotinamide adenine dinucleotide phosphate (NADP) by ADP ribosyl cyclase, however little is known as to how its enzyme activity is regulated by extra- or intracellular stimuli (Rutter 2003). The receptors for NAADP have recently been identified as the two- pore channels (TPCs) (Calcraft et al. 2009; Ruas et al. 2010), however, subsequent studies revealed that a linker protein is required for the NAADP-dependent activation of TPCs (Walseth et al. 2011). Patch-clamp studies reported that the TPC channels on their own are Na+-selective and are unresponsive to NAADP - hence the scientific attention shifted towards the identification of the proposed linker protein (Wang et al. 2012).

1.1.5.5. Nuclear calcium signalling

Local elevations of Ca2+ in the nucleus are crucial for the regulation of nuclear processes (Dolmetsch et al. 1998). The involvement of nuclear Ca2+ signals in cell proliferation was shown by the utilisation of a Ca2+ buffer that was artificially linked to a nuclear localisation signal (NLS) and upon its transfection to the cell it specifically reduced nuclear Ca2+ levels (Rodrigues et al. 2007). Although Ca2+ signals are registered throughout the nucleus, Ca2+ is likely to be stored within the nuclear envelope (NE), as it is able to freely diffuse through the nuclear pore complex (Eder and Bading 2007). The NE is continuous with the ER/SR which suggests a high similarity between their molecular strategies to handle Ca2+. Thus it

CHAPTER 1. INTRODUCTION

2+ is not surprising, that Ca release from the NE is directed by RyRs and IP3Rs (Humbert et al. 1996; Erickson et al. 2004). In addition, NE Ca2+ stores are also sensitive to NAADP and cADPR (Gerasimenko et al. 1995; Adebanjo et al. 2000). Moreover, the nuclear envelope is able to enter the nucleoplasm and form a reticular network which extends the surface of the NE and may allow the generation of local Ca2+ transients inside the nucleus (Wu and Bers 2006). There is still a lot to learn about nuclear Ca2+ signalling and hopefully more details will come to light in the near future.

1.1.5.6. Is the Golgi apparatus a calcium store?

Recently, the Golgi apparatus (GA) as a potential Ca2+ store has also been suggested. 2+ This idea is clearly supported by the presence of SERCA, IP3R and a unique Ca ATPase (secretory pathway Ca2+ ATPase, SPCA) on GA-related membranes (Gunteski-Hamblin et al. 1992; Taylor et al. 1997; Pinton et al. 1998). SPCA differs from other similar pumps in that it transports Mn2+ in addition to Ca2+ (Dode et al. 2005). It has been shown to be crucial for the maintenance of GA-Ca2+ homeostasis required for neuronal differentiation (Sepulveda et al. 2009). In addition, it has recently been revealed that transport of the Ca2+ via the SPCA is the main process to restore basal Ca2+ level between transients in human spermatozoa (Harper et al. 2005).

Interestingly, the free Ca2+ concentration gradually decreases with the distance of GA compartments from the ER (cis-, medial- and trans-compartments), but this effect seems to be due to the increasing buffering capacity down the secretory pathway (Pizzo et al. 2010). Nevertheless, the hypothesised differences suggest that the molecular composition of the Ca2+ signalling toolkit throughout the GA is not uniform.

CHAPTER 1. INTRODUCTION

1.1.5 Calcium dynamics of the cytosol

1.1.5.1. Cytosolic calcium buffers

Ca2+ buffers serve to modulate the temporal and spatial aspects of calcium signals by decreasing the amount of available free Ca2+. Buffering capacity of the cytosol is essential for the maintenance of normal cell homeostasis, especially in neurons (Baimbridge et al. 1992). Abnormal expression of Ca2+ buffers leads to disturbances in the Ca2+-handling of the cell. Decreased levels of the cytosolic buffer, calbindin D-28 were found in aging neurons and in Alzheimer`s disease, which may contribute to the elevated Ca2+ levels registered in these conditions (Kirischuk et al. 1992; Sutherland et al. 1993; Lally et al. 1997). Calretinin is involved in the modulation of neuronal excitability and long-term potentiation (LTP) (Schurmans et al. 1997) and parvalbumin contributes to the fine tuning of skeletal muscle cell Ca2+ levels (Muntener et al. 1995). These examples indicate the crucial role of the buffering capacity of the cytosol for the maintenance of cell physiology.

1.1.5.1. Calcium sensors of the cytosol

The diverse group of Ca2+ sensors convert changes in intracellular Ca2+ levels to the activation of their target proteins. This is usually directed by a conformational change in their protein structure upon Ca2+-binding. Calmodulin participates in the activation of a number of different proteins as a Ca2+ sensor. It stimulates downstream signalling elements, such as Ca2+/calmodulin-dependent protein kinases (CaMKs), adenylate cyclase, cyclic nucleotide phosphodiesterase and PLCβ enzymes (Huang et al. 1981; Eliot et al. 1989; McCullar et al. 2003). Stimulation of the myosin light chain kinase by calmodulin, is an essential step for actin-myosin interaction in skeletal muscle cells (Hathaway and Adelstein 1979). Interestingly, the activated calmodulin exerts a feedback effect to decrease the intracellular Ca2+ level by stimulating PMCAs, which extrude Ca2+ to the extracellular space (James et al. 1988). Another Ca2+ sensor, troponin C, is essential for skeletal muscle contraction.

CHAPTER 1. INTRODUCTION

When troponin C is activated by Ca2+, binding sites on myosin become accessible to the actin filament (Ogawa 1985). In the above mentioned molecules, Ca2+ triggers a conformational change upon binding, however, other sensors may undergo dimerisation. This mechanism regulates the function of synaptotagmin which is a molecule essential for vesicle exocytosis (Chapman et al. 1996). These molecules are just a subset of the many sensors identified to date. For instance, the neuronal calcium sensor family is comprised of 14 members possessing crucial roles in neuronal Ca2+ signalling, such as regulating neurotransmission, synaptic plasticity, receptor trafficking, neuronal growth and survival (Burgoyne 2007).

1.1.5.1. Calcium sensitive enzymes and processes - the effectors

Effectors of the Ca2+ signalling toolkit comprise Ca2+ sensitive enzymes, transcription factors and ion channels. Ca2+ sensitive enzymes are mostly kinases such as CaMKs which are activated by both calmodulin and Ca2+. Apart from many other of their targets, they phosphorylate CREB (cAMP responsive element binding protein) transcription factor to regulate the expression of genes containing CREB responsive elements in their promoters (Montminy and Bilezikjian 1987; Matthews et al. 1994). The protein kinase C family also contains Ca2+ sensors, although they are not directly stimulated by Ca2+. The effect of Ca2+ is translated by PLCs by generating the second messenger DAG which stimulates certain PKC isoforms (Shinomura et al. 1991). Nitric oxide synthase (NOS) produces the small diffusible hormone/transmitter, nitric oxide. It was shown that the various NOS subtypes are activated by Ca2+ directly or indirectly via calmodulin (Forstermann et al. 1991).

As mentioned, ion-channels are also effectors of the Ca2+ pathway. Ca2+-activated potassium channels which are regulated by calmodulin modulate neuronal excitability (Faber and Sah 2003; Schumacher et al. 2001). Moreover, calcium activated chloride channels play important roles in sensory transduction, neuronal depolarisation and muscle contraction (Hartzell et al. 2005).

CHAPTER 1. INTRODUCTION

PLCs are also effectors of the Ca2+-signalling toolkit. Whilst the purpose of the thesis is to characterise a PLC subtype, PLCη2, the following part of this Chapter describes the PLC family and introduces the unique features of PLCη2 that encouraged the scientific work on this topic.

1.2. The classification and comparative description of Phospholipase C enzymes

1.2.1. Phospholipids: their regulation and their importance in cellular signalling

Phospholipid metabolism is a highly organised process and involves the participation of multiple intermediary molecules, many of them possessing biological activity as second messengers. As the basic element, phosphatidate (or otherwise known from its acidic form, phosphatidic acid (PA)) serves as the substrate for reaction cascades resulting in the production of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG) and most importantly phosphatidylinositol (PI) (Kent 1995). The latter is produced by the PI-synthase from the PA derivative, cytidine diphosphate- (CDP-) diacylglycerol and myo-inositol (Figure 1.3) (Paulus and Kennedy 1960).

Enzymatic phosphorylation of PI provides the basis for the production of numerous phosphatidylinositol- (Figure 1.4.) and inositol phosphates (Figure 1.5.). Specific kinases join a phosphate group to the 3, 4 or 5 positions of the inositol ring to create PI-monophosphates, -bisphosphates or trisphosphate with different biological activities. Phosphatidylinositol 4-phosphate (PI(4)P) is enriched in trans-Golgi- membranes and regulates trafficking of membranes to cell surface by recruiting transport proteins (Godi et al. 2004). Some kinases exhibit important signalling functions by being activated in response to extracellular stimuli and producing PI- phosphates with important second messenger functions. For instance, PI 3-kinases

(PI3K) are stimulated by insulin or GPCRs and the generated PI(3,4,5)P3 serves as a

CHAPTER 1. INTRODUCTION docking site for proteins (Whitman et al. 1988; Murga et al. 1998; Brown et al. 1999). One example is the recruitment of AKT to the plasma membrane that allows its phosphorylation and consequently its activation by PDK1 (3-phosphoinositide dependent protein kinase-1) (Meier and Hemmings 1999).

Figure 1.3.: Biosynthesis of phosphatidylinositol. PI-synthase catalyses the production of PI from cytidine diphosphate diacylglycerol and myo-inositol. The figure does not demonstrate the subsequent exiting of citidine monophosphate (CMP) from the reaction. Figure is downloaded from: http://lipidlibrary.aocs.org/lipids/pi/index.htm.

The link between PI-phosphate and inositol phosphate metabolism is provided by the hydrolysing activity of phospholipase c enzymes (detailed in the next section).

Several molecule arise from I(1,4,5)P3 which are generated by the actions of numerous kinases and phosphatases (Figure 1.5.). A subset of these soluble inositol phosphates possesses regulatory roles on signalling molecules. For example, the inositol 3,4,5,6-tetrakisphosphate (I(3,4,5,6)P4) regulates plasma membrane chloride channels (Xie et al. 1996). Inositol-monophosphatases recycle the inositol-ring for phospholipid biosynthesis. Interestingly, their activity is inhibited by LiCl, a chemical traditionally given to treat manic depression, which effect is thought to be based on the preservation of the inositol phosphate-pool (Belmaker et al. 1996).

CHAPTER 1. INTRODUCTION

Although this is not the only effect of Lithium treatment as for example it downregulates Glycogen synthase kinase 3 β affecting gene expression levels (Yin et al. 2006).

As a whole, phospholipid metabolism plays a central role in cellular signalling by possessing multiple steps, some of which are regulated by unique external stimuli. Their cleavage by phospholipases introduces an additional level for the generation of lipid second messengers. The next section aims to describe these enzymes.

Figure 1.4.: Phosphatidylinositol metabolism. Phosphatidylinositol and its mono-, bis-, and trisphosphate forms are converted into each other by multiple phosphatases and kinases (indicated by numbers). Phospholipase C enzymes catalyse the cleavage of phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol 1,4,5- trisphosphate which latter serves as the basis of the inositol phosphate metabolism. The inositol ring is recycled for the synthesis of phosphatidyl-inositol. Note that abbreviation scheme of phospholipids is altered in the text. Figure is taken with permission from Portland Press Ltd, (Berridge 2012a).

CHAPTER 1. INTRODUCTION

Figure 1.5.: Inositol-phosphate metabolism. Inositol 1,4,5-trisphophate serves as the source of the various inositol phosphates that may carry a phosphate group on any of the 6 position of the inositol ring. According to this, a range of inositol phosphates from the monophosphates to the one hexaphosphate are known to exist. Moreover, formation of diphosphate groups are also possible (PPInsP4, PPInsP5 and [PP]2InsP4). Dephosphorylation of inositol-monophosphates produce an inositol ring which is recycled to phosphoinositol synthesis. Note that abbreviation scheme of phospholipids is altered in the text. Figure is taken with permission from Portland Press Ltd. (Berridge 2012a)

1.2.2. Phospholipase enzyme superfamily

The classification of phospholipases is based on their ability to hydrolyse phospholipids at different chemical bonds resulting in the generation of various

CHAPTER 1. INTRODUCTION products/second messengers (Figure 1.6). Accordingly, phospholipases have been divided into 5 groups: phospholipase A1, A2, B, C and D (PLA1, PLA2, PLB, PLC and PLD).

Figure 1.6.: The site of hydrolysis catalysed by different phospholipases. Description is found in the text. Figure is taken with permission from Wiley-VCH Verlag GmbH & Co (Wilton 2005).

PLA1 cleaves in the sn-1 position whereas PLA2 attacks the sn-2 bond to produce acyl-lysophospholipids and fatty acids (Aoki et al. 2007; Burke and Dennis 2009).

Diversity also occurs within the PLA1 family; depending on the substrate specificity of PLA1 the 2 main products are lysophosphatidic acid or lysophosphatidylserine cleaved from phosphatidic acid or phosphatidylserine, respectively (Aoki et al. 2007). AA is often sequestered at the sn-2 bond of phospholipids and therefore released to the cytosol upon stimulation of PLA2 (Perez-Chacon et al. 2009).

PLB enzymes possess the enzymatic activity of both PLA1 and PLA2 (Gassama- Diagne et al. 1992). In mammals, PLBs have important roles in the digestion of dietary lipids and participate in the molecular machinery of neutrophil immune response (Xu et al. 2009). PLDs hydrolyse the distal phosphodiester bond to produce phosphatidic acid and lipid headgroups such as choline (Liscovitch et al. 2000). PA is important regulator of cellular processes such as exocytosis/membrane trafficking

CHAPTER 1. INTRODUCTION

(Jones et al. 1999). With the generation of PA, PLDs also stimulate the membrane- associated PA-selective PLA1 enzyme group (Aoki et al. 2007).

PLCs catalyse the cleavage of the proximal phosphodiester bond to produce inositol-

1,4,5-trisphosphate (I(1,4,5)P3) and diacylglycerol (DAG) from phosphatidylinositol

4,5-bisphosphate (PI(4,5)P2, Figure 1.7..) (Fukami 2002). PLC mediated pathways are crucial for receptor mediated responses in a variety cell types throughout the life from the oocyte and to ageing neurons. The following sections will discuss their discovery, classification, domain structure, regulation and physiological role.

Figure 1.7.: Generation of second messengers by PLCs. PLC catalyzes the cleavage of PI(4,5)P2 at the proximal phosphodiester bond to generate the accumulation of DAG at the membrane and to release the diffusible I(1,4,5)P3 to the cytosol. Figure is taken with permission from Biochemistry and Molecular Biology (Suh et al. 2008).

1.2.3. Phospholipase C enzymes

1.2.3.1. A short history of the discovery of PLCs

The road towards the discovery and characterisation of PLCs began with the discovery by Hokin & Hokin in 1953. They measured increased P32 incorporation into phospholipids in pancreatic tissue slices in response to cholinergic drugs which was followed by increased amylase secretion (Hokin and Hokin 1953). At this time neither the transducing mechanism nor the identity of the lipid was known. After a long break, Abdel-Latif et al. and Allan and Michell reached the next breakthrough 32 in 1978. They reported the breakdown of P labelled PI(4,5)P2 in response to

CHAPTER 1. INTRODUCTION norephinephrin in rabbit iris muscle cells and determined the Ca2+-dependence of the phosphodiesterase activity (Abdel-Latif et al. 1978; Allan and Michelle 1978).

This study also identified I(1,4,5)P3 as the possible derivative of PI(4,5)P2. Following these findings a number of articles appeared reporting rapid breakdown of PI(4,5)P2 and elevation in I(1,4,5)P3 levels following treatments by various receptor agonists (Putney 1982; Weiss et al. 1982; Agranoff et al. 1983; Berridge et al. 1983). Berridge et al. combined the anion exchange and ionophoresis techniques to identify and measure inositol metabolites in the water soluble fraction of tissues with different origins. It was found that the primary product of activated cells is the I(1,4,5)P3 and other metabolites such as inositol 1-phosphate or inositol 1,4-bisphosphate are produced subsequently by phophatases (Berridge et al. 1983). I(1,4,5)P3 was then found to initiate Ca2+ release from the ER in permeabilised pancreatic cells (Streb et al. 1983) but the exact mechanism was still a debate. Soon after, a number of 32 publications revealed the presence of [ P]I(1,4,5)P3 in the microsomal cell fraction indicating the existence of specific I(1,4,5)P3 receptors (Baukal et al. 1985; Spat et al. 1986a; Spat et al. 1986b; Guillemette et al. 1987). In the following 2 years the receptor was purified from rat cerebellum (Supattapone et al. 1988) and the cDNA of the first variant was cloned (Furuichi et al. 1989a; Furuichi et al. 1989b).

In parallel, other studies focused on the membrane bound derivative of PI(4,5)P2, DAG, which also rapidly increases following external stimuli (Kennerly et al. 1979; Takai et al. 1979). DAG participates in the activation of several PKC isoforms (Takai et al. 1979). Interestingly, the initial PLC stimulated rapid accumulation of DAG is followed by a short decline and a delayed increase which is not derived from PLC activity directly (Nishizuka 1995).

Subsequently, proteins possessing the proposed phospholipase activity were also identified. The first successful purification of a PLC was in 1981 by Takenawa et al. from rat liver by using multiple chromatography steps (Takenawa and Nagai 1981). In addition, several biochemical properties, such as Ca2+ sensitivity, pH optimum and lipid preference were assessed. This study was followed by the identification of other

CHAPTER 1. INTRODUCTION isozymes in sheep seminal vesicular gland (Hofmann and Majerus 1982), bovine brain (Ryu et al. 1986; Katan and Parker 1987; Rebecchi and Rosen 1987; Ryu et al. 1987), guinea pig uterus (Bennett and Crooke 1987), rat brain and liver (Fukui et al. 1988; Homma et al. 1988) and human platelets (Banno et al. 1988). The accelerated occurrence of new isoforms made it necessary to establish a system for their classification PLCs. Rhee et al. was the first to standardise the nomenclature of PLCs and to group them into α, β, γ, δ and ε classes sorted by immunoblotting comparisons with the existing antibodies (Rhee et al. 1989). The order of the groups was consistent with the chronology of their purification date. Subsequently, the DNA sequence of various PLC isoform was published (Bennett et al. 1988; Katan et al. 1988; Suh et al. 1988). After cloning and expression in mouse fibroblast cells, PLCα did not exhibit phospholipase activity and its transfer into Trp-Cys-Gly-His-Cys-Lys motif containing proteins was required (Hirano et al. 1994). Currently six distinct classes of PLCs are known to exist in mammals; PLC-β, -γ, -δ, -ε, -ζ and –η (Table 1.1.). They have been classified on the basis of amino acid sequence, domain structure, and by the mechanism through which they are recruited in response to activated receptors (Suh et al. 2008).

CHAPTER 1. INTRODUCTION

η2)

1,7

3,3

6,7

3,3

1,7

18,3

13,3

26,7

(PLC

PLCeta2 PLCeta2

η1)

1,5

4,5

1,5

4,5

9,1

24,2

37,9

13,6

(PLC

PLCeta1 PLCeta1

of of the total

ζ1)

1,9

98,1

PLCzeta1 (PLC PLCzeta1

ε1)

0,7

2,8

0,7

7,7

4,2

3,5

9,2

0,7

5,6

0,7

1,4

0,7

1,4

0,7

28,9

21,1

(PLC

PLCepsilon1 PLCepsilon1

δ4)

1,5

7,5

1,5

4,5

1,5

4,5

expressed expressed as percentage

10,4

11,9

23,9

(PLC

PLCdelta4 PLCdelta4

δ3)

1,2

1,8

1,2

4,2

1,2

9,7

7,3

7,9

4,2

1,8

2,4

1,2

1,8

5,5

2,4

10,3

31,5

(PLC

PLCdelta3 PLCdelta3

δ1)

5,9

0,3

2,4

0,3

0,6

0,9

2,1

0,3

1,5

8,9

0,3

3,6

2,7

0,3

4,1

0,6

0,9

0,3

7,1

2,4

2,7

22,5

(PLC

PLCdelta1 PLCdelta1

γ2)

1,2

0,6

2,9

0,6

1,2

0,6

1,2

0,6

2,3

6,4

4,1

1,2

1,8

0,6

3,5

2,9

8,2

0,6

1,8

0,6

9,4

1,2

42,7

(PLC

PLCgamma2 PLCgamma2

γ1)

0,9

0,6

0,3

1,6

0,6

2,2

3,5

0,3

3,5

2,8

9,8

7,6

0,3

7,6

1,9

0,3

2,5

0,6

3,8

1,5

4,4

21,5

10,4

related related EST clones in a certain tissue

(PLC

PLCgamma1 PLCgamma1

β1)

3,4

0,4

0,9

3,9

5,2

0,4

7,7

2,6

5,6

7,3

6,4

4,3

1,7

2,6

1,7

2,1

0,9

6,9

2,1

13,3

18,9

PLCbeta1 (PLC PLCbeta1

β3)

3,4

1,9

3,8

1,9

3,8

4,8

2,9

13,5

14,4

12,5

19,2

11,5

PLCbeta3 (PLC PLCbeta3

β2)

2,7

9,5

0,7

1,4

0,7

7,4

2,7

0,7

3,4

4,1

0,7

3,4

1,4

0,7

18,2

10,8

24,3

oform. Data was collected by Suh by (2008). al. was collected et Data oform.

PLCbeta2 (PLC PLCbeta2

/is

β1)

Tissue Tissue specific distribution of the different PLC isoforms collected from the UniGene Database. The

2,5

0,8

1,6

3,3

2,5

5,7

2,5

4,1

0,8

1,6

5,7

1,6

13,1

12,3

36,1

PLCbeta1 (PLC PLCbeta1

ear

eye

skin

lung

liver

bone

brain

heart

nerve

blood

uterus

spleen

kidney

mouth

muscle

ganglia

thyroid

thymus

Tissues

stomach

pancreas

intestine

numbers numbers show the of occurrence PLC isoform Table 1.1.: ESTs of number

parathyroid

lymph node lymph

bone marrow bone

embryo tissue embryo

adipose tissue adipose

testis,prostate

ovary, placenta, placenta, ovary, mammary gland mammary connective tissue connective

CHAPTER 1. INTRODUCTION

1.2.3.2. Domain structure of PLCs

1.2.3.2.1. General overview

Five conserved domains are found in almost all PLC isozymes; the pleckstrin homology domain (PH domain), the EF-hand like domain, X and Y catalytic domains and the C2 domain (Figure 1.8). Apart from these, other PLC subtype specific domains also exist such as SRC homology domains in PLCγ or Ras activation related domains in PLCε (Suh et al. 2008).

PLCβ PH EFL X Y C2

PLCγ PH EFLEF X P SH2 SH2 SH3 H Y C2

PLCδ PH EFLEF X Y C2

PLCε RasGEF PH EFL X Y C2 RA1 RA2

PLCζ EFLEF X Y C2

PLCη PH EF EFLEF X Y C2 variable C-term

Figure 1.8.: Domain structure of PLCs. Abbreviations: PH: Pleckstrin homology domain, EF: EF-hand domain, EFL: EF hand-like domain, X: Catalytic X domain, Y: Catalytic Y domain, C2: C2 domain, SH2,SH3: SRC homology domains, RasGEF: guanine nucleotide exchange factor domain for Ras-like small GTPases, RA: Ras association domain, variable C-term: variable C-terminal domain. Figure was produced by Dr. Alan Stewart.

1.2.3.2.2. Pleckstrin homology domain

The approximately 100-120 amino acid long PH domain appears in a range of proteins that possess roles in cell signalling and cytoskeletal function (Haslam et al. 1993). The domain was named after its first identification as an internal repeat in pleckstrin (Tyers et al. 1989). The domain is formed by a 7 stranded β-sandwich and a C-terminal amphipathic α-helix (Yoon et al. 1994). This structure enables the PH domain-containing proteins to bind phospholipids and the small variations between

CHAPTER 1. INTRODUCTION the domains allow the development of selectivity towards a specific phospholipid in certain proteins (Wang and Shaw 1995).

Apart from PLCζ, all PLC isozymes possess at least one PH domain on the N- terminus. In PLCγ, an additional PH domain occurs between the X and Y catalytic domains (Burgess et al. 1990; Haslam et al. 1993). This PH domain is split by SH2 and SH3 domains but the insertion does not prevent the formation of the canonical PH domain structure (Wen et al. 2006). Although the phospholipid binding is lost by this domain, it was identified as an interaction site for the regulatory protein, Rac2 (Walliser et al. 2008).

The N-terminal PH domains of the different PLC isozymes often have a preference towards different phospholipids which is directed by 3 positively charged regions of the domain (Singh and Murray 2003). PH domains of PLCβs seem to bind phospholipids non-specifically (McCullar et al. 2007). The membrane binding dynamics and the subcellular localisation of PLCβs might be also regulated by the C- terminal extension which also possesses a nuclear localisation signal (Kim et al. 1996). In contrast, the N-terminal PH domains of PLCγs specifically target phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3, Falasca et al. 1998) whereas for PLCδ1 PI(4,5)P2 is the preferred phospholipid (Lomasney et al. 1996). This latter interaction is so specific and strong that the construct consisting of the PH domain of PLCδ1 fused to green fluorescent protein (GFP) became a general tool to specifically visualise PI(4,5)P2 in cells (Varnai and Balla 2008). Little is known about the lipid binding properties of the PLCε PH domain. Computerised modelling predicted that this PH domain mainly tethers non-specifically with membrane phospholipids (Singh and Murray 2003). The sperm-specific PLCζ lacks the PH domain although it is still able to bind phospholipids by the X-Y linker region (Nomikos et al. 2011a). The binding of the PLCδ1 PH domain to the enzymatic product, I(1,4,5)P3, was also shown implicating the existence of a feedback mechanism whereby PLCδ1 is liberated from the membrane after a sustained activation (Ferguson et al. 1995).

CHAPTER 1. INTRODUCTION

1.2.3.2.3. The EF-hands

The EF-hand is a domain frequently found in Ca2+-binding molecules. One EF-hand consists of 2 α-helices and a Ca2+-binding loop in-between. The name EF-hand was given by Kretsinger and Nockolds based on the mechanism by which the E and F helices move between the open and closed conformations in parvalbumin (Figure 1.9.) (Kretsinger and Nockolds 1973). In this model, the helices are creatively described as the forefinger and the thumb on a hand. The Ca2+- binding of the loop induces the movement of the “thumb-helix” away from the “forefinger-helix” which transforms the EF-hand from the closed (apoprotein) to the open conformation (holoprotein, Figure 1.9.) (Lewit-Bentley and Rety 2000). To attain structural stability, usually two EF-hands team up to induce the formation of a short antiparallel β-sheet between the Ca2+-binding loops (Grabarek 2006). This connection, termed EF-hand β-scaffold, also plays an important role in the Ca2+ dependent structural changes of the domain (Grabarek 2005).

EF-hands have different selectivities toward Ca2+ and Mg2+ although the binding of Mg2+ generates a conformation which is more related to the closed form (Malmendal et al. 1999). Ca2+-binding ligands are provided by negatively charged amino acids such as aspartic acid or glutamic acid. Canonical EF-loops consist of 12 residues and provide 7 oxygen ligands to form a septadentate Ca2+-binding site (Figure 1.9.) (Gifford et al. 2007). The glycine in the 6 position is a highly conserved residue which produces a 90o turn in the loop essential for the coordination of the Ca2+ with the 7 ligands. Moreover, the NH and CO groups of the hydrophobic residue in the 8. position constitute the previously mentioned short β-sheet with the paired EF-hand (Gifford et al. 2007).

CHAPTER 1. INTRODUCTION

Figure 1.9.: Structure of the EF-hand. A: The hand model of the EF-hand describing the structural change in the domain upon Ca2+-binding as moving one of the helices away from the other like the movement of the thumb in a hand. This open conformation (holoform) returns to the closed state (apoform) when the Ca2+ dissociates from the binding site. Figure is taken with permission from Elsevier Ltd. (Lewit-Bentley and Rety 2000). B: Schematic representation of Ca2+-coordination of the EF-hand-loop in relation to the helices (red). Residues supplying ligands for Ca2+ are shown in blue. The 9. residue asparagine coordinates Ca2+ by the substitution of a water molecule (W). A glycine residue which directs an essential turn in the loop is shown in green. The hydrophobic residue constituting the short β-sheet with the parallel EF-loop, is shown in purple. Figure is taken with permission from Portland Press Ltd. (Gifford et al. 2007).

The EF-hand like motifs found in PLCs are similar to the canonical EF-hands although their involvement in Ca2+-binding is not certain (Bairoch and Cox 1990). The deletion of the EF-hand in PLCδ1 resulted in a decrease in enzyme activity but this effect was independent from Ca2+ concentration (Nakashima et al. 1995). Although, a later study suggested that Ca2+-binding of this domain in PLCδ1 regulates membrane-association of the PH domain (Yamamoto et al. 1999). It is likely that the EF-hand has lost its Ca2+ -binding ability in most of the PLCs and functions only as a hinge-like domain between the PH domain and the catalytic site (Stewart et al. 2007).

CHAPTER 1. INTRODUCTION

In contrast, much more is known about the functionality of the EF-hands in PLCζ (Kouchi et al. 2005; Nomikos et al. 2005). Distinctively, PLCζ exhibits a 100-fold higher Ca2+ sensitivity than PLCδ1 which is due to the functional EF-hands (Nomikos et al. 2005). PLCζ is only expressed in sperm and it is a crucial factor for the initiation of Ca2+ oscillations upon fertilisation (Saunders et al. 2002).

1.2.3.2.4. The catalytic domains

The X and Y domains fold to form the catalytic site which is responsible for the cleavage of the substrate, PI(4,5)P2. The catalytic core is highly conserved across mammalian PLCs (Rhee and Choi 1992). Apart from PI(4,5)P2, it is able to catalyse the enzymatic cleavage of phosphatidylinositol (PI) and phosphatidylinositol 4- monophosphate (PI(4)P) but it never cleaves 3-phosphoinositides. Moreover, Ca2+ is essential to be present at the catalytic site and the substrate preference may be slightly altered at different Ca2+ concentrations (Ellis et al. 1998). In contrast, the phospholipid hydrolysis of bacterial PLCs is independent from [Ca2+] and prefers PI and glycerophospholipids (Griffith et al. 1991). The X and Y domain forms one structural unit of the catalytic core composed of alternating α-helices and β-strands. This structure is analogous to the common enzyme core known as TIM-barrel (triose phosphate isomerase-barrel) (Essen et al. 1997b). The X and Y domains are usually separated by a linker region which may contain other functional domains. These are not integral parts of the TIM-barrel but rather they possess regulatory function on the enzyme activity and are sometimes referred to as Z-region (Rebecchi and Pentyala 2000). This region in PLCγ contains a PH domain split by two SH2 and one SH3 domains and phosphorylation events on Tyr residues result in the rise of the accessibility of the enzyme core to the substrate (Humphries et al. 2004; Ozdener et al. 2002). In general, the XY-linker (lacking any additional domains in PLCβ, δ, ε and η) possesses an autoinhibitory function on PI(4,5)P2 hydrolysis (Hicks et al. 2008). Whereas this part is mainly negatively charged in other PLCs, it consists of positively-charged amino acids in PLCζ and contributes its non-specific association of the protein to phospholipids (Nomikos et al. 2011b).

CHAPTER 1. INTRODUCTION

1.2.3.2.5. The C2 domain

The C2 (constant 2) domain was first described in PKCs as the second conserved domain (Xu et al. 1997). Since then, the domain was identified in a wide range of proteins with phospholipid binding capability, such as phosphatidylinositol 3-kinase (Hiles et al. 1992), RasGAP (Yamamoto et al. 1995), synaptotagmin (Perin et al. 1990) and PLCs (Rhee et al. 1989). The C2 domain is the second most frequent lipid binding domain after the PH domain (Cho and Stahelin 2006), and binds phospholipids either selectively or non-selectively. This interaction is Ca2+- dependent although some exceptions do exist (Davletov and Sudhof 1993; Cho and Stahelin 2006). The domain is composed of eight parallel β-strands connected by variable loops at the margins (Figure 1.10.) (Sutton et al. 1995). Three loops on one side of the molecule represent the Ca2+ binding site where 1-3 Ca2+ are able to bind depending on the C2 domain subtype (Sutton et al. 1995; Perisic et al. 1998; Sutton and Sprang 1998).

The C2 domain is a basic element of all PLCs. In PLCδ1, two Ca2+ is coordinated by ligands supplied mainly by aspartates (Grobler et al. 1996). Interestingly, the lipid specificity is not similar between the PLCδ isoforms. Whereas the C2 domain of PLCδ1 and PLCδ2 prefers the Ca2+-directed binding of phosphatidylserine, a substitution of an asparagine Ca2+ coordinating residue (Asp717) to serine in PLCδ4 renders the domain less selective and the requirement of Ca2+ for lipid-binding is diminished (Ananthanarayanan et al. 2002). It was suggested in PLCδ1, that Ca2+ ligands are also supplied by the phospholipids and the interaction of the C2 domain with membranes are strengthened by the developing “Ca2+-bridge” (Grobler et al. 1996). A PLCζ mutant lacking the C2 domain possesses no detectable enzyme activity. This supports the idea that binding of the membrane via the C2 domain is essential for the enzymatic cleavage of the substrate. PLCζ-C2 domain possesses the highest sensitivity towards PI(3)P and PI(5)P and the interaction is not Ca2+- dependent (Kouchi et al. 2005).

CHAPTER 1. INTRODUCTION

Figure 1.10.: Structure of the rat PLCδ1 C2 domain. The domain is composed of 8 parallel β-sheets. The Ca2+ binding site consists of three loops found on one side of the β-sheets. Ca2+ ions are represented as red spheres. Structure was modelled by the author of this thesis with Accelrys Discovery Studio 3.1 from the structural data found at PDB: 1DJI, Essen et al. 1997a).

1.2.3.2.6. PLC isozyme-specific domains

Some PLC enzymes possess functional domains that are specific to individual classes. PLCβ enzymes contain a C-terminal extension lacking any identifiable domains. Although this extension may seem unimportant at first sight, it was shown to be a protein interaction site and was termed as C-terminal domain (CT). PLCβ enzymes function as homodimers through CT-CT interactions which are regulated by

Gαq (Singer et al. 2002). Moreover, an autoinhibitory segment was also found in this region in a cephalopod PLCβ homolog, which interacts with the catalytic site to negatively regulate the enzyme activity. According to this study, Gαq binding requires the dissociation of the CT from the catalytic site and its stimulatory effect resides in the inhibition of the autoinhibitory feedback (Lyon et al. 2011).

CHAPTER 1. INTRODUCTION

As previously mentioned, PLCγ contains a split PH domain separated by two SH2 and an SH3 domain. PLCγ is phosphorylated by tyrosine kinase receptors (RTKs) between the second SH2 and the SH3 domains. It seems that the binding affinity to receptors is regulated by the N-terminal SH2 domains (Rotin et al. 1992). From the three tyrosine residues which are phosphorylated upon activation (Y771, Y783, and Y1253), Y783 interacts with the C-terminal SH2 domain and this intramolecular interaction stimulates the enzyme activity by an unknown mechanism (Poulin et al. 2005).

PLCε is uniquely involved in the signalling mechanism of a group of small GTPases known as Ras-proteins. The RA (Ras association) domains found on the C-terminus of PLCε serve as the binding site for Ras proteins (Song et al. 2001). This interaction induces the translocation of PLCε to the perinuclear area upon EGF (epidermal growth factor) receptor stimulation (Jin et al. 2001). Additionally, a Ras guanine nucleotide exchanger domain (RasGEF) is located on the N-terminus to facilitate the exchange of GDP to GTP on Ras proteins (Song et al. 2001). By this mechanism, upon interaction, both molecules are activated (Lopez et al. 2001).

Apart from these domains, there are additional differences in the sequences of the conserved domains which lead to the diversity of regulatory mechanism affecting the different PLC isoforms. These alterations will be discussed in detail in the following section.

1.2.3.3. Regulation of PLCs

After the discovery of the first PLCs, the classical PLC activation was thought to be driven by GPCR-stimulated release of Gq/11, however, with the subsequent identification of other subtypes, a far greater complexity for the regulation of PLCs has been revealed (Figure 1.11.). PLCβ activation is highly dependent on Gq/11 activation as it is stimulated by several members of the GPCRs linked to this G- protein (Taylor et al. 1991; Wu et al. 1992b; Offermanns and Simon 1995; Singer et al. 2002). It seems that none of the other PLC classes are activated by Gq/11 proteins,

CHAPTER 1. INTRODUCTION

however some data suggest that transglutaminase/Gh which binds to atypical GPCRs is able to translate GPCR activation onto PLCδ1 (Murthy et al. 1999; Xu et al. 2006).

The regulatory mechanism of Gα variants on PLC activity may also be directed by the insertion of non-receptor coupled small G-proteins into the activation cascade.

G12 and G13 trigger the activation of PLCε via the stimulation of the small G-protein,

Rho which binds to the Y catalytic domain (Wing et al. 2003). Gβγ dimers dissociated from activated G-proteins are also able to act as messengers of GPCR activation and contribute to PLC stimulation. A comparison of Gβγ stimulation on the activity of various PLCs showed that PLCβ1, PLCβ2, PLCβ3 and PLCδ1 activities were upregulated (PLCδ

Gαq they are possibly differently regulated by various GPCR signals (Park et al.

1993). The release of Gαq is always paired with an increase in Gβγ levels, however by using specific scavenger molecules their individual effect could be revealed. One 2+ such experiment reported that Gβγ generates Ca oscillations whereas Gαq induces a prolonged elevation in intracellular Ca2+ level of pancreatic acinar cells. This effect might be directed by different subsets of PLC isotypes and highlights the importance of the fine tuning of PLCs to G-protein activation (Zeng et al. 1996). Gβγ regulates the enzyme activity of PLCβ by binding to its PH domain, a feature that is conserved when the domain is inserted into PLCδ1 (Wang et al. 2000). As mentioned before, the stimulatory effect of Gβγ seems to reside in its ability to dissociate inhibitory intramolecular connections of the PH and catalytic domain (Drin et al. 2006). PLCε is also stimulated by various Gβγ subtypes although the mechanism of binding is uncertain (Wing et al. 2001).

PLCγ is activated by receptor tyrosine kinases. As earlier described, upon ligand induced autophosphorylation of the receptor, PLCγ is tethered to RTKs and subsequently phosphorylated and activated by the receptor (Poulin et al. 2005). Additionally, receptors which do not possess intrinsic tyrosine kinase activity may still be able to stimulate PLCγ by phosphorylation. For instance, this occurs by the

CHAPTER 1. INTRODUCTION activation of the B-cell antigen receptor which stimulates PLCγ via activating the Bruton's tyrosine kinase (Takata and Kurosaki 1996). Alternatively, other PLC subtypes may be phosphorylated by specific kinases. The phosphorylation of PLCβ3 by PKA or PKC inhibits Gα and Gβγ stimulated activity (Yue et al. 1998; Yue et al. 2000). In contrast, phosphorylation of PKCδ1 by PKC seems to potentiate its enzyme activity in vitro (Fujii et al. 2009).

Small GTPases are able to translate receptor activation to PLCs (Figure 1.6b).

Gs/cAMP coupled GPCRs stimulate PLCε via the activation of Rap small GTPase (Schmidt et al. 2001). Upon growth factor-dependent stimulation, both Ras and Rap are activated. Whereas Ras stimulation is transient, Rap is responsible for a sustained activation of PLCε (Song et al. 2002). Rac contributes to the regulation of PLCβ and PLCγ. PLCγ2 but not PLCγ1 is regulated by Rac by its association with the split PH domain (Walliser et al. 2008). Only PLCβ2 of PLCβs is activated by Rac2 by their interaction through the N-terminal PH domain (Illenberger et al. 2003).

2+ All PLC subtypes require Ca for PI(4,5)P2 hydrolysis which is directed via its binding to the catalytic site. The mutation of the metal-binding residues to alanine in the PLCδ1 catalytic core decreased basal enzyme activity to a great extent and based on this finding, Ca2+ dependence of all subtypes has been described by this mechanism (Ellis et al. 1998). In contrast to being a cofactor for enzyme activity, in PLCδ and PLCζ, Ca2+ is also able to directly activate the hydrolysis of the substrate possibly via the EF-hand although the exact mechanism is uncertain (section 2+ 1.2.3.3.). The half maximal concentration of Ca which stimulates PI(4,5)P2 hydrolysis by PLCδ1 and PLCδ3 was determined between 100 nM and 1 µM Ca2+ which corresponds to the physiological range of cytosolic Ca2+ signals (Cheng et al. 1995; Ghosh et al. 1997). In contrast, the sperm-specific PLCζ is activated between 10 nM and 100 nM Ca2+ which suggests that it is activated by basal cytosolic Ca2+ levels. This Ca2+-induced activation and the subsequent inactivation and depletion of 2+ I(1,4,5)P3 sensitive Ca stores may be responsible for the developing oscillations in

CHAPTER 1. INTRODUCTION

Ca2+ concentration upon the administration of PLCζ into oocytes (Nomikos et al. 2005).

Figure 1.11.: Various activation of PLC isoforms. A: Receptor induced activation of PLCs. GPCR activation triggers the dissociation and the subsequent activation of Gα and Gβγ. Gα (from Gq/11 or transglutaminase/Gh) stimulates PLCβ and PLCδ whereas the Gβγ dimer increase PLCβ, PLCδ and PLCε activity. RTKs directly phosphorylate and consequently activate PLCγ. Phosphorylation of PLCβ by PKA or PKC negatively regulates PLCβ activity, whereas PKC potentiates PLCδ activity. PLCδ and PLCζ are directly activated by Ca2+ signals which may arise from Ca2+ influx or Ca2+ release from intracellular stores. They might be involved in the amplification of Ca2+ signals by their activation in response to small Ca2+ levels and the Ca2+ releasing effect of the consequently generated I(1,4,5)P3. Solid and dashed lines represent activation or inhibition, respectively. B: Activation of PLCs by small G proteins. Rac stimulates PLCβ and PLCγ activity. PLCγ, in which the main regulation of enzymatic cleavage is regulated by the small GTPases, is activated by Rho, Rap and Ras, but the mechanism of activation greatly differs (see text for description). Abbreviations: GPCR: G protein-coupled receptor, PI(4,5)P2: phosphatidylinositol 4,5-bisphosphate, I(1,4,5)P3: inositol 1,4,5-trisphosphate, DAG: diacylglycerol, PKC: protein kinase C, PKA: protein kinase A, IP3R: inositol 1,4,5- trisphosphate receptor, RTK: receptor tyrosine kinase, ER/SR: endo/sarcoplasmic reticulum. Figure was produced by the author of the thesis

CHAPTER 1. INTRODUCTION

In conclusion, PLC isozymes have distinct upstream regulators that may also differ between closely related members of a subfamily. These alterations allow the PLC family members to take part in alternative physiological processes. In the next section, the role of PLCs in neurophysiology will be discussed.

1.2.3.4. The role of PLCs in neuronal physiology

1.2.3.4.1. Neuronal pattern of PLC expression

Various PLC isoforms are distributed in the brain which strongly suggests that this enzyme family play crucial roles in neuronal physiology (a summary of the existing literature is found in Table 1.1.). The mRNAs of all four PLCβ isoforms are expressed in the brain with alternating distribution in different brain tissues. PLCβ1 is abundant in the cerebral cortex, striatum and hippocampal formation whereas PLCβ2 is expressed in structures of the white matter. PLCβ3 specifically appears in cerebellar Purkinje cells. PLCβ4 is abundant in regions opposite to PLCβ1; it is found in the olfactory bulb, thalamus, brainstem and cerebellum (Watanabe et al. 1998). Only low levels of PLCγ1 appear in the brain, mainly in the olfactory bulb, hippocampal formation, cerebellum, piriform- and frontal cortex (Ross et al. 1989). In addition, PLCγ2 is found in a small portion of the cerebellum (Tanaka and Kondo 1994). PLCδ1 mRNA is expressed in all brain areas tested (Lee et al. 1999). In a comparative study, the expression of all PLCδ isoforms were studied in cerebellar granule cells and PLCδ3 was found to be the predominant form. In addition, high levels of PLCδ3 were also detected in the cerebral cortex (Kouchi et al. 2011). The latest PLCδ isoform, PLCδ4 is also abundant in the brain, however, there has not been any attempt in the literature to assess its regional specific distribution (Lee and Rhee 1996). Limited expression of PLCε1 was also detected in human brain lysates by Northern blot analysis (Lopez et al. 2001). In the developing mouse, PLCε1 seems to be upregulated in neuronal precursor cells which are committed to become neurons indicating its role in neuronal differentiation (Wu et al. 2003). PLCζ

CHAPTER 1. INTRODUCTION expression is restricted to the sperm and therefore has no functions in brain physiology (Saunders et al. 2002).

expression isoform brain regions Reference in the brain

cerebral cortex, striatum, hippocampal PLCβ1 yes formation, amygdala, lateral septum

white matter structures (corpus callosum, PLCβ2 yes fimbria and medulla of the cerebellum) Watanabe et al., 1998 PLCβ3 yes Purkinje cells of the cerebellum

Purkinje and granule cells of the PLCβ4 yes cerebellum, olfactory bulb, medial septal nucleus, most thalamic nuclei

olfactory bulb, hippocampal formation, Ross et al., PLCγ1 yes cerebellum, piriform- and frontal cortex 1989

Tanaka and PLCγ2 yes no data available Kondo, 1994

all areas tested: cerebellum, cortex, Lee et al., PLCδ1 yes hipocampus, hypothalamus, mid-brain, 1999 pons and striatum

all areas tested: cerebral cortex, Kouchi et PLCδ3 yes cerebellum, hippocampal formation al., 2011

Lee and PLCδ4 yes no data available Rhee, 1996

Lopez et al., PLCε yes no data available 2001

Saunders et PLCζ no - al., 2002 Table 1.2.: Summary of known information relating to the distribution of PLC- isoforms in the brain.

CHAPTER 1. INTRODUCTION

Apart from the above mentioned spatial differences, temporal changes in PLC- activity and expression levels have also been detected. In the rat cerebellum, overall

I(1,4,5)P3 levels increase with age with the subsequent decrease in IP3R sensitivity and binding (Igwe and Filla 1995). Receptor-induced I(1,4,5)P3-accumulation is also augmented by ageing in the striatum and the cortex (Mundy et al. 1991; Igwe and Filla 1997). A study by Shimohama et al., compared the age-related changes in the protein levels of PLCβ1, PLCγ1 and PLCδ1 in separated cytosolic and particulate fractions of brain extracts. PLCβ1 protein levels elevated after birth, reached maximum at the age of 4 months and then slowly declined. Whereas until the 8. week, PLCβ1 was predominant in the cytosolic fraction, after this time-point the particulate fraction was found to be more enriched in this isoform. PLCγ1 and PLCδ1 were always found primarily in the cytosolic fraction. PLCγ1 expression gradually decreased with age. In contrast, PLCδ1 expression increased after birth and only declined at late ages (18 month-old rats) (Shimohama et al. 1998). To summarise, the levels of the numerous PLC isoforms are tightly regulated both spatially and temporally.

1.2.3.4.2. PLC activity-linked neuronal functions

Various neuronal processes are linked to PLC activation from neuronal differentiation to synaptic plasticity. A number of studies reported the importance of 2+ phospholipase activity and IP3R-driven Ca release in neuronal transduction. PLC activity is coupled to various neurotransmitter receptors, such as serotonergic, adrenergic, histaminergic, muscarinic and cholinergic receptors (Wallace and Claro

1990; Xu and Chuang 1987). In addition, decrease in the PI(4,5)P2-pool produced by NMDAR-stimulated PLC activity was implicated in the generation of long-term potentiation by liberating the protein kinase A anchoring scaffold protein, AKAP79/150 from the membrane, and downregulating the level of F-actin and the membrane distribution of AMPAR (Horne and Dell'Acqua 2007). In the absence of

PLC activation, the actin depolymerising protein cofilin is bound to PI(4,5)P2. Upon stimulation of PLCs in cortical neurons, PI(4,5)P2 is cleaved, releasing cofilin, which

CHAPTER 1. INTRODUCTION in-turn potentiates the rearrangement of the actin cytoskeleton. The internalisation of NMDAR is also accelerated by this process due to its connection to the actin cytoskeleton (Mandal and Yan 2009). Binding of the brain-derived neurotrophic factor (BDNF) to its tyrosine kinase receptor (TrkB) on both pre- and postsynaptic membranes activates PLCγ by phosphorylation. In hippocampal neurons, this process is important for the generation of LTP via the stimulation of CREB-dependent transcription and the phosphorylation of TRPC3 (Li et al. 1999; Gartner et al. 2006; Cunha et al. 2010). In addition, gabaergic stimulation of Purkinje cells is enhanced by BDNF-PLCγ signalling (Cheng and Yeh 2005).

Apart from playing important roles in neurotransmission within the adult brain, PLC activity also contributes to the regulation of neuronal differentiation. A study by Kouchi et al. highlighted the importance of PLCδ3 in directing neurite outgrowth and migration of cerebral and cerebellar neurons by in vivo and in vitro knockdown experiments. PLCδ3 acts on neurite outgrowth through the inhibition of RhoA/Rho kinase signalling (Kouchi et al. 2011). In contrast, high levels PLCγ1 inhibited the NGF-induced differentiation of a pheochromocytoma cell line (PC12) by altering the expression pattern of cell cycle regulators (CDK1, CDK2 and cyclin D1) and promoting cell proliferation (Nguyen Tle et al. 2007). As mentioned before, PLCε expression has been suggested to play important roles in cell fate decision of neuronal precursors based on its specific expression in only MAP2 positive cells during embryogenesis (Wu et al. 2003). Neurogenesis also occurs in restricted regions of the adult brain, namely in the olfactory bulb and the dentate gyrus of the hippocampal formation (Kaneko and Sawamoto 2009). Interestingly, this is influenced by PLCβ1 activity as shown by the elevated rate of neurogenesis in the hippocampus of the PLCβ1-knockout mice (Manning et al. 2012). PLC-signalling is important for chemoattractant-directed migration of granule cells. The SDF-1

(stromal derived factor-1) receptor CXCR4 (CXC chemokine receptor 4) is a Gi- coupled GPCR which has been suggested to stimulate PLCβ1 activity by the released

Gβγ dimer (Busillo and Benovic 2007).

CHAPTER 1. INTRODUCTION

1.2.3.5. The latest PLC family, PLCηs

1.2.3.5.1. First characterisation of PLCη enzymes

The latest PLC family with two members (PLCη1 and PLCη2) was independently reported in 2005 by four groups. Identification of this distinct PLC group was a result of a screen of the existing sequence databases such as the HUGE database (Kazusa DNA Research Institute) the Celera and the NCBI databases (Hwang et al. 2005a; Nakahara et al. 2005; Stewart et al. 2005; Zhou et al. 2005). Although the PLCη- domain structure is most related to that of PLCβs, their sequence is highly similar to PLCδs (Hwang et al. 2005a; Nakahara et al. 2005; Stewart et al. 2005). All PLC- specific domains were identified in PLCη structure (PH, EFL, X, Y, and C2 domains, Figure 1.12.) with an additional C-terminal extension lacking any known functional domain organisation. A His-tagged PH domain of PLCη1 was purified by a Ni-NTA agarose followed by cation-exchange chromatography. Circular dichroism spectra of this PH domain suggested that the secondary structure is composed of β-sheets similarly to other PLCs (Imai et al. 2007). Alignment of the catalytic site of PLCηs with other PLCs showed that the enzymatically necessary residues are conserved suggesting that their basic catalytic functions are equivalent. From the two histidines which are crucial in the specific binding of phospholipids (Essen et al. 1997b), one in each PLCη (mutations: H358Q in PLCη1 and H341Q in PLCη2) was mutated to generate a catalytically-inactive variant (Hwang et al. 2005a; Nakahara et al. 2005).

Figure 1.12.: Domain structure of PLCη enzymes. Both isoforms contain the PLC- specific structural composition with an N-terminal PH domain, EF-hands, X and Y catalytic domains connected by a short link (157 residues in hPLCη1 and 151 residues in PLCη2), a C2 domain, and the alternatively spliced C-terminus. Figure is taken with permission from The American Society for Biochemistry and Molecular Biology (Nakahara et al. 2005).

CHAPTER 1. INTRODUCTION

At the C-terminus, a class II PDZ-binding motif was identified in both PLCη1 and PLCη2 which provides the possibility for interactions with PDZ-domain containing proteins (Zhou et al. 2005). In addition, the C-terminus of PLCη2 contains a number of proline rich motifs which is a known target of SH3 domain containing proteins (Cohen et al. 1995). SH3 domains frequently occur in signalling proteins such as the Ras GTPase activating protein (RasGAP), phosphatidylinositol 3-kinase (PI3K) and PLCγ (Kohda et al. 1993; Pamonsinlapatham et al. 2009; Batra-Safferling et al. 2010). The C-terminal domain is also rich in serine/threonine residues suggesting possible regulation via protein kinases/phosphatases (Figure 1.13.). In the light of these data, the variable C terminus is potentially a major protein interaction site.

Figure 1.13.: Amino acid motifs of the PLCη2 C-terminal that might be targeted by kinases/phosphatases. The sequence shown is the C-terminus of the longest splice variant of PLCη2 (21b/23). Yellow: two proline intercepted by two other amino acids, green: serine and threonine residues, blue serine/threonine residues are integral to the PxxP motif. Figure was produced by the author of this thesis.

Alternative splicing of the C-terminus may possess an important regulatory role as this mechanism gives rise to a number of splice variants in both PLCη1 and PLCη2. This idea is supported by the appearance of the previously mentioned PDZ-binding motif only in certain spliceforms (Zhou et al. 2005). Three splice variants of PLCη1 (with sizes: 1002, 1693 and 1035 amino acids of the human isoform) (Hwang et al. 2005a), whereas five spliceforms of PLCη2 (with sizes 989, 1156, 1211, 1416 and 1583 amino acids in human) (Zhou et al. 2005) were suggested to be processed.

CHAPTER 1. INTRODUCTION

Interestingly, the different splice variants of PLCη2 exhibit tissue specific distribution, however, consequences of this process are largely unknown (Zhou et al. 2005). PLCηs are generally thought to be brain-specific, however, their presence in a limited number of other tissues were also shown. Both PLCη1 and PLCη2 are expressed in the lung, whereas only PLCη1 is represented in the spinal cord (Hwang et al. 2005a; Nakahara et al. 2005). High levels of PLCη2 expression were also found in the retina (in photoreceptors, and in horizontal-, bipolar- and amacrine cells) and in neuroendocrine cells (Stewart et al. 2007; Kanemaru et al. 2010). PLCη1 is expressed throughout the brain but it is most abundant in the olfactory bulb, hippocampal formation, cerebellum, cerebral cortex, zona incerta, habenula and hypothalamus (Hwang et al. 2005a). High amounts of PLCη2 were also detected in these regions with the exception of the zona incerta. Interestingly, PLCη2 protein levels gradually increase during postnatal development in both mouse brain and retina (Nakahara et al. 2005; Kanemaru et al. 2010). In primary cell cultures, PLCη was abundant in hippocampal pyramidal cells whereas in astrocyte-enriched cultures it was absent (Nakahara et al. 2005). These results highlight the importance of PLCη enzymes in neuronal physiology.

1.2.3.5.2. Biochemical properties

The capability of PLCη enzymes to hydrolyse PI(4,5)P2 was confirmed by using purified enzymes. PLCη1 catalytic activity possesses high sensitivity towards Ca2+ (10-100 nM) and its maximal activation is achieved by a ten-fold lower concentration compared to PLCβ1 (Hwang et al. 2005a). Ca2+ sensitivity of PLCη2 mirrors PLCη1 and its highest activity is reached at a ten-fold lower Ca2+ concentration than that of PLCδ1. These results indicate that the threshold for Ca2+- activation is much lower in the PLCη than in PLCβ or PLCδ1 families. ηUnfortunately, the purified PLCη2 showed decreased, 10-fold lower maximal activation (at any [Ca2+] ) than what was registered with PLCδ1 However, this difference could be due to the altered requirements of enzyme activity in PLCη2 and PLCδ1 which were not equally supported during the in vitro measurement.

CHAPTER 1. INTRODUCTION

Hydrolysing activity of PLCη2 is also dependent on pH with an optimum of pH 7 (Nakahara et al. 2005).

To describe the regulatory mechanism of PLCηs, several known PLC-regulating proteins were co-administered with PLCηs in cells that was followed by the measurement of PI(4,5)P2-hydrolysisng activity. PLCη2 activity was not stimulated by any of the Gα subunits examined or by the numerous small G-proteins tested

(Zhou et al. 2005). Of those examined, only Gβγ dimers (most efficiently Gβ1γ2) were able to stimulate PLCη2 activity. In contrast, PLCη1 enzymatic activity was not affected by the co-expression of Gβ1γ2 and therefore it seems that the two isoforms are differently regulated by GPCRs (Zhou et al. 2008). Consequently, the mechanisms by which PLCη1 is activated are less understood. Currently, its only known activator is Ca2+ and it has been implicated in the interplay with other PLCs by amplifying Ca2+ transients (Kim et al. 2011).

The cellular localisation of PLCη1 and PLCη2 was assessed in human immortalised cell-lines (HEK-293 and HeLa cells) using green fluorescent protein (GFP) tagged PLCη constructs. PLCη1 possessed equal distribution between membranes and the cytosol, whilst PLCη2 predominantly localised to membranes prior to stimulation (Hwang et al. 2005a; Nakahara et al. 2005).

1.4. Thesis outline

As highlighted in this Chapter, PLCηs are new members of the PLC family and our knowledge on their regulation and their involvement in cellular processes is relatively poor. The aim of the thesis is to characterise the second PLCη isoform, PLCη2 from several biochemical and functional aspects. Key questions addressed are how enzyme activity and cellular localisation are regulated by the different domains. In addition, the specific role(s) that this enzyme plays in neuronal physiology is also examined.

CHAPTER 1. INTRODUCTION

The activation of PLCη2 by mitochondrial Ca2+ release is demonstrated in COS-7 cells overexpressing PLCη2 by the administration of various Ca2+-mobilising agents and inhibitors. This is followed by the investigation of the PH domain, EF-hand and the C2 domain from several aspects. Deletion of the PH domain results in the loss of membrane binding capability. To further characterise this domain, its preference towards different phospholipids is also assessed. It is hoped that this study improves our understanding on the regulation of PLCη2 shuttling inside the cell. Ca2+- dependent secondary binding to the membrane by the C2 domain is also shown to be critical for enzyme activity, however this does not contribute to the Ca2+-sensitivity of the enzyme. In addition to an EFL-domain (which is found in all PLCs), a unique EF-hand was identified in PLCη enzymes. Using site directed mutagenesis it was found that a Ca2+-binding loop is responsible for the unique Ca2+-sensitivity of PLCη2 catalytic activity. Collectively, these results assist the formation of the better understanding of how PLCη2 is regulated.

In addition, acquiring information as to the physiological function(s) of PLCη2 in neuronal cells is of great importance. A previous study by Nakahara et. al. (2005) reveals that PLCη2 expression increases during postnatal development. This led me to investigate whether this enzyme is involved in neuronal differentiation. Using an established cell model for neuronal differentiation/neurite growth (Neuro-2a cells), PLCη2 expression and the role of the enzyme in this process were assessed. It was found that when PLCη2 levels are reduced Neuro-2a cell differentiation is compromised, implying its importance in this process. PLCη2 may direct differentiation by influencing retinoic acid regulated gene expression as implicated by a dual-luciferase assay. A possible interaction partner (LIMK1) was also identified by a bacterial two hybrid screen which links PLCη2 function to the regulation of actin rearrangement. It is hoped that this work will generate further interest relating to the role of PLCη2 in the brain.

Chapter 2.

Materials and Methods

CHAPTER 2. MATERIALS AND METHODS

2.1. Cell Culture Methods

2.1.1. Cell lines and their maintenance

o Cell lines were maintained at 37 C in a humidified atmosphere at 5% CO2 in appropriate medium as shown in Table 2.1. (media unless otherwise stated are obtained from Sigma-Aldrich, Poole, UK). Cells were split twice a week into a new 75 cm2 flask. The medium was removed and the cells were washed twice with 10 ml sterile PBS. Cells were harvested with 2 ml of pre-warmed (37oC) Trypsin-EDTA solution (Life Technologies, Paisley, UK). Trypsin was inactivated by 8 ml of complete medium and a small portion (0.5-2.5ml) was added to a new flask containing 25 ml medium (1:50- 1:10 ratio). COS-7 cells were a gift from Prof. Robert P. Millar (Queen’s Medical Research Institute, Edinburgh, UK) and Neuro-2a cells were obtained from The European Collection of Cell Cultures (ECACC).

Cell type Origin Maintenance

COS-7 immortalised kidney cells DMEM (Dulbecco`s modified Eagle`s from African green monkey medium) with high glucose (Gluzman 1981) (4500mg/L) supplemented with 2 mM L-glutamine and 50 units/ml of penicillin/streptomycin mixture (Invitrogen). Cells were maintained at o 37 C, 5% CO2.

Neuro-2a murine neuroblastoma cell EMEM (Eagle's minimal essential line (Klebe and Ruddle 1969) medium) supplemented with 2mM L- glutamine, 50 units/ml of penicillin/streptomycin mixture. Cells o were maintained at 37 C, 5% CO2.

Table 2.1.: Cell-lines and there maintenance.

CHAPTER 2. MATERIALS AND METHODS

2.1.2. Cryopreservation of cell lines

A cell suspension acquired after trypsinisation of a confluent 75 cm2 flask was spun at 800 rpm (Neuro-2a) or at 1,500 rpm (COS-7) for 3 minutes at 4oC in a Beckman JS-4.2 rotor. The cell pellet was resuspended in 4 ml freezing medium (Life Technologies) and was aliquoted into 4 cryovials (Nunc, Thermo Fisher Scientific, Waltham, MA, USA). The cells were kept at -20oC for 24 hours then at -80 oC for 24 hours and were transferred to a liquid nitrogen filled container for long-term storage.

2.1.3. Rescue of frozen cells

Liquid nitrogen-stored cells were quickly thawed at 37oC. The cell suspension was diluted with 10 ml fresh growth medium and cells were harvested as previously by centrifugation. The cell pellet was resuspended in 25 ml fresh medium and was transferred into a 75 cm2 flask.

2.1.4. Transfection by electroporation

Electroporation was used to transiently transfect COS-7 cells or to transfect and select stable clones from Neuro-2a cells (antibiotic selection will be described later). The following method was used for both cell types. Cells were trypsinised and seeded into 14 cm diameter tissue culture dishes with 25 ml growth medium which were allowed to grow for 3 days prior to transfection. Approximately 1.5 × 107 cells were used for each transfection (2/3 of a dish). When multiple transfections were required, they were done in parallel. Cells were trypsinised and collected with 10 ml growth medium as described previously. Cells were harvested by centrifugation and the pellet was washed with 10 ml Optimem/transfection (Life Technologies). Centrifugation was repeated and the pellet was resuspended in 0.7 ml Optimem/transfection and each 0.7 ml was aliquoted into an electroporation cuvette containing 15-20 µg plasmid. Cells were electroporated by using a Bio-Rad Gene Pulser at 230 V, 950 μF. Cells were recovered for 10 minutes, and the suspension

CHAPTER 2. MATERIALS AND METHODS was mixed with growth medium to seed into cell culture dishes for subsequent experiments as appropriate.

2.1.5. Lipofection of Neuro-2a cells

Lipofectamine 2000 (Life Technologies) was used to transfect Neuro-2a cells with constructs encoding fluorescently-tagged proteins (GFP-PLCη2, GFP-PLCη2 PH domain or mCherry-PLCδ1, more information is given later in this Chapter). Cells (100 cells/mm2) were seeded in antibiotic-free EMEM into a 6-well plate containing coverslips. The following day, 2 µg of plasmid and 5 µl Lipofectamine were mixed in 500 µl Optimem, respectively for each well. After 5 minutes, the two solutions were mixed and further incubated for 30 minutes. Each mixture was added to one well in the 6–well plate dropwise and mixed gently by rocking the plate. Cells were returned to the incubator and the growth medium was changed to fresh EMEM after 6 hours.

2.1.6. Generation of stable PLCη2 knock-down cell lines

The expression of PLCη2 was downregulated in Neuro-2a cells by the transfection of psi-H1 vectors encoding shRNAs designed to target PLCη2 expression (obtained from GeneCopoeia, Rockville, MD, USA). Four different shRNA constructs were tested and two stable clones were selected (shRNAPLCη2-1 and shRNAPLCη2-1). Target and the control sequences are shown in Table 2.2. Plasmid maps are presented in Chapter 7. Cells were transfected by electroporation as described previously. Stably transfected cells were selected after 48 hours by adding 3 µg/ml puromycin to the growth medium (InvivoGen, San Diego, CA, USA). Growth medium was renewed every second day until colonies appeared. One cell derived colonies were cloned with custom-made metal cloning rings and were transferred into 6-well plates. Knock-down efficiency in the different clonal lines was detected by western blot analysis.

CHAPTER 2. MATERIALS AND METHODS

construct 19-mer target sequences and control shRNAcontrol 5`-TCAAGAG-3` shRNAPLCη2-1 5`-TGACTGCAAGCTCCTCAAT-3` shRNAPLCη2-2 5`-GGACCTAGTGAAATATACC-3` shRNAPLCη2-3 5`-TCTGCAGCTGCTACATAAA-3` shRNAPLCη2-4 5`-TCTGCGCCTTCTACAAGAT-3` Table 2.2.: PLCη2-specific sequences in shRNA constructs.

2.2. Cell assays and treatments

2.2.1. Immunocytochemistry and fluorescent microscopy

For microscopic analysis cells were grown on coverslips in 6-well plates. For the specific staining of mitochondria, MitoTracker Red CMXRos (Life Technologies) was diluted in DMSO and was added to the culture medium (to a final concentration of 250 nM) an incubated at 37°C for 30 minutes. Cells were fixed with altered methods afterwards.

COS-7 cells transfected with constructs encoding GFP-fused proteins were fixed in ice-cold methanol for 10 minutes and dried on air. Fixed cells were washed twice with PBS and nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) in PBS. Coverslips were mounted in Mowiol solution (Sigma- Aldrich) and left to dry in the dark at 4oC.

Neuro-2a cells transfected with GFP- or mCherry-tagged constructs were fixed with 4% formaldehyde in PBS for 10 minutes and were washed with PBS three times. Coverslips were incubated in 2.1% citrate buffer containing 0.5% Tween-20 and in 11.8% citrate buffer containing DAPI for 5-5 minutes. The remaining of the last solution was removed by PBS and coverslips were mounted with Mowiol solution.

For the detection of endogenous proteins cells were fixed and permeabilised in one step with 4% formaldehyde and 0.2% Triton-X containing PBS. This solution was removed after 5 minutes and cells were washed with PBS 3 times. Coverslips were

CHAPTER 2. MATERIALS AND METHODS incubated with blocking buffer containing 10% FBS in PBS for 1 hour. A custom rabbit primary antibody raised against a short peptide of PLCη2 (SKVEEDVEAGEDSGVSRQN) was used to stain endogenous PLCη2. Other primary antibodies were obtained from Abcam (LIMK1, dilution 1:100, Cambridge,

UK) or Echelon Biosciences Inc. (PI(3,4,5)P3 dilution: 1:100, Salt Lake City, UT, USA). Actin was labelled with tetramethylrhodamine B isothiocyanate-conjugated phalloidin (TRITC-phalloidin, Sigma-Aldrich) which eliminated the requirement of a secondary antibody. Primary and secondary antibodies were diluted in 5% FBS in PBS. Coverslips were incubated with primary antibody solution for an hour followed by 3 washing steps with PBS. Secondary antibodies (anti-rabbit Dylight 488 or 594 and anti-mouse Dylight 594 from Jackson, West Grove, PA, USA) were diluted 1:300 and applied for an hour. When incubation time finished, cells were washed with PBS 3 times and incubated in 2.1% citrate buffer containing 0.5% Tween-20 in PBS for 5 minutes. DAPI was added in 11.8% citrate in PBS and cells were incubated for 5 minutes. Coverslips were washed once in PBS and mounted in Mowiol solution.

To verify the specificity of the PLCη2 antibody the short peptide which was used to raise the antibody in rabbit (SKVEEDVEAGEDSGVSRQN) was resynthesised (Peptide Protein Research Ltd., UK). One µl of PLCη2 antibody was incubated with 1 or 10 µg or without the peptide in 500 µl primary antibody solution overnight prior to addition to the cells. Rest of the immunostaining was conducted as described before. Loss of the antibody binding in the presence of the peptide verified its specificity towards PLCη2 and its suitability for further experiments.

Slides were examined with a DeltaVision deconvolution microscope (Applied Precision, GE Healthcare, Little Chalfont, UK) or with a multiphoton confocal microscope (TCP SP2 system, Leica Microsystems GmbH, Wetzlar, Germany). The confocal microscope is equipped with a Leica DM IRE2 inverted microscope and HeNe (633 nm), another HeNe (594 nm), Arg (488 nm) and diode (405 nm) lasers

CHAPTER 2. MATERIALS AND METHODS for excitation. A 60x and a 63x oil objectives were used with the DeltaVision and the Leica confocal systems, respectively.

2.2.2. Differentiation of Neuro-2a cells

Differentiation of Neuro-2a cells was initiated as described previously by Zheng and Zhou (2008). Cells were seeded at a density of 100 cells/mm2 and medium was changed the next day to differentiation medium (EMEM with 2% FBS, 2 mM L- glutamine and 20 µM retinoic acid). Cells were treated for 4 days by changing the medium on each day. To detect morphological changes during differentiation, micrographs were taken every day by a Zeiss Axiovert 40 CFL microscope with a 10× objective (Carl Zeiss Ltd., Cambridge, UK). Four micrographs were taken by random selection from all experimental conditions. Experiments were repeated 3 times. Cells possessing at least one neurite with a length at least twice the cell body were accepted as differentiated and their ratio was expressed as a percentage of the total cell number. Neurite outgrowth was calculated based on a previously described stereological approach (Lucocq 2008; Ronn et al. 2000). Briefly, an unbiased counting frame was superimposed on the micrographs containing 100 µm x 100 µm squares. This was followed by counting the number of neurite intersections on both vertical and horizontal lines of the frame (one marginal line was excluded from both) and the cell number. The length of neurites was estimated with the equation:

in which L is the total length of neurites, d is the distance between lines in the frame (100 µm) and l is the total number of neurite intersections (vertical + horizontal lines). In order to calculate the total length of neurites/cell L was divided by the cell number.

CHAPTER 2. MATERIALS AND METHODS

2.2.3. [3H]Inositol-phosphate release assays (IP-assay)

COS-7 cells were electroporated as previously described. The transfection mixture was diluted in serum free DMEM and cells were seeded into 12-well plates (1 ml/well). The medium was changed the following day to serum- and inositol-free DMEM (MP Biomedicals, Illkirch, France) containing 1 μCi/ml myo-D-[3H]inositol (GE Healthcare) and was incubated for 24 hours. On the third day, cells were pretreated with 10 mM LiCl (inhibitor of inositol monophosphatases) containing HEPES-buffered DMEM to enable the accumulation of inositol phosphates. Thereafter, cells were stimulated at 37oC for 3 hours with various drugs in HEPES- buffered DMEM containing 10 mM LiCl. These drugs and their dilution are listed in Table 2.3. name of agent effect dilution vehicle monensin (Sigma-Aldrich) Na+-ionophore 0-70 µM ethanol ionomycin (Tocris *) Ca2+-ionophore 0-1 µM DMSO A23187 (Tocris *) Ca2+-ionophore 0-5 µM DMSO thapsigargin (Tocris *) inhibitor of the SERCA pump 0-5 µM DMSO bafilomycin A1 (Tocris *) inhibitor of vacuolar H+-ATPases 0-0.5 µM DMSO ethylene glycol tetraacetic acid (EGTA, Sigma-Aldrich) Ca2+-chelator 0-2.5 mM water U73122 (Sigma-Aldrich) inhibitor of PLCs 0-10 µM DMSO antagonist of the mitochondrial CGP-37157 (Tocris *) 0-5 µM DMSO Na+/Ca2+ exchanger 2+ SKF-96365 (Tocris *) non-specific Ca -channel inhibitor 0-10 µM water Table 2.3.: Agents used in combination with [3H]inositol phosphate release assays. *: Tocris Biosciences, Bristol, UK.

Treatments were terminated by the removal of the medium and addition of 1 ml 10 mM formic acid for 30 minutes. Formic acid, containing 3H-labelled inositol phosphates, was transferred onto Dowex AG 1 resin (0.5 ml slurry containing 50 % water, Bio-Rad Laboratories, Inc., Hemel Hempstead, UK). Resin was washed with water and 60 mM ammonium formate/5 mM sodium tetraborate. Inositol phosphates were eluted with 1 ml of 1 M ammonium formate/0.1 M formic acid, from which 800 µl was transferred into scintillation tubes. Subsequently, 2.5 ml Optiphase Hisafe

CHAPTER 2. MATERIALS AND METHODS

3 (Perkin Elmer) was mixed with the content of the tubes and activity was measured with a MicroBeta2 scintillation counter (PerkinElmer, Waltham, MA, USA).

A modified IP-assay was also conducted to assess phospholipid-turnover in stably transfected Neuro-2a cells. Cells were seeded into 14 cm diameter dishes at a density to yield ~ 70-80% confluence the next day. After 24 hours, medium was changed to serum- and inositol-free DMEM containing 1 μCi/ml myo-D-[3H]inositol. On the third day, cells were trypsinised and counted with a Z1 Coulter particle counter (Beckman Coulter, Brea, CA, USA). Cells were harvested by centrifugation and resuspended to equal cell density in Buffer A containing 140 mM NaCl, 20 mM

HEPES, 8 mM glucose, 4 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 1 mg/ml BSA. Equal numbers of cells (5 x 105) were split into tubes, containing 1 ml final volume of Buffer A with 10 mM LiCl and were incubated for 1 hour. Cells were harvested by centrifugation at 800 rpm and medium was changed to fresh Buffer A with LiCl containing 5 µM A23187 or its vehicle (DMSO). Incubation was terminated by the addition of a final 10 mM formic acid and cell debris was removed by centrifugation at 5,000 rpm for 3 minutes. The rest of the assay was conducted as described above.

2.2.4. Permeabilisation of COS-7 cells to Ca2+

To investigate the Ca2+-sensitivity of wild type and mutant PLCη2, the encoding plasmids were transiently transfected into COS-7 cells which were permeabilised in the presence of various free Ca2+ concentrations. Cells transfected by electroporation were transferred into 14 cm diameter dishes containing 25 ml complete DMEM and were treated with myo-D-[3H]inositol as previously described. On the third day, cells were trypsinised and washed once in 50 ml Buffer 1 (/ construct) containing 145 mM NaCl, 5.6 mM KCl, 5.6 mM glucose, 15 mM HEPES, 0.1 % BSA, pH 7.4. Cells were harvested by centrifugation at 1500 rpm, at 4oC for 3 minutes and the pellet was resuspended in 50 ml Buffer 2 containing 20 mM PIPES (K+ salt), 129 mM + glutamate (K salt), 5 mM glucose, 5 mM ATP, 5.31 mM MgCl2, 5 mM EGTA, 10 mM LiCl, 0.1% BSA, pH 6.6. At this point, cell number was counted and the cells

CHAPTER 2. MATERIALS AND METHODS were harvested with centrifugation again. The pellets were resuspended in Buffer B to equal cell densities. One ml of each cell suspension was permeabilised by their exposure to 5 discharges of 3 kV/cm3 at both +ve and –ve polarities using a custom- made device as previously described (Guild 1991). Suspension was aliquoted (40 µl) into eppendorf tubes containing 960 µl Buffer 2 and various free concentrations of Ca2+ in equilibrium with 5 mM EGTA (Table 2.4, Portzehl et al. 1964). Cell suspensions were incubated at 37ºC for 2 hours. The incubation was terminated by the addition of formic acid in a final concentration of 10 mM. Cell debris was removed by centrifugation at 5,000 rpm for 3 minutes. One 800 µl of supernatant was transferred onto Dowex AG 1- resin and the [3H]inositol phosphate release assay were performed as described before.

2+ [CaCl2] (mM) [MgCl2] (mM) Free [Ca ] logM 0 5.31 - 9 0.020 5.30 - 8 0.197 5.30 - 7 1.460 5.29 - 6 4.050 5.25 - 5 5.159 5.10 - 4 7.340 4.11 - 3

Table 2.4.: Concentrations of CaCl2 and MgCl2 required to obtain various concentrations of free Ca2+ in equilibrium with 5 mM EGTA.

2.2.5. Dual-Luciferase reporter assay

Dual luciferase assay was conducted in order to measure retinoic acid stimulated gene-transcription in stably transfected Neuro-2a cells. Luciferase reporter vectors (Figure 4.16.) were obtained from SABiosciences (Qiagen Ltd., Crawley, UK). Two plasmids were transfected simultaneously; one containing an inducible firefly luciferase gene under the regulation of the retinoic acid responsive element (RARE, sequence: AGGTCACCAGGAGGTCA), and one encoding the constitutive

CHAPTER 2. MATERIALS AND METHODS expression of the Renilla luciferase. This latter was used as an internal control of transfection efficiency. On the first day of the experiment, equal numbers of cells (5 x 104/well) were seeded onto a 96-well plate in antibiotics-free medium. Twenty- four hours later, transfection mixture was prepared as follows (calculated for each well): 100 ng plasmid mixture was diluted with 25 µl Opti-MEM and in parallel 0.3 µl Lipofectamine 2000 was also mixed with 25 µl Opti-MEM. These were incubated for 5 minutes and then pooled together to be further incubated for 30 minutes. The 50 µl transfection solution was added to the medium dropwise. The medium was changed 6 hours later to antibiotic- and serum-free EMEM. Cells were treated with retinoic acid or DMSO for 4 hours the next day. To terminate the treatment, cells were washed once with PBS. Cells were lysed by the Passive Lysis Buffer (20 µl) of the Dual Luciferase Reporter Assay Kit (DLRA, Promega, Southampton, UK) for 15 minutes. This kit also provides the materials required for the rest of the luciferase assay. Samples were stored at -20oC until further analysis. DLRA was conducted step-wise by the manual addition of the reagents and the activity was read out by a Fluostar Optima plate reader (BMG Labtech, Ortenberg, Germany). The DLRA is supplied with two substrates to allow the subsequent measurement of the activities of both luciferases in the same lysate. The reaction of the firefly luciferase gives a stable luminescent signal when 100 µl Luciferase Assay Reagent II is added to the 20 µl cell lysate. After the appropriate measurement of the signal, it is quenched by the Stop & Glo reagent which contains a different substrate for the Renilla luciferase. Activity was measured by a 2-second pre-measurement delay and a continuous measurement for 10 seconds. Triplicates were used in all experimental conditions.

CHAPTER 2. MATERIALS AND METHODS

2.3. Molecular biology methods

2.3.1. RNA and DNA isolation, analysis and modification

2.3.1.1. RNA isolation

RNA work was routinely preceded by the cleaning of the lab surfaces and pipettes as well as the illumination of pipettes with UV light to avoid RNAse contamination. RNA was extracted by the Isolate RNA Mini Kit (Bioline, London, UK) according to the manufacturer`s instructions. RNA concentration was measured by a NanoVue spectrophotometer (GE Healthcare) at 260 nm and purity was tested with the 260 nm/280 nm ratio. RNA was kept at -80oC until further analysis.

2.3.1.2. DNA digestion and Reverse Transcription (RT)

DNA contamination was removed by DNAse digestion (RQ1 RNase-Free DNase, Promega, Southampton, UK) in accordance with the manufacturer`s instructions. Half µg of digested RNA was subjected to a two-step RT reaction. First the RNA was incubated with 100 pmol oligodT18 and 0.5 mM dNTP (when in the final 20 µl, Fermentas, St. Leon-Rot, Germany) in 14.5 µl (completed with DEPC-treated water) at 65oC for 5 minutes and then chilled on ice. Next, 200 units of RevertAid Premium Reverse Transcriptase, 20 u Thermo Scientific RiboLock RNase Inhibitor and 1x RT-buffer (Fermentas) was added. Tubes were incubated at 50oC for 30 minutes, at 60oC for 10 minutes and the reaction was terminated by heating up the samples to 85oC for 5 minutes.

2.3.1.3. Conventional (semi-quantitative) Polymerase Chain Reaction (PCR)

One µl of the RT sample was subjected to PCR analysis with the addition of

1.25 units GoTaq polymerase, 1× buffer, 1.5 mM MgCl2 (Promega), 0.2 mM dNTPs (Fermentas) and 250 nM of each primer. Cycling profile was: 95oC for 15 s, 60oC for

CHAPTER 2. MATERIALS AND METHODS

30 s and 72oC for 1 min, preceded by a 2-minute enzyme activation at 95oC. Primers used throughout the thesis for PCR analysis are listed in Table 2.5.

Primer Sequence RARα FW 5`-CACTTTTGCAGAGCGCGGTGCGG-3` RARα RW 5`-GCGTTTGCTGGTGATGAAGACGTGGC-3` RARβ FW 5`-GGAAGGAGAAGGCAGTGACTCTGTGG-3` RARβ RW 5`-CGGTGCTGCCATTCGGCCTGG-3` RARγ FW 5`-GCGGAGTCAGTGTGCGGTTTGGG-3` RARγ RW 5`-GGTCTCGGGGATGGAGCACCGC-3` RPL0 FW 5`-GAGTGATGTGCAGCTGATAAAGACTGG-3` RPL0 RW 5`-CTGCTCTGTGATGTCGAGCACTTCAG-3` PLCɳ2 FW 5`-GGCTACACTCTGACCTCCAAGATCC-3` PLCɳ2 RW 5`-GGAAGCATGGTGGCATCTTCGCTGC-3` 21a FW 5`-GGCCCCTACTCAAGAGAGGTCAGG-3` 21b FW 5`-CCTGCCAGCCGGAGGGTGC-3` 21c RW 5`-GCATGCCAGGTGTAGAGAGCATGGG-3` 22 RW 5`-GGCACTCGCAGGTCTCAATCCC-3` 23 RW 5`-CTGGGAGCTGGTCACTTTGGCTCC-3`

Table 2.5.: Primer sequences used for RT-PCR. FW: forward, RW: reverse.

2.3.1.4. Quantitative PCR (qPCR)

Gene-expression levels were accurately measured by qPCR relative to the level of the large ribosomal protein P0 (RLP0) mRNA with Brilliant III Ultra-Fast SYBR Green mix (Agilent Technologies, Cheshire, UK). Each reaction contained cDNA product of 5 ng RNA, 250 nM of each primer, 1x qPCR mix and RNAse free ultrapure water in 20 µl. PCR was conducted in a Rotor-Gene Q PCR machine (Qiagen, Crawley, UK). Parameters were: 95oC for 3 min., and then 40 cycles of 95oC for 5 s and 60oC for 20 s. PCR product was analysed by adding a melting curve at the end of the qPCR program (temperature gradient between 50oC and 99oC). Expression levels were calculated by the ΔΔCt method (Livak and Schmittgen 2001).

CHAPTER 2. MATERIALS AND METHODS

2.3.1.4. Agarose Gel Electrophoresis

PCR products were analysed on agarose gels composed of 1% or 2% (w/v) agarose, melted in Tris-acetate/EDTA-buffer (TAE-buffer, from 40 mM Tris acetate and 1 mM EDTA). SyberSafe dye was added to the melted agarose which enables the detection of DNA in the gel. Gel was left to set and then was transferred into an electrophoresis tank containing TAE-buffer. Samples were prepared with a gel loading dye and Hyperladder I DNA-ladder was also loaded at least in one lane (Bioline). Gels were run at 150 V for as long as required for the separation of bands. DNA was visualised in a Gel Doc XR+ Imager and analysed using an Image Lab 2.0 software (Bio-Rad Laboratories, Inc.).

2.3.1.5. Purification of DNA-bands from agarose gels

DNA was visualised by a Blue Light Transilluminator (Life Technologies) and DNA bands were excised by sterile blades. DNA was purified by the QIAquick Gel Extraction Kit (Qiagen) from the gel block according to the manufacturer`s protocol. Purified DNA was eluted in 30 µl of sterile water.

2.3.1.6. Digestion with restriction endonucleases

Digestion of DNA by restriction enzymes was used as a diagnostic tool to detect the presence of the correct insert in vectors or to generate compatible overhanging DNA ends for cloning. All enzymes were obtained from New England Biolabs (NEB, Ipswich, MA, USA) and were supplied with their compatible buffer. As a general protocol, 1 µg of DNA was mixed with 2 units of enzyme, 2 µg BSA and 1x buffer in a final volume of 20 µl completed with sterile water. Double digestion was conducted in a buffer which was optimal for both enzymes. When needed, restriction enzyme was inactivated by incubating the reaction at 65 oC for 20 minutes.

CHAPTER 2. MATERIALS AND METHODS

2.3.1.7. Molecular cloning

The human LIMK1 plasmid fused to a c-myc tag on the C-terminus and cloned into pcDNA3.1 was kindly provided by Prof. Michael Olson (Beatson Institute, Glasgow, UK). The 75–1238 residues of mouse PLCη2 (isoform a; NP_780765) cloned into pcDNA3.1 was a gift from Prof. Kiyoko Fukami (Tokyo University of Pharmacy and Life Science, Japan). The mCherry-conjugated PLCδ1 PH domain in pmCherry-C1 vector (Clonetech, Saint-Germain-en-Laye, France) was provided by Dr. Alexander Gray (College of Life Sciences, University of Dundee, UK). GFP-tagged constructs (GFP-PLCη2, GFP-ΔPH-PLCη2 and GFP-PH- PLCη2) were cloned by the insertion of sequences into pcDNA3.1/NT-GFP-TOPO vector (Life Technologies) using the TA-cloning method according to the manufacturer`s instructions. Briefly, desired insert sequences were amplified by Taq polymerase which encompasses a terminal transferase activity linking a single deoxyadenosine to the 3` ends of PCR products. Reactions were run on agarose gels and DNA was gel purified as described before. One µl of the purified DNA was mixed with a ligation mixture containing 1 µl salt solution, 1 µl vector (10 ng) and 2 µl sterile water. Mixture was incubated for 5- 30 minutes. During this time, PCR products are integrated by joining to the overhanging 3´ deoxythymidine residues in the vector.

PH-lacking PLCη2 (ΔPH-PLCη2) was cloned into pcDNA3.1 (Life Technologies) vector by directional cloning in accordance with the manufacturer`s instructions. Briefly, sequence encoding residues 228–1238 of PLCη2 was amplified by a forward primer providing the addition of the CACC sequence and the start codon (ATG) onto the 5` end and a reverse primer linking a stop codon at the 3` end. PCR was conducted by a proofreading DNA polymerase (Velocity DNA polymerase, Bioline) according to the manufacturer`s protocol. Reactions were analysed on agarose gels and gel purified as described before. One µl of the purified DNA was mixed with a ligation mixture containing 1 µl salt solution, 1 µl vector (10 ng) and 3 µl sterile water. Mixture was incubated for 5-30 minutes. The amplified sequence integrates into the plasmid by annealing to the GTGG 3` overhang on the reverse strand.

CHAPTER 2. MATERIALS AND METHODS

The GST-tagged PLCη PH domains were generated by inserting sequences corresponding to residues 14-137 and 115-238 of PLCη1 and PLCη2, respectively into the bacterial expression vector, pGEX6 using EcoRI and NotI restriction sites. Cloning was conducted by Dr. Alan Stewart.

Ligation mixtures were transformed into E. coli in all cases as in section 2.3.1.9. The full-length sequences of the inserts were verified by sequencing, conducted at the Dundee Sequencing Service, Dundee, UK.

2.3.1.8. Site-directed mutagenesis

Point-mutations in PLCη2-sequence integral to plasmids pcDNA3.1 and pcDNA/NT-GFP were generated by QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturer`s instructions. The kit contains a proofreading DNA polymerase (PfuTurbo) and buffer composition allowing the enzyme to amplify the whole plasmid with primers containing the desired mutation during PCR cycles. Amplification is followed by a digestion of the parental plasmid DNA by DpnI restriction enzyme which targets methylated DNA. Mutations were verified by sequencing as previously.

2.3.1.9. Transformation of vectors into E. coli

One to four µl of the ligation mixture was transformed into 50 µl Alpha-select chemically competent cells (Bioline) by heat shock according to the manufacturer`s protocol. This was followed by the addition of SOC medium (Bioline) and its subsequent incubation at 37oC for 1 hour and shaking at 210 rpm. The whole mixture was spread on agar plates contaning the appropriate antibiotic resistance (100 μg/ml ampicillin). Plates were incubated at 37oC overnight and positive clones were isolated the next day. Stocks of the transformed bacteria were made by mixing 800 µl of an overnight culture with 200 µl glycerol in cryovials. Tubes were stored at -80oC.

CHAPTER 2. MATERIALS AND METHODS

2.3.1.10. Isolation of plasmid DNA

Single-cell derived colonies or a small portion of glycerol stocks were used as the source to grow E.coli in 5 ml terrific broth (TB, Sigma-Aldrich) overnight (for miniprep) or in 1 ml Luria broth (LB, Sigma-Aldrich) for 6-8 hours from which 100 µl was transferred into 200 ml LB for overnight incubation (for maxiprep). Cultures were shaken at 220 rpm and 37oC. Plasmids were isolated by the Plasmid MiniPrep Kit I for diagnostic procedures, or by the Endofree Plasmid Maxi Kit for transfection of mammalian cells according to the manufacturer`s instructions (Biomiga, San Diego, CA, USA). Plasmid DNA was stored at -20oC.

2.3.2. Protein analysis

2.3.2.1. Western-blot analysis

Cells in a 6-well plate were washed two times with PBS and were scraped in 50- 100 µl RIPA buffer (150 mM NaCl, 2 mM EDTA, 1% IGEPAL CA-630, 0.1% SDS in 50 mM TrisHCl, pH 7.4 with protease inhibitor, Roche, Burgess Hill, UK) depending on the cell density. During this procedure plate was kept on ice. Cell lysates were spun at 14,000 rpm in a JA-18.1 rotor (Beckman-Coulter) for 10 minutes to remove cell debris. To generate membrane enriched fractions, cells were scraped in RIPA buffer free of NP-40 and SDS. This was homogenised and spun at 14.000 rpm (JA-18.1) for 10 minutes. Pellet was washed once with the modified RIPA buffer, centrifugated and was resuspended in complete RIPA buffer.

When it was required, protein concentration was measured by the BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer`s protocol. Protein samples were prepared with gel running buffer (final concentrations: 50 mM Tris– HCl, 10% glycerol, 2% SDS and 0.1 M DTT, 0.01% bromophenol blue, pH 6.8). Samples were incubated at 95oC for 10 minutes before loaded onto NuPAGE precast polyacrylamide gradient gels (4–12% Bis–Tris). The Hyperpage prestained protein marker was used as a reference for protein mass (Bioline). Samples were separated at

CHAPTER 2. MATERIALS AND METHODS

150 V for 1 hour to 1.5 hours in a MES buffer system (SDS-PAGE, Life Technologies).

Proteins were transferred onto PVDF membranes (GE Healthcare) in NuPage transfer buffer (Life Technologies, supplemented with 0.01% SDS and 11% methanol) at 30 V for 75 minutes. Before and following the transfer, membranes were treated with methanol for 2×5 minutes. Blots were incubated in blocking buffer (5% non-fat milk in TBS, for 1 hour) and in 1% non-fat milk and 0.5% Tween-20 in TBS for 1.5 hours containing primary antibody. Primary antibodies used in the thesis were: goat polyclonal anti-PLCη2 antibody (1:250 dilution; Santa Cruz Biotechnology, Heidelberg, Germany), rabbit polyclonal anti-PLCη2 antibody (1:5000 dilution; custom), mouse polyclonal anti-LIMK1 or rabbit anti-phospho- LIMK1 (Thr508) antibodies (1:1,000 dilution; Abcam), rabbit anti-CREB or anti- phospho-CREB (Ser133) antibodies (1:1,000 dilution; Cell Signaling Technology, Danvers, MA, USA) and mouse monoclonal anti-β-actin antibody (1:10,000 dilution; Sigma-Aldrich). The incubation was followed by three washing steps with TBST (TBS containing 0.5% Tween). Secondary antibodies were diluted in blocking buffer containing 0.5% Tween (1:10,000 dilution) which was added to the blots for 1.5 hours at room temperature. Blots were then washed 3 times in TBST for 15 minutes each. Two types of species-specific secondary antibodies were used. Alkaline phosphatase (AP)-linked secondary antibodies (anti-mouse, anti-rabbit and anti-goat) were used in combination with a colorimetric detection whereby the AP catalysed the diversion of the substrate (Western Blue Stabilized Substrate, Promega) into a purple product which was detected after drying the membrane. Otherwise, blots were incubated with horseradish peroxidase-conjugated antibodies (1:10,000 dilution; Thermo Fisher Scientific) and antibody-binding was detected by the SuperSignal West Dura chemiluminescent substrate (Thermo Fisher Scientific) with an LAS-3000 phosphoimager (Fujifilm, Düsseldorf, Germany). Densitometry was performed using ImageJ software (NIH, Bethesda, MD, USA).

CHAPTER 2. MATERIALS AND METHODS

2.3.2.2. Purification of GST-tagged PH domains

The pGex6 plasmids containing PLCη1 and PLCη2 PH domains were transformed into chemically competent E.coli strain BL21 (kindly provided by Dr. Jo Parish, College of Medical and Dental Sciences, University of Birmingham) by heat shock and plated as described earlier. A single colony from each was transferred into 5 ml LB and was incubated overnight at 37oC with shaking at 250 rpm. From this culture,

2 ml was transferred into 100 ml LB and was incubated until the OD600 reached 0.7- 0.9 (approx. 2.5-3 hours). At this point, IPTG was added in a final concentration of 0.1 mM and protein expression was induced for an additional 2.5 hours. Bacteria were harvested in 50 ml centrifugation tubes at 2,500 rpm for 10 minutes at 4oC in a JS.4.2 rotor. Pellets were resuspended in 10 ml GST-binding buffer containing 25 mM Tris pH 7.5, 150 mM NaCl, 1mM EDTA and protease inhibitor cocktail (Roche). Triton X100 was mixed in a final 0.5% to lyse the cells and the tubes were transferred to -80oC for 2 hours. Cells were then thawed and sonicated. Remaining cell debris was removed by high-speed centrifugation at 10,000 rpm in a JA-20 rotor (Beckman Coulter). Supernatants were collected into a 50 ml tube and 500 µl of 50% glutathione-linked agarose beads (in GST binding buffer) were added. This was incubated for 2 hours at 4oC with mixing. Beads were collected at 1,000 rpm for 1 minute and were washed with 20 ml GST-binding buffer. This was repeated once. The agarose slurry was then transferred into 1.5 ml microcentrifuge tubes, beads were pelleted by a quick spin and the remaining of the liquid phase was discarded. Proteins were eluted by 500 µl Elution buffer containing 10 mM reduced glutathione, 50 mM Tris-HCl and 5 % glycerol, pH 8.0 which was incubated for 15 minutes. Elution was repeated but eluates were not pooled. Purities of eluates were tested by SDS-PAGE followed by staining the whole protein content of the gel. This latter included the incubation of the gel with excess amounts of Coomassie staining (0.003% Coomassie brilliant blue-R, 50% methanol and 10% acetic acid) for 30 minutes followed by two washes in ultrapure water and repeated incubation in de-

CHAPTER 2. MATERIALS AND METHODS stain solution (40% methanol and 10% glacial acetic acid in ultrapure water). Gel was photographed in the Gel Doc XR+ Imager (Bio-Rad Laboratories, Inc.).

2.3.2.3. Förster resonance energy transfer (FRET)-based detection of protein-lipid interactions

The assay was conducted by Dr. Alexander Gray (College of Life Sciences, University of Dundee, UK) similarly as described before (Gray et al. 2003). Briefly, PLCη-PH domains (14 μl of 0.62 mg/ml) were mixed with 4 μl APC-streptavidin (2 mg/ml, Prozyme Ltd. Hayward, CA, US), 80 μl Eu-anti-GST (80 μg/ml) (Perkin- Elmer) in 2.5 ml assay buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 5 mM DTT, 0.05% Chaps). The assay was conducted in a 96-well format as follows: 25 μl of the assay mixture were added to the same volume of biotinylated lipids (elevating concentrations of 0-50 pmol in final volume) and the association of biotinylated phosphoinositide/PH domain complexes were measured in an LJL Analyst microplate reader. In addition displacement of GST-PLCη2 PH was also measured with the addition of elevating concentrations of phosphoinositides.

PI(4,5)P2-PH domain sensor complexes were developed by mixing 4 μl (2 mg/ml)

Streptavidin APC, 200 pmol biotin PI(4,5)P2, 14 μl (0.62 mg/ml) PH domain and 50 μl Eu-anti GST (80 μg/ml) in 2.5 ml assay buffer. Complexes were dissociated by the addition of serially diluted non-biotinylated phosphoinositides.

2.3.2.4. Detection of protein-lipid interactions by lipid-adsorbed membranes (PIP strips)

Membranes pre-adsorbed with phospholipids; lysophosphatidic acid, lysophosphocholine, phosphatidylinositol, phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate, phosphatidylinositol 5-phosphate, phosphatidylethanolamine, phosphatidylcholine, sphingosine 1-phosphate, phosphatidylinositol 3,4-bisphosphate, phosphatidylinositol 3,5-bisphosphate, phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol trisphosphate,

CHAPTER 2. MATERIALS AND METHODS phosphatidic acid and phosphatidylserine were obtained from Echelon Biosciences Inc. and were processed according to the manufacturer`s instructions. Briefly, membranes were blocked and then incubated by the addition of purified GST-PH domains in the concentration of 0.5 µg/mL in 3% BSA and 0.1% Tween-20 in phosphate-buffered saline (PBS). This was followed by two incubation steps with a monoclonal mouse-anti-GST (Sigma-Aldrich) and anti-mouse HRP-linked secondary antibodies diluted in the same buffer, respectively. Antibody-binding was detected by the SuperSignal West Dura chemiluminescent substrate in the LAS-3000 phosphoimager.

2.3.2.5. Identification of protein-protein interactions by bacterial two-hybrid screen

The BacterioMatch II Two-Hybrid System (Agilent) was used to identify interaction partners for PLCη2 C-terminal domain in accordance with the manufacturer`s instructions. The cDNA corresponding to the C-terminal domain of PLCη2 splice variant 21a/22/23 (residues 967-1070) was cloned into the pBT bait vector (work was conducted by Dr. Alan J. Stewart). This was co-transfected into the reporter E.coli strain with a human pancreas cDNA library encoded by the pTRG plasmid (cloned by Dr Kevin Morgan, MRC Human Reproductive Sciences Unit, Edinburgh, UK). When an interaction occurs between the C-terminal domain and the protein encoded by the pTRG vector, it allows the expression of the His3 and Aad3 genes in the reporter E. coli strain. That permits the bacteria to grow in the presence of 3- aminotriazole (3-AT, an inhibitor of histidine synthesis) or streptomycin, which are used for selection of interaction partners. The pBT and pTRG plasmids were co- transformed into reporter cells according to the manufacturer’s protocol. Transformed cells were spread onto plates containing 5 mM 3-AT and growing colonies were transferred onto dual-selective plates containing 5 mM 3-AT and 12.5 μg/ml streptomycin. The plasmids were isolated from a single colony-derived culture, and sequenced to identify the interaction partner using NCBI-BLAST.

CHAPTER 2. MATERIALS AND METHODS

2.3.2.6. Myc-tag co-immunoprecipitation (pull-down)

Experiment was conducted by using the Pierce Mammalian c-Myc Tag IP/Co-IP Kit (Thermo Fisher Scientific) in accordance with the manufacturer`s instructions. Vectors, expressing PLCη2 and myc-tagged LIMK1 were transfected into COS-7 cells by electroporation as previously. As controls, cells transfected with only one of the constructs were used. Transfected cells were seeded onto 6-well plates. After two days, cells were washed twice with TBS and lysed with the Mammalian Protein Extraction Reagent of the kit, by adding 200 µl to each well of 5 wells. One well of cells was scraped and collected in RIPA buffer for the subsequent detection of the expression levels of transfected proteins by Western blot. Other wells were pooled together, prepared as suggested by the protocol and protein concentration was measured by the BCA protein assay kit (Thermo Fisher Scientific). From each transfection, 500 µg of protein was added to 5 µg antibody-linked agarose slurry. This was incubated overnight and protein complexes were purified and eluted the next day according to the manufacturer`s instructions. Proteins were detected by Western blot.

2.4. Statistical analysis

Experiments were repeated independently at least three times and all assays were performed in triplicate. Data are presented as mean values ± the standard error of the mean (SEM). Three levels of statistical significance were used according to the confidence values: p<0.05, p<0.01 or p<0.001, which were indicated by asterisks in figures. Analyses were performed using the SigmaPlot software (Systat Software Inc, Chicago, IL, USA) with one-way ANOVA followed by the Holm-Sidak post hoc test or Student`s t-test depending on the type of the experiment.

Chapter 3.

Biochemical characterisation of PLCη2

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

3.1. Introduction

Following the identification of PLCη2 the general attention was focused on the characterisation of its enzyme activity. Nakahara et al. generated GST-tagged PLCη2 using a baculovirus/Sf9 cell expression system in parallel with GST-PLCδ1 (Nakahara et al. 2005). These proteins were treated with various amounts of Ca2+ in a reaction mixture containing reconstituted membranes composed of 3 phosphatidylethanolamine, PI(4,5)P2 and [ H]PI(4,5)P2. The reaction was followed 3 2+ by the measurement of [ H]I(1,4,5)P3 accumulation. Although the threshold for Ca activation of recombinant PLCη2 was 10-fold lower than that of PLCδ1, its maximal rate of PI(4,5)P2 hydrolysis was measured at much smaller values (~10-fold) (at any concentrations of Ca2+) (Nakahara et al. 2005). Reduced enzymatic activity of PLCη2 might be due to the lack of essential factors that are required for effective hydrolysis and are normally found in the cell. One of those factors might be the presence of membranes with physiological phospholipid content that might be required for the efficient docking of the enzyme. A primary aim of this Chapter is to therefore study the activation of the enzyme in a cellular context. The overexpression of PLCη2 in COS-7 cells offered an excellent tool for this purpose.

PH domains of PLCs are important for membrane targeting and possess different sensitivities towards phospholipids (Singh and Murray 2003). This latter feature might be of unique importance in determining subcellular localisation of PLCη2. In this Chapter, the importance of the PH domain for membrane binding as well as its lipid-binding specificity is assessed. A secondary, Ca2+-dependent binding of the membrane by the C2 domain is crucial for optimal phospholipase activity in PLCδ1 (Grobler et al. 1996). The importance of the C2 domain in PLCη2 was therefore also examined by site-directed mutagenesis.

PLCζ was previously demonstrated to contain a functional EF-hand which is unique among PLCs. This domain provides the structural background for sensing Ca2+ in the nanomolar levels. The sperm-specific PLCζ uses this exceptional feature to evoke

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

Ca2+ transients upon its transfer to the oocyte during fertilisation (Kouchi et al. 2005; Nomikos et al. 2011a). High Ca2+-sensitivity of PLCηs inspired the examination of the molecular properties of its EF-hand and to evaluate the Ca2+-binding properties of this domain also by site directed mutagenesis studies.

3.2 Characterisation of PLCη2 Ca2+-activation

3.2.1. PLCη2 is activated by monensin

PLCη2 was expressed in COS-7 cells as described in Materials and Methods. This particular cell line was selected for two main reasons. Firstly, COS-7 cells are often used as mammalian cellular models for expression studies due to their high transfection rate by electroporation (Parham et al. 1998). In addition, COS-7 cells possess no endogenous PLCη2 expression and therefore adequately report the differences upon their transfection with the PLCη2-encoding vector (Stewart et al. 2007). Numerous agents were used known to increase intracellular Ca2+ levels through different mechanisms (monensin, ionomycin, A23187, thapsigargin and bafilomycin, Figure 3.1.). Empty vector transfected cells were used as controls in all experiments. Monensin is a Na+/H+ antiporter which was previously shown to activate Na+/Ca2+ exchangers to remove the excess Na+ from the cell and consequently increase intracellular Ca2+ level (Wang et al. 1999). Ca2+ transients evoked by this mechanism greatly stimulated PI(4,5)P2 hydrolysis in PLCη2- expressing cells (~6-fold, p<0.001, one-way ANOVA, Figure 3.1a) at maximal monensin-concentration (70µM). Treatment of a 10-fold lower concentration also resulted in a significant, ~2.8-fold upregulation of phospholipid turnover (p<0.01, one-way ANOVA). A slight increase (less than 55% of the untreated control) in

PI(4,5)P2 hydrolysis was observed in empty vector transfected cells possibly due to the activation of the endogenous PLC-pool. Next the effects of the Ca2+-ionophores, ionomycin and A23187, were investigated (Figure 3.1b and 3.1c). Unexpectedly, none of the agents significantly increased PI(4,5)P2 hydrolysis in PLCη2 expressing cells relative to the empty vector containing cells (one-way ANOVA). A small

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2 difference between PLCη2 and empty vector transfected cells was observed when 5 µM A23187 was used, however as stated above this was not significant. Potency of these drugs to stimulate PLC-activity is implicated by the dose-dependent activation of endogenous PLCs in the empty vector transfected cells.

2+ Figure 3.1.: The effects of various Ca -mobilising agents on PI(4,5)P2 hydrolysis in COS-7 cells transfected with PLCη2. Cells were treated with monensin (0-70 µM, A), ionomycin (0-1 µM, B), A23187 (0-5 µM), thapsigargin (0-5 µM, D) or bafilomycin (0-500 nM, E) for 3 hours. Empty vector transfected cells were used to detect the activity of the endogenous PLCs of COS-7 cells. All agents stimulated phospholipid-turnover in a dose-dependent manner in the control cells. However, only the Na+/H+ antiporter monensin induced a significant elevation in PLCη2- transfected cells compared to the activity in the control cells. A representative western blot shows (F) that PLCη2 is only expressed in transfected cells. Blot was probed with PLCη2 (PLCη2-AB) and β-actin antibodies (β-actin-AB). Error bars represent ± S.E.M. (one-way ANOVA, p<0.05:*, p<0.01:**, p<0.001:***, n=3). Figure is reproduced with permission from Elsevier (Popovics et al. 2011).

The highest concentration of ionomycin (1 µM) induced a ~65% increase whereas

5 µM A23187 elevated PI(4,5)P2 hydrolysis by 3-fold in empty vector transfected

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2 cells. Thapsigargin is a well-defined inhibitor of SERCA which prevents the Ca2+- uptake to the ER/SR leading to its Ca2+-depletion (Sagara and Inesi 1991). Although thapsigargin increased basal PLC activity to some extent (~30% at 5 µM), PLCη2- expressing cells showed no significantly different rate of [3H]inositol phosphate accumulation. Bafilomycin (A1) is a potent stimulator of lysosomal Ca2+ release. This is directed by its inhibitory effect on the vacuolar H+-pump leading to increased pH. Alkalinisation mobilises Ca2+ previously bound to proteins of the lysosome and the consequent increase in free lysosomal Ca2+ triggers a transient Ca2+-release from the organelle (Camacho et al. 2008). However, bafilomycin had no significant stimulatory effect upon PLCη2 activity in the transfected COS-7 cells. The lack of the effects of the Ca2+ ionophores ionomycin and A23187 on PLCη2 activity raises the possibility that it is not directly activated by the monensin-induced Ca2+ rise – rather it could be induced by non-direct, Ca2+-dependent processes. The following experiments were designed to provide evidence that the monensin-induced changes in PLCη2 activity are clearly Ca2+-dependent.

3.2.2. Monensin-induced activation of PLCη2 is directed by Ca2+

Monensin-induced activation of PLCη2 was further investigated by applying various agents that affect PLC activity or intracellular Ca2+ level (U73122, EGTA, SKF- 96365 and CPG-3757) during the treatment of the maximal concentration of monensin (70 µM). Firstly, the non-PLC specific inhibitor, U73122 was used to verify that the stimulation of inositol phosphate release is dependent on PLC activity in both empty vector and PLCη2-transfected cells. This inhibitor strongly reduced the phospholipid turnover in both conditions in a dose-dependent manner (Figure 3.2a, p<0.01 and p<0.001 at 1 and 10 µM, one-way ANOVA).

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

Figure 3.2.: Inhibition of monensin-induced PI(4,5)P2 hydrolysis in PLCη2- transfected COS-7 cells by various inhibitors. Cells were treated simultaneously with 70 µM monensin and elevating concentrations of the inhibitors: U73122 (0-10 µM, A), EGTA (0-2.5 mM, B), SKF-96365 (0-10 µM, C) and CPG-3757 (0-5 µM, D). In one experiment two inhibitors were administered simultaneously (SKF-96365 and CPG-3757, E). Black bars show the endogenous phospholipase activity in empty vector- and white bars in PLCη2-transfected cells. Error bars indicate ±S.E.M. Statistical significance of inhibition is shown relative to monensin-induced PLCη2 transfected cells (one-way ANOVA, p<0.05:*, p<0.01:**, p<0.001:***). Figure is reproduced with permission from Elsevier (Popovics et al. 2011).

In the following experiment, to demonstrate that the action of monensin is clearly dependent on Ca2+, a specific chelator (EGTA) was used. EGTA decreased the monensin-induced stimulation of PI(4,5)P2 hydrolysis in a concentration-dependent manner to reach the control level at the highest dose (2.5 mM, p<0.001, one-way ANOVA, Figure 3.2.b). A non-selective Ca2+-channel inhibitor, SKF-96365 (Merritt

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2 et al. 1990) also abolished the monensin-induced PLCη2 activity further indicating the Ca2+-dependence of this process. However, it has to be mentioned that in certain conditions SKF-96365 may inhibit off-target proteins and might affect the transport of other ions than Ca2+ (Rae et al. 2012). Interestingly, a selective inhibitor of the

NCXmito, CPG-37157 (Cox et al. 1993) reduced inositol-turnover in PLCη2- transfected cells to a similar extent (~40%, p<0.01, one-way ANOVA) to the maximal concentration of SKF-96365 (10 µM, p<0.001, one-way ANOVA). When the maximal concentration of SKF-96365 and CPG-37157 (10 µM and 5 µM, respectively) was applied simultaneously, only a slight additional reduction was administered in the [3H]inositol phosphate accumulation of the PLCη2-transfected cells (~50% total inhibition, p<0.001, one-way ANOVA). This suggests that a substantial extent of SKF-96365-inhibition is derived from its action on the NCXmito. Moreover, this leads to the observation that mitochondrial Ca2+-release –either directly or indirectly- is involved in the activation of PLCη2.

3.2.3. PLCη2 is activated by physiological levels of Ca2+ in permeabilised COS-7 cells

To determine the sensitivity of PLCη2 towards Ca2+, PLCη2-expressing COS-7 cells were permeabilised and were incubated for two hours in the presence of various free Ca2+ concentrations (with 10-fold dilution steps from 10-3 to 10-9M Ca2+). This was followed by the measurement of [3H]inositol phosphate release (Figure 3.3.). Empty vector-transfected cells were used as controls. PLCη2 activity displayed a strong increase between 0.1 and 1 μM free Ca2+. Maximal stimulation was acquired at 10-5 M Ca2+. Endogenous PLC activity registered in empty vector-transfected cells only exhibited a slight elevation and reached maximal activation at 10-4 M Ca2+. This is consistent with the previous finding which examined the Ca2+-sensitivity of PLCη2 and determined its maximal activity to be at ten-fold less Ca2+ concentration than that of PLCδ1 (Nakahara et al., 2005). It is noteworthy that above 10-4 M Ca2+ the activity of PLCη2 declines which event might suggest the presence of an inhibitory

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2 mechanism and could have an important role in the regulation of PLCη2. This assay set up is used in further experiments to show alterations in Ca2+-sensitivity of PLCη2 mutants.

Figure 3.3.: Measurement of [3H]inositol phosphate release in permeabilised COS-7 cells transfected with empty vector (dotted line) or PLCη2 (solid line) in the presence of various concentrations of free Ca2+. Results are expressed ± S.E.M, n=3.

3.2.4.: Localisation of PLCη2 in COS-7 cells

In section 3.2.2., monensin-induced PLCη2 activation was linked to mitochondrial Ca2+-release. This suggests that PLCη2 is localised to membranes which are in communicating distance from these organelles. To reveal the cellular localisation of PLCη2, two experiments were conducted. Firstly, an N-terminally tagged GFP- construct of PLCη2 was generated by TA-cloning using the 1/NT-GFP-TOPO vector as described in Chapter 2. Cellular localisation of the GFP-fused PLCη2 was assessed in transfected COS-7 cells. Two days after transfection, cells were treated with a mitochondrial-specific fluorescent dye, mitotracker (Red CMXRos, Life Technologies) according to the manufacturer`s instructions. Upon entering the cell, the dye undergoes oxidation, accumulates in the mitochondria and reacts with thiol protein groups. These steps provide the chemical background for the development of fluorescence which is retained after fixation (Macho et al. 1996). Distribution of the GFP-PLCη2 protein was somewhat variable, usually manifesting as punctate staining

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2 throughout the cell indicating its association to organelle membranes. Unexpectedly, it only partially co-localised with the mitochondrial staining (Pearson coefficient: 0.4433, S.E.M.=0.0387, Figure 3.4., co-localisation is indicated by yellow colour on the merged image).

Figure 3.4.: GFP-PLCη2 (green) possesses a low co-localisation rate with the mitochondria (red) in COS-7 cells. Transfected cells were treated with a mitochondrial marker (mitotracker) before fixation. Nuclei were stained with DAPI (blue). Images show little co-localisation of GFP-PLCη2 with mitochondria. This is supported by the co-localisation coefficient (0.4433, S.E.M.=0.0387, Pearson coefficient) calculated from 3 images. Images are representative and bars correspond to 5 µm. Images were taken by a Leica confocal system (TCP SP2).

For the detection of PLCη2 intracellular localisation, the protein was tagged on its N- terminus by GFP just before the PH-domain. Disturbance in target-binding and consequently in enzyme activity is a possible outcome when protein structure is altered by a fluorescent protein tag. Enzyme integrity of GFP-PLCη2 was assessed by measuring monensin-induced [3H]inositol-phosphate accumulation in transfected

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

COS-7 cells that was compared to wild-type PLCη2-expressing cells (Figure 3.5.). The GFP-tag highly reduced the PLC-activity which only reached a 2.3-fold elevation at maximal monensin concentration (70 μM).

Figure 3.5.: Measurement of monensin-induced accumulation of [3H]inositol phosphate in COS-7 cells transfected with constructs encoding wild-type and GFP- PLCη2. Empty vector transfected cells were used to detect the activity of the endogenous PLC activity. PLCη2 tagged with GFP on its N-terminus showed reduced activity compared to the untagged wild-type protein. Results are expressed ± S.E.M., n=3.

To exclude possibility that the GFP-tag altered the targeting-specificity of the protein, exogenously expressed untagged-PLCη2 was labelled by immunostaining (Figure 3.6.). Untagged-PLCη2-overexpressing COS-7 cells were again treated with mitotracker red, fixed, permabilised and probed with a rabbit antibody against PLCη2, and an anti-rabbit Dylight-488 secondary antibody. The green staining only occurred in certain cells which were considered transfected. Surprisingly, PLCη2 was highly co-localised with the mitochondrial dye (Pearson coefficient: 0.7921, S.E.M.=0.0629). On the other hand, PLCη2 expressing cells underwent a structural change and abnormal condensation of the mitochondria at the perinuclear region was observed. The origin of the altered morphology is unknown, however the mitochondrial localisation of PLCη2 uncovers why it was specifically activated by

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2 mitochondrial Ca2+-release. It is also intriguing that the number of mitochondria seems to be substantially increased in PLCη2-transfected cells. This could be due to the presence of PLCη2 which might have altered basal Ca2+ concentrations and has led to increased mitochondrial fissure.

Figure 3.6.: Exogenously introduced PLCη2 exhibits a high co-localisation rate with mitochondria and induce morphological changes in COS-7 cells. PLCη2 was transfected into COS-7 cells by electroporation. Cells were treated with mitotracker before fixation to label mitochondria (red) and PLCη2 was immunostained by a specific primary antibody and an anti-rabbit Dylight-488 secondary antibody (green). Nuclei were stained with DAPI (blue). Images show high co-localisation rate of PLCη2 with mitochondria. This is supported by the co-localisation coefficient (0.7921, S.E.M.=0.0629, Pearson coefficient) calculated from 3 images. Images are representative and bars correspond to 10 µm. Images were taken by a Leica confocal system (TCP SP2).

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

3.3. Investigation of the PLCη2 PH domain

3.3.1. PLCη2 PH domain is required for membrane-binding

Membrane-targeting of PLC enzymes (with the exception of PLCζ) is directed by the N-terminal PH domain (section 1.2.3.2.). To ascertain whether PLCη2 is able to associate with the membranes in the absence of the PH domain, a deletion mutant of PLCη2 lacking this domain (residues 228–1238, ΔPH-PLCη2) was inserted into 1/NT-GFP-TOPO vector as described in Chapter 2. Wild-type and ΔPH-PLCη2 were then overexpressed in COS-7 cells and the distribution of each protein (as visualised by the GFP-tag) was examined by confocal microscopy (Figure 3.7.).

Figure 3.7.: Localisation of GFP-PLCη2 and its PH domain lacking deletion mutant (GFP-ΔPH) in COS-7 cells. The two GFP-fused constructs (shown in green) were transfected by electroporation and fixed after 2 days. Nuclei were stained with DAPI (blue). GFP-PLCη2 was localised to the plasma membrane and exhibited a punctate staining throughout the cell indicating its presence on extra- and intracellular membranes. GFP-ΔPH-PLCη2 has lost its ability to bind membranes revealed by its diffused staining in the cytosol and the nucleoplasm. Bars correspond to 10 µm. Images were taken by a Leica confocal system (TCP SP2). Figure is reproduced with permission from Elsevier (Popovics et al. 2011).

The GFP-PLCη2 protein localised to cellular membranes with punctate staining throughout the cell body only lacking in the nucleus. A similar study by Nakahara et

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2 al. using HeLa S3 cells found that GFP-PLCη2 predominantly localises at the plasma membrane (Nakahara et al., 2005). This suggests that PLCη2 might exhibit a cell- specific localisation pattern that may be directed by the alterations in phospholipid- composition of membranes in these cells. The GFP-ΔPH-PLCη2 protein exhibited diffuse staining throughout the cytosol indicating its inability to associate with cellular membranes. This is consistent with the findings of the above mentioned study (Nakahara et al., 2005), which similarly highlighted the importance of the PH domain in membrane binding.

Figure 3.8.: Membrane-binding ability of PLCη2 is reduced in its PH-lacking mutant as it was shown by western blot from membrane enriched fractions. A: Western blots showing the protein levels of wild-type and ΔPH-PLCη2 in whole cell lysates (wcl) and in membrane-enriched fractions (mef) of transfected COS-7 cells. To collect membranes, cells were scraped and homogenised in RIPA buffer (lacking IGEPAL and SDS) and the homogenate was centrifugated at 14,000 g for 10 min. The pellet was washed and resuspended in complete RIPA buffer. Blots were also probed with antibodies specific to β-actin and the α1 subunit of Na+/K+-ATPase to show equal loading. B: Protein levels were calculated relative to wild-type PLCη2 after their normalisation to β-actin Na+/K+-ATPase levels. Results are expressed ± S.E.M, n=3. Figure is reproduced with permission from Elsevier (Popovics et al. 2011).

The abilities of PLCη2 and ΔPH-PLCη2 proteins to bind to cellular membranes were also assessed by Western blotting. Both proteins were transfected into COS-7 cells which were lysed after 2 days and protein levels were detected by Western blot (Figure 3.8a). Firstly, the expression of the two proteins in whole cell-lysates was

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2 calculated relative to β-actin levels. Membrane enriched cell lysates were also produced from the same transfected cells by an altered preparation of cell lysates (see Chapter 2 for more information on preparation). In these samples the level of PLCη2 and ΔPH-PLCη2 were detected and expressed relative to the expression of the membrane marker Na+/K+ ATPase. Figure 3.7b demonstrates the relative levels of PLCη2 and ΔPH-PLCη2 after their normalisation with their levels in whole cell membranes. This indicates that ΔPH level is ~75% reduced compared to wild type PLCη2 providing further support of the importance of this domain for membrane- binding.

3.3.2. Membrane association of PLCη2 is essential for its activity

To assess whether the PH domain-dependent membrane association is essential for the enzymatic activity of PLCη2, monensin-induced activation was studied in COS-7 cells transfected with empty vector, wild type or ΔPH-PLCη2 constructs (Figure

3.9.). PI(4,5)P2 hydrolysis is reduced to the control level (vector) in ΔPH-PLCη2- expressing cells indicative of very little or no enzyme activity.

Figure 3.9.: Measurement of monensin-induced accumulation of [3H]inositol phosphate in COS-7 cells transfected with constructs encoding wild-type and ΔPH- PLCη2. Empty vector transfected cells were used to detect the activity of the endogenous PLC activity. PLCη2 lacking the PH domain possessed no additional activity compared the level of the vector-transfected cells. Protein expression levels are shown on the previous figure. Results are expressed ± S.E.M., n=3. Figure is reproduced with permission from Elsevier (Popovics et al. 2011).

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

In a separate experiment, activation of the wild-type and ΔPH-PLCη2 at various concentrations of free Ca2+ were assessed in COS-7 cells as previously (Figure 3.10.). This revealed that Ca2+ was unable to activate the PH-lacking protein. Collectively these results show that membrane attachment of PLCη2 by the PH domain is crucial for its enzymatic activity.

Figure 3.10.: Measurement of PI(4,5)P2 hydrolysis in wild-type and ΔPH PLCη2- expressing permeabilised COS-7 cells in the presence of various free Ca2+ concentrations. Empty vector transfected cells were used to detect the activity of endogenous PLCs. PLC-activity in ΔPH PLCη2-transfected cells was similar to the control level indicating that this construct is defective in PI(4,5)P2 cleavage. Results are expressed ± S.E.M., n=3. Figure is reproduced with permission from Elsevier (Popovics et al. 2011).

3.3.3. Purification of GST-tagged PH domains of PLCη1 and PLCη2

A further step towards understanding the properties of PLCη PH domains is to evaluate their phospholipid binding selectivity. When the mouse sequences of PLCη1 and PLCη2 PH domains (residues corresponding to 14-137 and 115-238, respectively) are aligned (Figure 3.11.) a high degree of similarity can be observed between these sequences. This suggests that their phospholipid-binding specificities may be similar.

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

Figure 3.11.: ClustalW alignment of protein sequences corresponding to the PH domains of PLCη1 and PLCη2. The high rate of conserved residues suggests that the two PH domains possess similar phospholipid-binding properties. Symbols used to express the rate of similarity: “*” identity, “:” high similarity, “.” weak similarity. Colour coding corresponds to the clustal color scheme.

Figure 3.12.: Polyacrylamide gel demonstrating the purity of GST-PH-PLCη in the elution buffer. Bacterial cell lysates, first wash (from multiple washes) and the 1. and 2. elutions were separated on a 4-12% polyacrylamide gradient. The first elution was used in subsequent experiments. Recombinant PH domains were detected at approximately 40 kDa.

For the subsequent analysis, PLCη PH domains (encoding residues 14-137 and 115- 238 of PLCη1 and PLCη2, respectively) were cloned into the bacterial expression vector, pGEX6 by using EcoRI and NotI restriction sites from the full-length cDNA containing pcDNA3.1. (conducted by Dr. Alan Stewart). This vector has the

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2 advantage of tagging the inserted PH domains with GST (Glutathione S-transferase). Sequence verified plasmids were transformed into E. coli strain BL21. Expression of PH domains was induced by IPTG. Cells were pelleted, lysed and recombinant proteins were purified on glutathione-conjugated agarose beads. Purity of eluates was tested by PAGE followed by Coomassie staining (Figure 3.12.). Protein mass was calculated by an online software (www.bioinformatics.org/sms/prot_mw.html) and was estimated to be: 41.36 kDa for GST-PLCη1-PH, and 41.22 for GST-PLCη2-PH. Expressed proteins were identified according to their weights. Samples from the first elution were at least 90% pure and were subjected for further examination.

3.3.4. Determination of the specificity of PLCη PH domains towards phosphoinositides by a FRET-based assay

The following experiment was conducted by Dr. Alexander Gray (University of Dundee, Dundee, UK) from the materials which were produced in the previous section. Purified GST-PLCη2 PH domains were subjected to a Förster resonance energy transfer (FRET) assay which aimed to measure the dose-dependent binding of the PH domains to various phospholipids. Simplified description of this experimental approach is described in Figure 3.13. FRET signal is generated when a sensor complex is formed by the interaction of the PH domain and the biotinylated lipid.

By using the above described FRET approach, first the ability of both PH domains to form sensor complexes with biotinylated phosphatidylinositol 3,4,5-trisphosphate

(PI(3,4,5)P3), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), phosphatidylinositol

3,4-bisphosphate (PI(3,4)P2), phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 4-phosphate (PI(4)P) was examined (Figure 3.14). PLCη PH domains possessed the highest sensitivity towards PI(3,4,5)P3 with EC50: 0.7611 pmol for PLCη1 and 0.7761 pmol for PLCη2. In addition, PI(4,5)P2 also associated with PH domains with slightly lower affinity (3.0576 pmol for PLCη1 and 3.1131 pmol for PLCη2). None of the other biotinylated-lipids were able to form FRET- active complexes.

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

Figure 3.13.: Principles of detecting PH domain-phospholipid interactions by FRET. In the reaction, GST-PH domains associate with the Eu-tagged anti-GST antibody and with the preferred phospholipid. The allophycocyanin (APC) also binds biotinylated phospholipids through its streptavidin tag. FRET-signal (upon excitation at 340 nm) is generated when the Eu chelate and the APC gets to a close distance which is achieved when PH domain-phospholipid interactions are formed. Figure was taken with permission from Elsevier (Gray et al. 2003).

Figure 3.14.: Measurement of the association of PLCη1 and PLCη2 PH domains with biotinylated phosphoinositides by using TR-FRET detection. Both PLCη1 and PLCη2 PH domains exhibited the highest affinity towards PI(3,4,5)P3 (EC50: 0.7611 pmol for PLCη1 and 0.7761 pmol for PLCη2) but also bound to PI(4,5)P2 at slightly higher biotin lipid concentration (3.0576 pmol for PLCη1 and 3.1131 for PLCη2). TR-FRET was conducted as described in Materials and Methods. Biotinylated lipids used to build sensor complexes were as follows: PI(3,4,5)P3, PI(4,5)P2, PI(3,4)P2, PI(3)P and PI(4)P. EC50 values were calculated by the SigmaPlot software. Data are expressed as mean ± S.D., n=3.

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

As a parallel approach, the ability of non-biotinylated phosphoinositides to dissociate already formed sensor complexes formed by PLCη-PH and biotinylated PI(4,5)P2 was also assessed (Figure 3.15.). Apart from the phosphoinositides used in the previous experiment, inositol phosphates were also included such as I(1,3,4,5)P4

(inositol 1,3,4,5-tetrakisphosphate), I(1,3,4)P3 (inositol 1,3,4-trisphosphate),

I(1,4,5)P3 (inositol 1,4,5-trisphosphate). IC50 values are summarised in Table 3.1.

Almost all lipids tested were able to displace biotinylated PI(4,5)P2 in the complex.

PI(3,4,5)P3 was the strongest competitor with IC50 values 35.70 pmol and 41.60 pmol for PLCη1 and PLCη2, respectively. This was followed by PI(4,5)P2 (EC50 130.60 pmol and 198.40 pmol for PLCη1 and PLCη2, respectively). The list of the rest of the phospholipids arranged by their potency to compete with the PLCη2-

PI(4,5)P2-binding contained some differences for the two PLCη PH domains. For

PLCη1-PH the order was: PI(3,4)P2 (442.10 pmol), 1,3,4,5 IP4 (671.70 pmol), PI(3)P

(1051.80 pmol), 1,4,5 I(1,4,5)P3 (1066.10 pmol). However, when PLCη2-PH was subjected to the assay it was I(1,3,4,5)P4 (301.10 pmol), I(1,4,5)P3 (542.90 pmol),

PI(3,4)P2 (919.80 pmol) and PI(3)P (2256.40 pmol). This might indicate that the PH domain of PLCη2 is more vulnerable to dissociation from its membrane-binding state (achieved by binding PI(3,4,5)P3 or PI(4,5)P2). This process is recognised as a negative feedback of PH domains possessing proteins (Jia et al. 2007; Lomasney et al. 1996). Only PI(1,3,4)P3 was unable to compete with the PLCη-PH-PI(4,5)P2 complexes.

These results collectively indicate the major role of PI(3,4,5)P3-binding as a regulator for the localisation of PLCη-PHs. Although there were slight differences in the potency for competition of inositol phosphates it seems that their role in negatively regulating the association of PLCη-PH complexes with PI(3,4,5)P3 or PI(4,5)P2 is negligible. In the case of the PLCδ1, the enzymatic activity is greatly reduced by the addition of I(1,4,5)P3 to PI(4,5)P2 (main target of this PH domain) in a ratio of 1:10 suggesting an important regulatory mechanism. However, in the previous experiments, it was revealed that there is at least a 10-fold difference in the sensitivities of PH-PLCηs towards PI(3,4,5)P3 and I(1,4,5)P3 or its derivatives. When

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2 the PH domains of PLCηs were aligned with that of PLCδ1 an interesting structural detail was revealed (Figure 3.16.). Conserved residues of the inositol-binding loop, which projects from the β-structure of the PH domains were identified in all three proteins. However, the first two residues of this loop which were shown to be important for the binding of inositol phosphates are excised in PLCηs (Ferguson et al. 1995). This deletion might be the causative of the altered lipid-binding properties of the PH domains in the PLCη enzyme family.

Figure 3.15.: FRET measurement of the dissociation of biotinylated PI(4,5)P2-PLCη PH domain sensor complexes by unbiotinylated phosphoinositides. PH domains were pre-incubated with PI(4,5)P2 (200 pmol/reaction) before the addition of serially diluted lipids: PI(3,4,5)P3, PI(4,5)P2, PI(3,4)P2, PI(3)P, I(1,3,4,5)P4, I(1,3,4) or I(1,4,5)P3. The strongest competitor of the PI(4,5)P2-PLCη PH domain complex was PI(3,4,5)P3 for both PLCηs followed by PI(4,5)P2. Only I(1,3,4)P3 was unable to dissociate the sensor complex.

IC50 (pmol) IC50 (µM) PLCɳ1 PLCɳ2 PLCɳ1 PLCɳ2

PI(3,4,5)P3 35.70 41.60 0.71 0.83

PI(4,5)P2 130.60 198.40 2.61 3.97

PI(3,4)P2 442.10 919.80 8.84 18.40 PI(3)P 1051.80 2256.40 21.04 45.13

1,3,4,5 IP4 671.70 301.10 13.43 6.02

1,3,4 IP3 <10000 <10000 <200 <200 1,4,5 IP3 1066.10 542.90 21.32 10.86

Table 3.1.: Half-maximal inhibitory values (IC50) calculated from the FRET-assay measuring the dissociation of the PI(4,5)P2-PLCη PH domain sensor complexes by unbiotinylated phosphoinositides. Values were calculated by SigmaPlot software and are shown both in pmoles/sample and in µM concentrations.

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

Figure 3.16.: Unique structure of PLCη PH domains suggested by their alignment with PLCδ1. I(1,4,5)P3-binding residues were previously identified from the crystal structure of the PLCδ1-PH-I(1,4,5)P3 complex (residues are indicated by black triangles, Ferguson et al., 1995). The sequence of an elongated loop which contains 4 from the above mentioned I(1,4,5)P3-binding residues is bordered with a black box. Sequence alignment revealed that the first two residues of this loop which bind I(1,4,5)P3 in PLCδ1 are absent in PLCηs. This might represent the structural basis for the inability of I(1,4,5)P3 to effectively compete with the PI(1,4,5)P2-PLCη complex as revealed in the FRET assay. Structure of the PLCδ1 PH domain was taken with permission from Elsevier (Ferguson et al. 1995). Alignment was produced by the author of this thesis.

3.3.5.: Detection of PLCη PH domain-phospholipid interactions by lipid-absorbed membranes

Phospholipid-protein interactions may also be detected by comercially available lipid-absorbed membranes known as PIP-strips. The advantage of this method that it provides a wider array of lipids containing others than just phosphinositols. Membranes containing the following lipids were used: lysophosphatidic acid (LPA), lysophosphocholine (LPC), (PI), PI(3)P, PI(4)P, PI(5)P, PE, PC, sphingosine 1- phosphate (S(1)P), PI(3,4)P2, PI(3,5)P2, PI(4,5)P2, PI(3,4,5)P3, PA and PS. Membranes were incubated with purified GST-PLCη1-PH or GST-PLCη2-PH, a mouse anti-GST antibody and horseradish peroxidase-conjugated anti-mouse antibody. Antibody complexes were visualised by an enhanced chemiluminescent

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2 substrate (ECL, Figure 3.17.). The binding of PH-PLCη1 and PH-PLCη2 to

PI(3,4,5)P3 was confirmed by this method, however, it was unable to detect their association to PI(4,5)P2. Additionally, in contrast to the FRET-analysis binding of both PH domains to PI(3)P and PI(4)P was detected. Strong associations of PH domains to phosphatidylserine were also observed. This phospholipid has been previously shown to localise to the plasma-, endosomal- and lysosomal membranes (Yeung et al. 2008).

Figure 3.17.: Investigation of PLCη PH domain phospholipid selectivity by lipid- protein overlay assay. GST-tagged PLCη PH domains were probed with hydrophobic membranes spotted with 15 phospholipids. These were as follows: LPA: lysophosphatidic acid, LPC: lysophosphocholine, PI: phosphatidylinositol, PI(3)P: phosphatidylinositol 3-phosphate, PI(4)P: phosphatidylinositol 4-phosphate, PI(5)P: phosphatidylinositol 5-phosphate, PE: phosphatidylethanolamine, PC: phosphatidylcholine, S(1)P: sphingosine 1-phosphate, PI(3,4)P2: phosphatidylinositol 3,4-bisphosphate, PI(3,5)P2: phosphatidylinositol 3,5-bisphosphate, PI(4,5)P2: phosphatidylinositol 4,5-bisphosphate, PI(3,4,5)P3: phosphatidylinositol 3,4,5- trisphosphate, PA: phosphatidic acid, PS: phosphatidylserine. Both PH domains showed strong binding to PS. In addition the binding of PI(3,4,5)P3, PI(3)P and PI(5)P was also registered.

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

The quality of these membranes and their ability to show phospholipid-protein interactions were poor in these experiments. Moreover, a repeat of this experiment did not provide any detectable results (data not shown). However, strong binding of PS by the PH domains may be significant and worthwhile following up in future experiments.

3.3.6.: Localisation of PH-PLCη2 in Neuro-2a cells in relation to the distribution of PI(3,4,5)P3 and PI(4,5)P2

Previous experiments investigated the phospholipid-binding properties of PLCη PH domains, however, in an artificially constituted environment. A further step towards understanding the role of the interactions between PLCη2 PH domain and phospholipids is to study their role in the regulation of cellular localisation. Inspired by this, a GFP-tagged construct of the PH domain of PLCη2 in pcDNA3.1 was produced by TA-cloning (performed by Mohammed Arastoo, Ph.D. student). This was cotransfected with the PI(4,5)P2-specific PH domain of PLCδ1 (Watt et al. 2002) tagged with the fluorophore, mCherry. Alternatively, a specific antibody was used to detect the cellular localisation of PI(3,4,5)P3. In this study, Neuro-2a cells were used which are derived from murine neuroblastoma cells (Klebe and Ruddle 1969). GFP- PH-PLCη2 exhibited limited co-localisation with mCherry-PH-PLCδ1 on the plasma membrane and the cytosol (Pearson coefficient: 0.0953, S.E.M.=0.033555, n=2) but the highest extent of the GFP-PH-PLCη2 was restricted to the nucleus similarly to the PI(3,4,5)P3-immunostaining (Pearson coefficient: 0.7475, S.E.M=0.0355, n=2,Figure 3.18.). Cellular distribution of PLCη2 in neuron-like cells will be further assessed in Chapter 4.

100

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

Figure 3.18.: Localisation of GFP-PH-PLCη2 (green) in Neuro-2a cells in relation to PI(4,5)P2 and PI(3,4,5)P3 (both shown in red). Nuclei are stained with DAPI (blue). GFP-PH-PLCη2 was transfected by lipofectamine and cells were fixed the next day. PI(4,5)P2 was labelled with the cotransfection of mCherry-PH-PLCδ1 whereas PI(3,4,5)P3 was immunostained with a specific antibody. GFP-PH-PLCη2 possessed a high co-localisation rate with PI(3,4,5)P3 in the nucleus (0.7475, S.E.M=0.0355, n=2, Pearson coefficient). In contrast, PLCη2 colocalised less markedly with mCherry-PH-PLCδ1 (0.0953, S.E.M.=0.033555, n=2, Pearson coefficient). Images are representative and were taken from a deconvoluted Z-stack. Bars correspond to 10 µm.

3.4. An EF-hand directs the Ca2+-sensitivity of PLCη2 – the description of a novel EF-hand structure

EF-hand-related sequences in other PLCs have been implicated in Ca2+-binding and enzyme activation (Yamamoto et al 1999; Nomikos et al. 2005). Accordingly, these domains are distinguished as EF-hand like domains in other PLCs (described in section 1.2.3.2.3.). On the other hand, PLCζ possesses a unique functional EF-hand which is responsible for its high Ca2+-sensitivity compared to other PLCs (Nomikos et al. 2005). As revealed from the Ca2+-permeabilisation experiment, PLCη2 also exhibits a high sensitivity towards Ca2+ which might indicate that this PLC also carries an active EF-hand domain. To investigate this possibility, PLCη2 sequence was aligned with proteins with known functional paired EF-hand-loops (Figure

101

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

3.19.). Ca2+-binding loops are formed by 12 residues in canonical EF-hands which is true for the first loop, however, insertion of a residue in the second loop resulted in a loop consisting of 13-residues. This insertion is expected to be at either H296 or Q297 (at poitions 5 or 6) which assumption is based on the conservity of the other residues in the loop and is highlighted by the alignment with other EF-hand- containing proteins (Figure 3.19b). In the alignment calbindin D9K is also included which contains a non-canonical first loop containing two insertions (alanine between the 1. and 2. and a proline between 5. and 6.) however this insertion does not prevent the coordination of the Ca2+ (Kordel et al. 1993).

Figure 3.19.: Coordination of Ca2+ in EF-hands (A) and the alignment of these domains from PLCη enzymes and other proteins (B). Ligands in positions Y, -Y, Z, -Z1 and -Z2 are arranged as a pentagon whereas ligands X and -X are placed in an axial position to the others that allows the formation of a pentagonal bipyramid structure. The sequences of murine and human PLCη2 (MmPLCη2 and HsPLCη2) are compared with the human forms of PLCη1 (HsPLCη1), calmodulin EF-hand domain 1 (HsCaMEF1), human calmodulin EF-hand domain 2 (HsCaMEF2), human troponin C type 2 (HsTropCII), human neuronal calcium sensor-1 (HsNCS-1), calcineurin B (HsCalcinB) and human calbindin D9K (HsCalD9K). The putative Ca2+-coordinating residues X, Y, Z, -Y ,-X and -Z are indicated based on the conserved residues. Residues which were mutated in the following experiments are bold. Figure is created by Dr. Alan Stewart.

102

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

To assess the role of the EF-loops in PLCη2, mutations in both loops were generated by site-directed mutagenesis. Aspartic acids in the first position of both loops are highly conserved in EF-hand-containing proteins and usually supply the “X” ligands for coordination of the Ca2+ in a pentagonal bipyramid structure (Figure 3.19.). Thus, these residues were the first targets of the mutagenesis studies and were both changed to alanine (D256A and D292A). Subsequently, three more similar mutations were generated in the second loop changing residues in positions 5, 6 and 13 to alanines (H296A, Q297A and E304A). Either H296 or Q297 has been inserted into the 12-residue loop and led to the constitution of this novel EF-loop. Hence, the mutation of these residues may provide information on how this insertion affects Ca2+-binding properties of the domain. In addition, E304 provides a bidentate ligand in canonical EF-hands and its mutation is expected to greatly impair the Ca2+- dependent conformational changes in the EF-loop. Wild type (wt) and mutated PLCη2 proteins were overexpressessed in COS-7 cells to study the role of the EF- hand in monensin-induced activation and Ca2+-sensitivity in permeabilised cells. Transfection of these constructs were conducted in two sets: one containing the vector, wt, D256A and D292A and one set with vector, wt, H296A, Q297A and E304A. Expression levels of recombinant proteins were detected by Western-blotting from cell lysates (Figure 3.20.).

Figure 3.20.: Western-blot showing that wild-type (wt) and mutants of PLCη2 are expressed in transfected COS-7 cells. Transfections were conducted in two series: one containing vector, wt, D256A and D292A and one containing vector, wt, H296A, Q297A and E304A. Equal amounts of protein lysates were subjected to SDS-PAGE and transferred onto PVDF membranes. Blots were probed with PLCη2- and β-actin-specific antibodies. β-actin serves as a loading control.

103

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

Only small variability was found in the transfection efficiencies. However, all experiments were repeated at least three times to ensure that differences in enzyme activities are not the consequences of altered expression levels.

Monensin treatment stimulated inositol phosphate accumulation by 4-5 fold in both experiments (Figure 3.21 and 3.22). Mutation of the D256A residue blocked the ability of the enzyme to respond to monensin. In contrast, mutation of the same aspartate in the second loop (D292A) only resulted in a 20% decrease in monensin- induced activation compared to the wild type construct. These results clearly demonstrate the importance of the first loop for activation, however, the involvement of the second loop is not certain.

Figure 3.21.: Measurement of monensin-induced accumulation of [3H]-labelled inositol phosphate in COS-7 cells transfected with constructs encoding wild-type or mutant PLCη2 (D256A and D292A). Cells transfected with the vector were used as controls. The D256A mutant exhibited no activity above the control level whereas mutation of the D292A residue only resulted in a slight decrease in PI(4,5)P2 hydrolysis. Results are representative of multiple experiments and expressed as ± S.E.M.

To further evaluate the role of the second EF-loop in Ca2+-dependent activation of the enzyme, other putative Ca2+-binding residues were mutated as stated above. Surprisingly, these mutations did not affect the monensin-stimulated enzyme activity (Figure 3.22.). These results indicate that only the first EF-loop possess regulatory role on the enzyme activity probably via a structural change triggered upon Ca2+- binding.

104

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

Figure 3.22.: Measurement of monensin-induced accumulation of [3H]-labelled inositol phosphate in COS-7 cells transfected with constructs encoding wild-type or mutant PLCη2 (H296A, Q297A and E304A). Cells transfected with the vector were used as controls. None of the mutations of the second EF-loop inhibited phospholipase activity. Results are representative of multiple experiments and expressed as ± S.E.M.

Figure 3.23.: Measurement of the accumulation of [3H]-labelled inositol phosphates in permeabilised COS-7 cells transfected with constructs encoding wild-type or mutant PLCη2 (D256A: A and D292A: B) in the presence of various free Ca2+- concentrations. Cells transfected with the vector were used as controls. Whereas D256A exhibited 10-fold less sensitivity for Ca2+, the D292A mutant mirrored the activation of the wild-type enzyme. Experiments were performed simultaneously. The activities of the mutants are shown with solid lines. The wild-type enzymatic activity and the endogenous level in control cells are also indicated by dashed and dotted lines, respectively. Results are representative of multiple experiments and expressed as ± S.E.M.

Ca2+ sensitivity of the EF-hand mutants were also assessed in permeabilised COS-7 cells in the presence of various free Ca2+ concentrations. Results for each mutant are shown separately by indicating the level of the empty vector- and wild-type- transfected cells. Experiments were conducted in two groups similarly to the

105

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2 monensin-treated cells. Interestingly, the first activation of D256A mutant was only acquired at a ten-fold higher Ca2+-concentration (10 µM) compared to wild-type. In contrast, the D292A mutant exhibited the same activation curve as the wild-type PLCη2 (Figure 3.23.). It is noteworthy that the D292A mutant displayed different activation when monensin or purely Ca2+-was administered. Although the nature of these experiments greatly differ, these results also raise the possibility that monensin activation of PLCη2 is not only directly derived from its effect on Ca2+-release. In FRTL-5 cells, monensin elevated intracellular pH and altered iodide transport (Smallridge et al. 1992). Thus, it is possible that monensin has a complex effect on cellular physiology and these changes collectively lead to the potentiation of PLCη2 activity.

Figure 3.24.: Measurement of the accumulation of [3H]-labelled inositol phosphates in permeabilised COS-7 cells transfected with constructs encoding wild-type or mutant PLCη2 (H296A: A Q297A: B and E304A: C) in the presence of various free Ca2+-concentrations. Cells transfected with the vector were used as controls. All mutants possessed similar Ca2+-sensitivities to the wild-type enzyme, however their activity was slightly decreased at 10 and 100 µM Ca2+-concentrations. Experiments were performed simultaneously. The activities of the mutants are shown with solid lines. The wild-type enzymatic activity and the endogenous level in control cells are also indicated by dashed and dotted lines, respectively. Results are representative of multiple experiments and expressed as ± S.E.M.

Further mutations in the second EF-loop (H296A, Q297A and E304A) did not alter the sensitivity of the enzyme towards Ca2+ but resulted in a decrease in enzyme activity at 10 and 100 µM Ca2+-concentrations (Figure 3.24.). This might indicate that for maximal activation the functionality of the second loop is also required. Although monensin-activation was unaffected by the mutations, it is possible that 70

106

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

μM monensin treatment has similar intracellular effects as when the cells are permeabilised in the presence of 10-6 M Ca2+. This could result in the inability of the monensin-activation to show mutation induced differences in enzyme activity when they are coupled to higher Ca2+ concentrations. Or otherwise, the complexity of monensin actions did not allow the differences to be revealed.

Collectively, these results indicate that Ca2+-binding to the first EF-loop in PLCη2 triggers a conformational change of the protein that translates to the catalytic site leading to increased enzymatic activity. Whether the second loop binds Ca2+ or not, remains uncertain. However, it was revealed that it does not contribute to the activation of PLCη2 by Ca2+ in the range of 100 nM-1 µM, which is the concentration window particular for PLCη enzymes. Hopefully in the future more structural data will be acquired by the purification and crystallisation of the domain in which the Ca2+-binding mechanism could be further investigated. Nevertheless, PLCη2- (and certainly PLCη1) EF-hand represents a novel type of this domain containing a canonical and a non-canonical Ca2+-binding loopfrom which the canonical first loop directs the unique Ca2+ sensitivity of the enzyme. .

3.5.: Ca2+-regulated membrane association of the C2 domain is essential for PLCη2 enzyme activity

All PLC isozymes contain a C2 domain of which only the Ca2+-bound structure of PLCδ1 has been solved to date by X-ray crystallography. Metal binding sites of this domain have been identified by using Ca2+, or its analogues Ba2+ and La3+ in the buffer used for crystallography. This revealed that two or three Ca2+ may associate to the three membrane-facing loops of PLCδ1-doman. Upon binding, the Ca2+ ions may form a bridge between the C2 domain and phospholipid headgroups or alter electrostatic features of the protein surface to enhance its association with the membrane. Coordination of the Ca2+ is mostly achieved by aspartic acid residues (Essen et al. 1997a). In Figure 3.25., murine sequence of C2 domain from PLCη2 is aligned with that of PLCη1, PLCδ1 and synaptotagmin 1 (Syt1). Synaptotagmin

107

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2 contains two C2 domains and also represents a structurally altered subtype of C2 domain formed by circular permutation (Essen et al. 1996). Sequence alignment revealed the existence of 5 conserved aspartates in PLCη2 C2 domain. Residues D861 and D920 were chosen as targets for site directed mutagenesis to study the role of Ca2+-dependent membrane association of the C2 domain for PLCη2 enzyme activity. These residues were replaced by alanines (D861A and D920A). In addition, it was found that the residue corresponding to D861 is an asparagine in PLCδ1. In order to detect if this natural mutation has important functions in PLCη2 enzymatic activity, an additional construct was generated with D861N mutation.

Figure 3.25.: Alignment of murine sequences corresponding to the C2 domains of PLCη2, PLCη1, PLCδ1 and synaptotagmin-I (Syt-1). Synaptotagmin-I contains two C2 domains (C2A and C2B). Five conserved aspartic acid residues which are implicated in Ca2+-dependent membrane binding of this domain are shown in bold. The alignment revealed that the first aspartate is altered to an asparagine in PLCδ1. For further analysis, the first and the third aspartate was mutated to alanine (D861A and D920A) or asparagine (D861N). Figure is reproduced with permission from Elsevier (Popovics et al. 2011).

Constructs encoding wild-type and mutant PLCη2 proteins were transfected into COS-7 cells by electroporation to study the effect of mutations on monensin-induced activation and Ca2+-sensitivity. As shown in Figure 3.26., constructs were successfully transfected and expressed in these cells with similar protein levels.

108

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

Figure 3.26.: Western blot showing equal protein levels of wild-type (WT) and mutant PLCη2 (D861A, D861N and D920A) in whole cell lysates from transfected COS-7 cells. Equal amounts of protein lysates were subjected to SDS-PAGE and transferred onto PVDF membranes. Blots were also probed with an antibody specific to β-actin as a loading control. Figure material is collected from the same membrane by joining two fragments indicated by a vertical line. Figure is reproduced with permission from Elsevier (Popovics et al. 2011).

Figure 3.27.: Monensin-induced accumulation of [3H] inositol phosphate in COS-7 cells transfected with wild-type PLCη2 or D861A, D861N and D920A mutants. Empty vector transfected cells were used to detect the activity of the endogenous PLC-pool. From the mutations generated in the C2 domain of PLCη2 only D920A showed no activity above the level of the control indicative of its defect in PI(4,5)P2- hydrolysing. In addition, the D861N mutation also resulted in the reduction of the enzyme activity. Data shown is representative of multiple experiments. Results are expressed ± S.E.M., n=3. Figure is reproduced with permission from Elsevier (Popovics et al. 2011).

Monensin (0-70 µM) induced the expected elevation in the wild-type enzyme activity and slightly stimulated endogenous PLCs (Figure 3.27.). The D861A

109

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2 mutation only yielded a ~23% decrease (after adjusting for basal PLC activity) compared to the wild-type PLCη2 activity. However, the mutation of the same residue to asparagine (D861N) resulted in a ~55% reduction (after adjusting for basal PLC activity). The D920A mutant activity was similar to that of the control level with all monensin concentrations tested.

Figure 3.28.: Measurement of the accumulation of [3H]-labelled inositol phosphates in permeabilised COS-7 cells transfected with constructs encoding wild-type or mutant PLCη2 (D861A, D861N and D920) in the presence of various free Ca2+- concentrations. Cells transfected with the vector were used as controls. Endogenous PLC-activity is indicated with a solid line on the first diagram and then with dashed lines on the rest. The D861A mutant activity was similar to that of the wild-type. In contrast, the D861N and D920A mutants exhibited much reduced activity compared to the wild-type PLCη2. This reduction affected the whole activation curve and did not change the sensitivity of the enzymes towards Ca2+. Results are representative of multiple experiments and were expressed as ± S.E.M. Figure is reproduced with permission from Elsevier (Popovics et al. 2011).

Ca2+-sensitivities of wild-type and C2 domain mutant PLCη2 enzymatic activities were investigated by permeabilisation of COS-7 cells in the presence of elevating

110

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

Ca2+-concentrations (Figure 3.28.). Wild-type PLCη2 activity exhibited an increase at concentrations between 0.1 and 1 μM free Ca2+ as was expected. The D861A mutant displayed activation curve similar to that of the wild-type. In contrast, the D861N and D920A mutants demonstrated much less activation by Ca2+ (~78% and 85%, respectively at 10 µM Ca2+ after adjusting for basal PLC activity). None of the mutants were differentially sensitive towards Ca2+ compared with the wild-type enzyme. This suggests, that the association of the C2 domain with the membrane (in 2+ the presence of Ca ) is a crucial step for the effective hydrolysis of PI(4,5)P2, however, it is not responsible for the activation of the enzyme by the submicromolar Ca2+ concentrations.

Figure 3.29.: Mutations in the C2 domain did not alter the membrane-binding properties of PLCη2 in COS-7 cells. Vectors encoding GFP-tagged wild-type (WT) and mutant PLCη2 (D861A, D861N and D920A) were transfected into COS-7 cells by electroporation and cells were fixed after 2 days of expression. All constructs localised to intra- and extracellular membranes. Image of the wild-type GFP-PLCη2 is taken from Figure 3.4. Bars correspond to 10 µm. Images were taken by a Leica confocal system (TCP SP2).

111

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

The subcellular membrane targeting of some PLCδ isoforms are regulated by the selectivity of their C2 domain to phosphatidylserine (Ananthanarayanan et al. 2002). To evaluate if the C2 domain of PLCη2 affects the cellular localisation of the full- length protein the previously introduced mutations were also generated in the GFP- PLCη2 construct, respectively. GFP-D861A, -D861N and –D920A were expressed in COS-7 cells as previously the wild-type GFP-PLCη2 (Figure 3.29.). All GFP- constructs exhibited similar cellular localisation to the wild-type construct with punctuate distribution inside the cell and limited appearance on the plasma membrane. This suggests that the C2 domain has little or no participation in the membrane-targeting of the full-length PLCη2 which mechanism is probably under the absolute control of the PH domain.

Figure 3.30.: Western blot showing the protein levels of wild-type and mutant PLCη2 (D861A, D861N and D920A) in membrane-enriched fractions of transfected COS-7 cells. Cells were scraped and homogenised in RIPA buffer (lacking IGEPAL and SDS) and the homogenate was centrifugated at 14,000 g for 10 min. The pellet was washed and resuspended in complete RIPA buffer. Equal amounts of protein lysates were subjected to SDS-PAGE and transferred onto PVDF membranes. Blots were also probed with an antibody specific to the α1 subunit of Na+/K+-ATPase (a marker for the plasma membrane) as a loading control. Figure material is collected from the same membrane by joining two fragments indicated by a vertical line. The blot is representative of three independent experiments.

The C2 domain directs the secondary attachment of the whole protein to the membrane which might increase the affinity of this association. This theory was investigated by the assessment of wild-type and mutant PLCη2 protein levels in membrane-enriched fractions from wild-type and mutant PLCη2-transfected COS-7 cells (WT, D861A, D861N and D920A, Figure 3.30.). Protein levels, however, were

112

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2 similar, indicative of the high affinity of the PH domain to the membrane rather than the C2 domain in this process.

3.6.: Discussion

3.6.1. The initial characterisation

The data presented within this Chapter provides information as to how the PH domain, C2 domain and EF-hand regulate the enzyme activity of PLCη2. The first step in the initial characterisation was the identification of a Ca2+-mobilising agent as a specific trigger for PLCη2 activity in a cellular environment (COS-7 cells). Unexpectedly, only monensin induced a significant elevation in PLCη2 activity of all Ca2+-mobilising agents tested (ionomycin, A23187, thapsigargin and bafilomycin). Kim et al. also demonstrated that ionomycin stimulation of the endogenous PLC- activity was retained after PLCη2 knock-down in Neuro-2a cells (Kim et al. 2011). Ca2+ ionophores ionomycin and A23187 might have been incapable of inducing the special spatial and/or temporal distribution of Ca2+ which was developed by monensin. It was demonstrated earlier that monensin is able to increase intracellular Ca2+ level by 2.3-fold in FRTL-5 thyroid cells by reversing the NCXs and this was coupled to increased PLC activity. This seems to confirm that the effect of monensin on PLCη2 is due to the elevated Ca2+ levels. Although, in this case the Ca2+- ionophores (ionomycin and A23187) should have had at least some effect on PLCη2 activity. To better understand the nature of Ca2+ dynamics following monensin stimulation Ca2+-chelation and non-specific inhibition of Ca2+ channels were used. In the presence of EGTA, PLCη2 activity was reduced to the control level. The compound SKF-96365, which is a non-specific inhibitor of the voltage- and receptor- operated Ca2+-entry (Merritt et al. 1990; Singh et al. 2010) also greatly reduced PLCη2 activity in transfected cells. These agents further demonstrated that the action of monensin on PLCη2 is clearly Ca2+-dependent. Depending on the localisation of PLCη2, its activation might be controlled by Ca2+release from a specific store. A specific inhibitor of the mitochondrial Na+/Ca2+-exchanger (CPG-3757) greatly

113

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2 interfered with the monensin-induced activity of PLCη2. This suggests that the monensin-induced Ca2+ release from mitochondria – either directly or indirectly- influenced PLCη2 activity. At least some of the inhibition of SKF-96365 on monensin-induced Ca2+-release was likely derived from its action on the Na+/Ca2+- exchanger. This was shown by the co-administration of SKF-96365 and CPG-3757 which resulted in only a slight additional inhibition. Collectively these results show that monensin is an activator of PLCη2 and in this activation mitochondrial Ca2+ release is likely to play an important role. However, it is plausible that monensin has no direct effect and other factors might transmit its effect on PLCη2 activity via an unknown mechanism.

The cellular distribution of the GFP-tagged PLCη2 protein highlighted that this enzyme may be present on both plasma- and organelle membranes. However this construct did not provide sufficient evidence that PLCη2 would primarily localise on mitochondrial membranes as was suggested by previous results. Interestingly, localisation of the same construct was found to be predominant on the plasma membrane in HeLa S3 cells (Nakahara et al. 2005). Immunostaining of the overexpressed PLCη2 yielded in a somewhat contradictory result placing the majority of PLCη2 onto the mitochondria. Moreover, mitochondrial labelling highlighted a morphological change in the intracellular structure of transfected COS- 7 cells. One possibility is that PLCη2 may act as a link between mitochondrial membranes directed by the homodimerisation of the molecule. PLCβ was previously found to compose dimers via its C-terminal extension (Singer et al. 2002). If PLCη2 is also able to form such structures, the dimer-partners may attack different membranes with their PH domains and the overexpression of PLCη2 may lead to the development of extended PLC-membrane complexes. In PLCη2-transfected COS-7 cells a considerably higher level of mitochondrial-staining was observed compared to untransfected cells. This suggests that the rate of mitochondrial fissure was elevated in transfected cells that might be caused by the high levels of PLCη2. Similar structural changes are detected when hFis1, a protein involved in mitochondrial

114

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2 fissure is overexpressed in HeLa cells (Frieden et al. 2004). The greater number of mitochondria and the high localisation of the overexpressed untagged PLCη2 on mitochondria-related membranes might partially explain why mitochondrial Ca2+ release possessed such a great effect on PLCη2 activity. ηηIn addition, the future assessment of the effect of PLCη2 on mitochondrial fissure might be of great importance and should be followed up in the future. Elevated mitochondrial fissure may be a hallmark of degenerative diseases such as Alzheimer`s Disease (Wang et al. 2009) so it is plausible that imbalanced PLCη2 activity also contribute to the development of age-related cell abnormalities.Nakahara et al. identified the first time that PLCη2 possesses a unique sensitivity towards Ca2+ in vitro. According to this study, PLCη2 is activated by a 10-fold lower Ca2+ concentration (1 µM) than PLCδ1. Unfortunately, results of this assay were not convincing due to the low level of activation and the high variance in values (Nakahara et al. 2005). My aim was to study Ca2+-sensitivity of PLCη2 in a cellular model providing the physiological environment necessary for the activation of the enzyme. Permeabilised COS-7 cells offered the desired tool whereby the Ca2+-sensitivity of wild-type and mutant PLCη2 proteins were quantitatively determined. PLCη2 enzyme activity displayed the greatest increase between 100 nM and 1 µM [Ca2+]. This renders the enzyme 10-fold less sensitive to Ca2+ than the sperm-specific PLCζ, although 10-fold more than PLCδ1 (Kouchi et al. 2005; Nakahara et al. 2005).

3.6.2. Targeting mechanism of the PH domain

PLC enzymes are typically distributed in the cytoplasm and are recruited to the membranes upon agonist-induced stimulation (Suh et al. 2008). In PLCδ1, the Ca2+- dependent binding of the PH domain to PI(4,5)P2 is directed via its EFL-domain by an unknown mechanism (Yamamoto et al. 1999). In contrast, PLCη2 does not require extracellular stimuli for membrane-association indicated by the punctuate staining of GFP- PLCη2 in unstimulated COS-7 cells. Considering this and the high Ca2+-sensitivity of the enzyme, PLCη2 has been proposed as the “primarily reacting PLC” (Popovics and Stewart 2012b). This is supported by the finding of Kim et al.

115

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2 showing that the closely related isoform, PLCη1, is an amplifier of the external stimuli which lowers the threshold for PLCβ1 activation (Kim et al. 2011).

The unique membrane-binding dynamics of PLCη2 is likely to depend merely on its specific PH domain. In the absence of this domain the protein lost the ability of membrane-binding. Similar results were obtained by Nakahara et al. in HeLa S3 cells with a few differences. In their experiments the full-length GFP-PLCη2 distribution was concentrated to the plasma membrane with a small extent remaining cytosolic (Nakahara et al. 2005). Moreover, without the PH domain PLCη2 loses its ability to associate with the membrane. Using a FRET-based method the lipid-specificities of both PLCη1 and PLCη2 PH domains were assessed. Both domains exhibited a high selectivity towards PI(3,4,5)P3, followed by the PLC enzyme substrate PI(4,5)P2 with approximately 4-5-fold lower EC50 values. The PLCγ1 PH domain also possesses specific affinity for PI(3,4,5)P3 but almost no binding to PI(4,5)P2. This allows the protein to be activated by the PI3K-pathway which is known to elevate PI(3,4,5)P3 levels (Falasca et al. 1998). PLCδ1-PH is highly specific to PI(4,5)P2 and this facilitated its usage as a marker for this phospholipid (Watt et al. 2002). The PLCη PH domain may participate in membrane-binding even in the absence of its preferred lipid, PI(3,4,5)P3 by associating to PI(4,5)P2. The association of PLCδ1-PH domain to membranes is regulated by the cleavage product of PI(4,5)P2 hydrolysis,

I(1,4,5)P3. It is also bound to the PLCδ1-PH domain although as I(1,4,5)P3 is released to the cytosol it liberates the associated PLCδ from the membrane by this mechanism. This represents a negative feedback mechanism on PLCδ. The PLCη PH domains showed approximately 10-fold lower IC50 toward I(1,4,5)P3 than for

PI(3,4,5)P3. For the dissociation of PLCδ1 from the membrane by the physiologically released I(1,4,5)P3, it was determined that its PH domain is 20-fold more sensitive to

I(1,4,5)P3 than PI(4,5)P2 levels (Hirose et al. 1999). In the light of these findings, it is improbable, that the PLCη PH domain-PI(3,4,5)P3 association is under the control of

I(1,4,5)P3.

116

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

The study of PH domain lipid-selectivity by the PIP-strip method provided more insight into this process. One interesting outcome was the high level of binding of the PLCη-PH domains to phosphatidylserine. This is not exceptional among PH domain containing proteins; in 3-phosphoinositide dependent kinase-1 (PDK1) a specific PS- binding groove was identified which is different from its phosphoinositide-binding site. Two mutations of this groove (R466A/K467A) prevented agonist-induced translocation of PDK1 to the membrane (Lucas and Cho 2011). PH domain of evectin-2 has a high specificity towards PS which directs the localisation of the protein to the ER (Uchida et al. 2011). In PLCδ1, PS-content of the cell membrane inhibits the association of its PH domain which is driven by electrostatic interactions (Uekama et al. 2007). PS-binding of PLCη-PH domains might be unique in the PLC family, although binding-properties should be assessed in the future by a quantitative method such as the FRET-assay used in this Chapter.

3.6.3. Identification of a novel EF-hand in PLCη2

An EF-hand-domain with two Ca2+-binding loops was identified in PLCη proteins based on the alignment of its sequence with other proteins with similar domains. This revealed that PLCη EF-loops are asymmetrical; whereas EF-loop 1 is similar to canonical EF-hand conserved amino acid composition possessing 12-residue, the second loop contains a one-residue insertion. This was mapped between position 4 and 5 (H296) or 5 and 6 (Q297). The distorted EF-loop 2 might still be able to co- ordinate Ca2+.For example, the extracellular Ca2+-binding protein, BM- 40/osteonectin contains an amino acid insertion in its first EF-loop and it is still able to develop the pentagonal bipyramid Ca2+-binding structure (Hohenester et al. 1996).

To assess the participation of each loop in Ca2+-activation, mutations were generated, changing key residues to alanine in both loops. The aspartic acid in position 1 of the loops provides a carboxylate group as the first ligand for coordination of the Ca2+ in canonical EF-hands (Gifford et al. 2007). The corresponding residue in each of the EF-loops was mutated (D256A and D292A) and the activities of each were

117

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2 examined. The D256A mutant transfected cells were unaffected by monensin treatment and exhibited a 10-fold decrease in its threshold for activation by Ca2+. In contrast, the D292A mutation did not induce any visible change in Ca2+ sensitivity and only slightly decreased the stimulatory action of monensin. Further mutations were generated in the second loop in order to reveal if Ca2+-coordination by other residues may affect the catalytic activity. These were the hypothesised insertions (H296A and Q297A) and the glutamic acid in the 12 position, which provides a bidentate ligand in the canonical EF-hand structure (E304A) (Gifford et al. 2007). None of these mutations affected monensin-induced activity and Ca2+-sensitivity of PLCη2. Although, it is worth mentioning that all three mutants exhibited reduced activity at 10 µM and 100 µM Ca2+ concentrations. Collectively, these results suggest that the allosteric changes upon Ca2+-binding of the first EF-loop is alone responsible for the activation of the catalytic domain whereas the second loop might only possess a little regulatory role on this process. Activity of the wild-type PLCη2 declines at high Ca2+-levels (100 nM and 1 mM). This suggests the existence of a negative feedback by high [Ca2+] which might be directed by the second EF-loop. Nevertheless, further studies are required to ascertain exactly how the PLCη2 EF- hand domain translates Ca2+ binding into the regulation of enzymatic activity.

3.6.4.: C2 domain supports the secondary membrane-docking of PLCη2

Putative Ca2+-binding residues of PLCη2 C2 domain were identified following alignment of its sequence with C2 domain sequences from PLCη1, PLCδ1 and synaptotagmin. In these proteins, similarly to other Ca2+-binding domains, ligands for Ca2+-coordination are predominantly provided by aspartic acids of the C2 domain (Sutton et al. 1995; Ananthanarayanan et al. 2002). Accordingly, conserved residues D861 and D920 in PLCη2 were selected for mutation. At position D861 in PLCδ1 (N647), Ca2+-coordination is directed by an asparagine. This residue is responsible for the selectivity of the C2 domain towards phosphatidylserine and regulates the intracellular localisation of the domain when it is expressed without other domains as

118

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2 a GFP-tagged protein (Ananthanarayanan et al. 2002). To assess the role of this variation, the D861 residue was mutated to both alanine and asparagine in PLCη2, respectively.

Measurement of PLC-activity in transfected cells following monensin stimulation revealed that the lack of Ca2+-coordination by the D861 residue has little effect on this process (23% reduction). However, the D861N mutation decreased monensin- induced activation to a much greater extent (55% reduction). As described before, this latter residue directs lipid sensitivity in PLCδ1 and therefore it might occur that the substitution of the D861 residue with an asparagine in PLCη2 mimicked the lipid-binding specificity of the C2 domain in a way that was deleterious for enzymatic cleavage (Ananthanarayanan et al. 2002). Nevertheless, inability of the D861A mutation to induce great changes in enzyme activity suggests that lack of Ca2+-coordination by the first conserved aspartate of PLCη2 C2-domain might be compensated by the other Ca2+-binding residues in the domain. Conversely, mutation of the D920 residue greatly reduced the enzyme activity in both assays implicating its specific role in providing ligand to Ca2+. Similar results were obtained by the permeabilisation of transfected COS-7 cells showing that D861A and D920A mutants were comparatively unresponsive to Ca2+. In addition, there was no change in Ca2+-sensitivity in any of the C2 domain mutants providing more evidence on the unique involvement of the EF-hand in this process. In conclusion, the D920A mutant revealed that the secondary membrane association of the C2 domain is essential for regular enzymatic activity of PLCη2.

119

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

effect of effect of Ca2+- PLCη2 domains mutations monensin (70 μM) permeabilisation

PH-domain ΔPH deleterious deleterious 10-fold decrease D256A deleterious in Ca2+- sensitivity no difference D292A ~20 % decrease from WT EF-hand no difference ~20 % decrease D296A from WT at 10 μM no difference ~20 % decrease Q297A from WT at 10 μM no difference ~30 % decrease E304A from WT at 10 μM no difference D861A ~20 % decrease from WT ~80 % decrease C2-domain D861N ~60 % decrease at 10 μM ~90 % decrease D920A deleterious at 10 μM

Table 3.2.: Summary of mutations generated in PLCη2 and their effects on enzyme activity.

3.6.5.: Conclusion

The main conclusions of this Chapter are presented in Figure 3.30. Enzymatic activity of the catalytic site is assisted by a two-point membrane-association of PLCη2. Firstly, PH domain selects the membrane subtype based on its lipid composition and secondly, the C2 domain also associates with the membrane possibly via a Ca2+-bridge. This highlights that the intracellular localisation of PLCη2 is primarily controlled by the PH domain. As determined in this Chapter, it depends on the presence of two molecules, PI(3,4,5)P3 and PI(4,5)P2 in membranes, respectively. In addition, the activation of the membrane-docked protein by submicromolar Ca2+concentration is directed by the EF-hand. This allows the enzyme to react to small changes in Ca2+ levels and amplify these signals by

120

CHAPTER 3. BIOCHEMICAL CHARACTERISATION OF PLCη2

2+ releasing more Ca from I(1,4,5)P3-sensitive stores. These findings describe the basic regulation of PLCη2. The following Chapter investigates the PLCη2 from a new, physiological aspect to determine its role in differentiating neurons.

Figure 3.30.: Illustration of the role of PLCη2 domains in supporting enzymatic cleavage. PH domain directs the primary association of PLCη2 to membranes. The 2+ EF-hand directs the activation of PI(4,5)P2 hydrolysis upon Ca -binding. A secondary, Ca2+-associated binding of the membrane is also crucial for normal enzymatic function which is regulated by the C2 domain. Stimulation of the activity leads to the generation of diacilglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). Figure is reproduced with permission from Elsevier (Popovics et al. 2011).

121

122

Chapter 4.

The role of PLCη2 in neuronal physiology

123

124

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

4.1. Introduction

Although some of the biochemical properties of PLCη2 have been characterised, its functional role and the answer to the question “why do we need another PLC?” is yet to be revealed. Hypothetical functions have been proposed by us and others largely based on high expression of PLCη2 in the brain and in neuroendocrine cells (Cockcroft 2006; Stewart et al. 2007; Popovics and Stewart 2012b). PLCη2 is expressed throughout the cerebral cortex but its highest levels are found in the hippocampus, habenula, olfactory bulb and cerebellum (Nakahara et al. 2005; Kanemaru et al. 2010). Its presence seems to be restricted to neurons suggested by its lack from astrocyte-enriched primary cultures (Nakahara et al. 2005). In the first part of this Chapter, the tissue specific expression of PLCη2 is examined through the use of in situ hybridisation data available within the Allen Brain Atlas (Lein et al. 2007, http://mouse.brain-map.org). Depending on the cellular localisation of the enzyme, it may be involved in various functions of the neuron. Unfortunately, until now the cellular localisation of PLCη2 had only been assessed in non-neuronal cells (COS-7 and HEK 293 cells) (Nakahara et al. 2005; Popovics et al. 2011). Therefore, one aim of this Chapter is to assess its distribution in neuronal cells.

Interestingly, PLCη2 levels gradually increase after birth (Nakahara et al. 2005). Changes in its level during intrauterine development have not previously been assessed. Data stored within the Allen Brain Database show a tendency for PLCη2 to elevate during both intrauterine and postnatal development (http://developinghumanbrain.org) indicating that it might possess an important role for neuronal differentiation or maturation. Here, the role of PLCη2 in neuronal differentiation is examined. For this study, a well-defined mouse neuroblastoma cell line (Neuro-2a) was selected as a model of neuronal differentiation that was isolated from a spontaneous tumour of murine neural crest cells (Klebe and Ruddle 1969).

125

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

4.2. Results

4.2.1. The distribution of PLCη2 in the mouse brain

The expression of PLCη2 in different areas of the mouse brain had been characterised earlier by in situ hybridisation, LacZ reporter knock-in or by western blot (Nakahara et al. 2005; Kanemaru et al. 2010). Additional information on PLCη2 expression is stored within the Allen Brain Atlas which was created by a non-profit medical research organisation known as the Allen Institute for Brain Science (AIBS). Their first large scale project was to map the expression of approx. 20,000 genes in the mouse brain by in situ hybridisation (Lein et al. 2007). The expression of these genes were detected, quantified and the data was collected into a database which is suitable for multi-level comparisons of gene expression patterns. To facilitate the correct localisation of gene expressions, a comprehensive atlas of the mouse brain was also produced. All data was collected from 8-week-old adult C57BL/6J male mice. In this Chapter, the database was used to reveal PLCη2 mRNA expression patterns of different brain regions and to compare it to the existing literature. The anatomical terminology used is identical to the atlas provided by the AIBS. After the digitalisation of gene expression levels, a so-called heat map was produced for all in situ hybridised brain slice. On these maps the level of gene expression is shown by different colours as follows: blue: low, green: medium, red: high expression level.

PLCη2 is expressed throughout the cerebral cortex and its highest levels are found in the olfactory bulb, hippocampus and cerebellum (Figure 4.1.). This is consistent with previous findings (Nakahara et al. 2005; Kanemaru et al. 2010).

126

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

Figure 4.1: The expression of PLCη2 mRNA in the mouse brain detected by in situ hybridisation. A: heat map representing the expression level of PLCη2 (blue: low, green: medium, red: high expression level), B: colorimetric detection of In situ hybridisation by digoxigenin labelled PLCη2 RNA probe;; C: Reference atlas of the mouse brain. Data was collected from 8 week old adult C57BL/6J male mice. Main anatomical structures: MOB: main olfactory bulb, AOB: accessory olfactory bulb, OT: olfactory tubercule, ACB: nucleus accumbens, CP: caudoputamen, CTX: cerebral cortex, TH: thalamus, HY: hypothalamus, HPF: hippocampal formation, MB: midbrain (SC: superior colliculus, IC: inferior colliculus, MRN: midbrain reticular nucleus, VTA: ventral tegmental area), CBX: cerebellar cortex, P: pons and MY: medulla. Pictures were taken with permission from AIBS (Lein et al. 2007).

Magnification of a cerebellar folium (Figure 4.2.) revealed the cell layer-specific localisation of PLCη2. PLCη2 expression is absent in the Purkinje- but pronounced in the granular layer. The most frequent cell type of this latter layer is the granule cell that is also present in the olfactory bulb and dentate gyrus of the hippocampus. In the cerebellum, granule cells receive input from the mossy fibers and their axon is terminated in the molecular layer to form synapses with the Purkinje cell arborisation (Nieuwenhuys 2007). PLC activity is particularly important in these cells as it

127

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY transforms the ligand-dependent activation of muscarinic, metabotropic glutamatergic, histaminergic and α-adrenergic receptors into downstream cellular responses (del Rio et al. 1999).

Figure 4.2: PLCη2 is highly abundant in the cerebellum. A small section of a cerebellar folium is shown with the indication of the different layers: molecular layer (mol. lay.), Purkinje layer (pur. lay.) and granular layer (gran. lay.). PLCη2-positive cells are predominantly found in the granular layer. A similar tissue section with Nissl staining (staining of the cell nuclei) is used as reference. In situ hybridisation was performed using a digoxigenin-labelled PLCη2 RNA probe. Data was collected from 8 week old adult C57BL/6J male mice. Pictures were taken with permission from AIBS (Lein et al. 2007).

The hippocampal formation is composed of two main structural entity; the Ammon`s horn (CA1-CA3 regions) and the dentate gyrus with the main cell types being pyramidal and granule cells, respectively. Whereas cells in the CA1-CA3 contain high amounts of PLCη2 mRNA, only a couple were PLCη2 positive in the dentate gyrus (Figure 4.3a). In the study by Nakahara et al., the presence of PLCη2 mRNA in the dentate gyrus granule cells was also detected by in situ hybridisation. Unfortunately, this study did not specify the age of the mice or the mouse lineage (Nakahara et al. 2005). In a separate report, Kanemaru et al showed only a weak expression of the gene in the CA1-CA3 region in PLCη2/ LacZ reporter transgenic

128

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY mouse and the in situ hybridisation conducted by the same study showed no staining in the dentate gyrus (Kanemaru et al. 2010). Variations in gene expression levels are likely to be the result of different ages of the animals. It is also possible considering that the hippocampal formation is highly involved in learning and memory formation, the difference observed might be related to the environmental enrichment of the different animals. The subgranular zone of the dentate gyrus is one of the major origins of neurogenesis in the adult brain (Eriksson et al. 1998). Here, granule cells are produced throughout life by a long maturation process (Ehninger and Kempermann 2008). The number of new born neurons is associated with the quality of the environment. For instance, exercise has a stimulatory action whereas stress possesses an inhibitory effect on adult neurogenesis (van Praag et al. 1999; Karten et al. 2005). PLCη2 expression in the dentate gyrus is restricted to small numbers of cells suggesting that it might encompass a specific temporal expression pattern during the maturation of granule cells.

Increased expression of PLCη2 was found in the medial habenula (MH), pineal gland (PIN,) and the suprachiasmatic nucleus (SCH, part of the hypothalamus, Figure 4.3). These structures are functionally interlinked by their involvement in the generation and organisation of circadian information. The major intrinsic rhythm generator is the SCH, which receives inputs from melanopsin containing cells of the retina to acquire information of the current phase of the day and night cycle (Hattar et al. 2006). The habenula and the SCH innervate the PIN and consequently regulate its melatonin secretion (Teclemariam-Mesbah et al. 1999). Interestingly, not only the intracranial structures of the circadian system but also the retina contain elevated PLCη2 levels (Zhou et al. 2005; Kanemaru et al. 2010). Suggested by these findings, PLCη2 might have a specific role in the physiology of the regions collectively known as the circadian system.

129

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

Figure 4.3.: High expression levels of PLCη2 were found in the hippocampus, medial habenula (A), suprachiasmatic nucleus (B) and pineal gland (C). Reference atlases were included for pictures A and B. Abbreviations of the anatomic structures are: Hippocampal formation (HPF): Ammon`s horn (CA1, CA2 and CA3) and dentate gyrus (DG). Epithalamus: medial habenula (MH), lateral habenula (LH) and pineal gland (PIN), the suprachiasmatic nucleus (SCH) as part of the hypothalamus (HY), found above the optic chiasm (och). PLCη2 only appears in the polymorph layer (po) of the dentate gyrus and it is almost absent in the granule cell layer (sg). In addition, PLCη2 is highly abundant in the CA1-CA3 regions, MH, SCH and PIN. In situ hybridisation was performed using a digoxigenin labelled PLCη2 RNA probe. Data was collected from 8 week old adult C57BL/6J male mice. Pictures were taken with permission from AIBS (Lein et al. 2007).

High mRNA levels of PLCη2 were also detected in the main olfactory bulb (MOB), pyriform cortex (PIR) and in the olfactory tubercule (OT). These regions are part of the system which processes olfactory information (Figure 4.4). The granule layer of the MOB and the cell-dense groups of the OT (isles of Calleja) are mainly composed of granule cells (Millhouse 1987). Hence, the presence of PLCη2 in this cell type was again shown but in regions associated with different functions. In addition, PLCη2 might specifically be involved in the procession of olfactory information.

130

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

Figure 4.4.: Increased PLCη2 mRNA levels are found in the main olfactory bulb (MOB), pyriform cortex (PIR) and the olfactory tubercule (OT). Layers of the main olfactory bulb are magnified and labelled as: mitral layer, inner plexiform layer and granule layer. The in situ hybridisation was performed using a digoxigenin labelled PLCη2 RNA probe. Data was collected from 8 week old adult C57BL/6J male mice. Main anatomical structures are: MOB: main olfactory bulb, AOB: accessory olfactory bulb, ACB: nucleus accumbens, OT: olfactory tubercule, PIR: piriform cortex, SI: substantia innominata, isl: isles of Calleja, lot: lateral olfactory tract. Pictures were taken with permission from AIBS (Lein et al. 2007).

4.2.2. Different splice variants of PLCη2 are expressed in Neuro-2a cells and in mouse brain

Splicing of the exons in PLCη2 C-terminal domain provides the mechanism for the generation of 5 different splice variants with large variations in their mass (from

131

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

119.2 kDa to 181.4 kDa of the mouse protein, Zhou et al. 2005). The terminology of the variants indicates their exon composition (Figure 4.5). The splice variant specific sequences are selected from 3 exons: 21, 22 and 23, however, exon 21 is excised at 3 different sites on its C-terminal end serving as the origin for more variation between spliceforms. The expression of the spliceforms is tissue-specific; among the five different splice variants of PLCη2, two (21b/22/23 and 21c/22/23) are expressed in the brain and 4 in the retina (Zhou et al. 2005).

Figure 4.5: Exon composition of the C-terminus in different splice variants of PLCη2. Coloured blocks represent exons: 21a, 21b, 21c, 22 and 23. Stop codons are indicated by arrows. Black lines symbolise protein segments encoded by maturated mRNA of the different spliceforms. Figure was produced by the author of this thesis.

Both PLCη1 and PLCη2 have been shown to be expressed in Neuro-2a cells although only with methods which did not discriminate between splice variants (Kim et al. 2011). Zhou et al employed splice variant-specific primers in an RT-PCR to detect differences between PLCη2 variant-composition of mouse tissues (Zhou et al. 2005). Unfortunately, the authors did not specify the PCR conditions for this experiment and when it was repeated in our lab the primers were not able to amplify PCR products in any of the conditions tested (data not shown). Therefore, new primers were designed (Table 4.1 contains the sizes of PCR products and the primer sequences are found in Chapter 2, Table 2.5.).

132

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

size of PCR products (bp) primer composition 21a/23 21a/22/23 21b/23 21b/22/23 21c/22/23

21aFW/23RW 214 241 718 740 1567 21bFW/23RW 287 not separated 314 not separated 1136 21aFW/22RW 66-not detectable 570 1392 21bFW/22RW 139 961 21bFW/21cRW 163 Table 4.1.: Detection of PLCη2 spliceforms by using specific primers. Various combinations of forward and reverse primers determine the amplification of splice- variant specific PCR products (see Materials and Methods for primer sequences). The Table reports the expected sizes (bp) of all potential PCR products. Two products of the 21bFW/23RW primer combination were undiscriminated due to the small differences in their sizes (287 bp and 314 bp). The smallest product of the 21aFW/22RW primer combination (66 bp) was not detectable.

Figure 4.6.: Detection of PLCη2 splice variants in Neuro-2a cells (A) and in mouse brain (B) by RT-PCR. Lanes: 1: 21aFW/23RW, 2: 21bFW/23RW, 3: 21aFW/22RW, 4: 21bFW/22RW, 5: 21bFW/21cRW, 6: PLCη2 (not spliceform specific), 7: RPLP0 (loading marker). Sizes of PCR products are found in Table 4.1. (A non-specific band is found in Lane 4 close to the 600 bp marker. In Lane 5 of Figure A, only non- specific products were amplified probably due to the lack of the 21c/22/23 variant in Neuro-2a cells). Summary of results is found in Table 4.2.

With these primers, cDNAs from both Neuro-2a cells and whole mouse brain were tested for splice-variant composition (Figure 4.6.). In Neuro-2a cell cDNA, the presences of 21a/23 and 21a/22/23 variants were confirmed. The 21b/22/23- and 21c/22/23- specific primers of the same sample only resulted in a product once (out of 4 and 3, respectively) therefore it was treated as false positive. In contrast, mainly

133

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY the 21b/22/23 and 21c/22/23 variants were detected in brain as previously found by Zhou et al (Zhou et al. 2005).

detected bands primer composition 21a/23 21a/22/23 21b/23 21b/22/23 21c/22/23

21aFW/23RW N2A N2A brain 21bFW/23RW brain? brain? N2A 21aFW/22RW brain 21bFW/22RW N2A,brain brain 21bFW/21cRW brain Table 4.2.: Splice variant-specific PCR products detected in Neuro-2a cells and in mouse brain. Mainly the 21a/23 and 21a/22/23 spliceforms were detected in Neuro- 2a cells (abbreviated as N2A), whereas in brain the 21b/22/23 and 21c/22/23 forms were predominant.

RT-PCR results were supported by employing a western blot on Neuro-2a cell- lysates with a PLCη2 specific antibody (Figure 4.7.). It is apparent from the blot, that the 21a/22/23 spliceform is the predominant variant as detected at its expected protein mass (119.2 kDa). An additional band, which possibly corresponds to the 21a/23 variant (164.3 kDa), was also detected. This latter finding might be important as it is one of the two splice variants which contain the PDZ-binding motif at the C- terminal end. However, as the 21a/22/23 spliceform is the dominant in Neuro-2a cells, the expression of this variant will be assessed in further experiments and will be referred to as PLCη2.

Figure 4.7.: Immunoblot of Neuro-2a cell lysates demonstrating the expression of the 21a/23 and 21a/22/23 splice variants of PLCη2. Bands corresponding to the 21a/23 and the 21a/22/23 splice variant have been detected at their expected sizes (164.3 kDa and 119.2 kDa, respectively). Whole cell lysate from Neuro-2a cells was separated on a 4-12% SDS-polyacrylamide gel.

134

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

4.2.3.: PLCη2 level gradually elevates during retinoic acid induced differentiation of Neuro-2a cells

Neuro-2a cells were previously described as neuronal stem cells being highly susceptible to differentiation initiating molecules. Zeng and Zhou et al. employed the combination of retinoic acid treatment (20 µM), reduced serum (2% FBS) and low cell plating density (100cells/mm2) (Zeng and Zhou 2008). Following this protocol, Neuro-2a cells were differentiated in 4 days. During this time, cells changed their stem-cell like morphology to possess extended neurites and an enlarged cell body (Figure 4.8.a). This 4-day model was used to examine PLCη2 protein levels during neuronal differentiation. Short-term treatment (4 hours) of retinoic acid already resulted in a significant increase in PLCη2 protein level (76%, t-test, p<0.01). Moreover, PLCη2 levels gradually increased throughout the differentiation process. At day 4, PLCη2 level was ~10-fold higher compared to its basal expression on day 0 (t-test, p<0.01, Figure 4.8.b,c).

As a first step towards investigating the specific role of PLCη2 in neuronal differentiation, the cellular localisation of the endogenous protein was investigated in undifferentiated and differentiated Neuro-2a cells by immunocytochemistry (Figure 4.9.). This was conducted with a PLCη2-specific antibody as described in Chapter 2.

135

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

Figure 4.8.: PLCη2 protein levels increase during retinoic acid-stimulated differentiation of Neuro-2a cells. A) Bright field images showing Neuro-2a cells differentiated with 20µM retinoic acid for 4 hrs, or for 1 to 4 days. Bars correspond to 100 µm. B) Western blot showing PLCη2 protein levels during differentiation (Day 0, 4 hrs, Day 1-4). The protein levels of β-actin were used as a loading control. The blot shown is representative of 3 independent experiments. Protein samples were separated on a 4-12% polyacrylamide gradient. C) Quantification of PLCη2 protein levels during Neuro-2a cell differentiation. Densitometry was conducted by using the ImageJ analysis software. PLCη2 protein expression was normalised to the β-actin protein level and the basal expression on Day 0 was defined 1. Error bars represent ± S.E.M. (t-test, p<0.05:*, p<0.01:**, p<0.001:***, n=3).

136

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

Figure 4.9.: Cellular localisation of endogenous PLCη2 (green) in undifferentiated (A) and differentiated (B) Neuro-2a cells counterstained with the nuclear marker, DAPI (blue). A) In undifferentiated Neuro-2a cells PLCη2 is localised to the nucleus, perinuclear area and plasma membrane (perinuclear and extracellular membrane localisation are indicated by arrows). B) In differentiated Neuro-2a cells, PLCη2 is also highly abundant in the nucleus and the perinuclear area. In the neurite, PLCη2 staining is displayed as local accumulations in bulge-like structures (indicated by arrows). Neuro-2a cells were seeded onto coverslips and fixed the next day (undifferentiated cells) or differentiated for 4 days with 20 µM retinoic acid (differentiated cells). Images are representative and were taken from a deconvoluted Z-stack by a DeltaVision deconvolution microscope (Applied Precision). Bars correspond to 10 µm.

In undifferentiated cells, PLCη2 was localised mainly in the nucleus but was also found in the perinuclear area and on the cell membranes (Figure 4.9.a). In

137

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY differentiated cells, a similar pattern was observed (Figure 4.9.b). In addition, PLCη2 also localised to the neurite where it concentrated to small bulge-like structures. Detection of PLCη2 in the nucleus was unexpected based on earlier findings. In non- neuronal cells the GFP-tagged enzyme was mainly detected at the cellular membrane (HEK-293) or on intracellular membranes (COS-7 cells in Chapter 3) but its transport to the nucleus was not indicated (Nakahara et al. 2005). Therefore the nuclear transport of PLCη2 in these cells might be facilitated by an unknown mechanism which would consequently involve the enzyme in the regulation of nuclear Ca2+ dynamics in neurons. Although the presence of a nuclear localisation signal was not yet characterised on PLCη2 these results highlight the importance of such investigation in the future.

Furthermore, PLCη2 was also observed in the neurites, often as small concentrated areas in bulge-like structures. These varicosities are likely to be the structures involved in axonal transport of packed ER tubules and mitochondria that has been described in regenerating neurons (Koenig et al. 1985; Hollenbeck and Bray 1987). The movement of these structures is Ca2+ dependent and therefore PLCη2 activity may contribute to the regulation of this process (Koenig et al. 1985).

Cellular localisation of PLCη2 was also assessed in Neuro-2a cells transfected with the GFP-tagged PLCη2 construct used in Chapter 3 (Figure 4.10.). Neuro-2a cells were transiently transfected by Lipofectamine and were fixed the next day (undifferentiated cells) or were treated with 20 µM retinoic acid for 2 days before fixation.

138

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

Figure 4.10.: Cellular localisation of GFP-tagged PLCη2 (green) in undifferentiated (A) and differentiated Neuro-2a cells (B) counterstained with the nuclear marker, DAPI (blue). A) GFP-PLCη2 is highly abundant in the nucleus but it is also present on the plasma membrane and in the cytoplasm (plasma membrane localisation is indicated by an arrow). B) In differentiated cells, GFP-PLCη2 is mainly localised to the nucleus, perinuclear region and the intra- and extracellular membranes. At the nerve ending PLCη2 is predominantly localised to the extracellular membrane. Neuro-2a cells were seeded onto coverslips, transfected with GFP-PLCη2 the next day and fixed after 24 hrs (undifferentiated cells) or transfected and differentiated for 2 days with 20 µM retinoic acid before fixation (differentiated cells). Images are representative and were taken from a Z-stack. Images were taken by: A) DeltaVision deconvolution microscope (Applied Precision) B) Leica confocal microscope. Bars correspond to 10 µm.

139

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

The advantage of the GFP-tag is that it provides a better signal-to-noise ratio, however, it always has to be taken into account that a protein which is overexpressessed might display altered cellular distribution than the endogenous protein. There is always a chance that the surplus GFP-tagged protein might associate to membranes which normally do not attract PLCη2. However, the cellular distribution GFP-PLCη2 largely mirrored the localisation of the endogenous PLCη2, although, a large pool of PLCη2 was found in the cytosol. This might be due to the excess amounts of PLCη2 produced by the overexpression of the protein. The main proportion of the GFP signal was found in the nucleus in both undifferentiated and differentiated cells. Apart from this, GFP-PLCη2 was also observed on extra- and intracellular membranes. In addition, the enhanced signal of GFP highlighted the localisation of PLCη2 in nerve endings where it was detected in the synaptic membrane and on tubular like intracellular structures.

Figure 4.11.: Endogenous PLCη2 (green) is partially colocalised with mitochondria (red) in non-differentiated and differentiated Neuro-2a cells. Co-localisation coefficient was 0.5875 in undifferentiated cells (S.E.M.: 0.0509, n=3, Pearson coefficient) indicating a tendency of PLCη2 to associate with mitochondrial membranes. In undifferentiated cells the co-localisation level was below 0.5 possibly due to the increased presence of PLCη2 in the nucleus. Cells were seeded onto coverslips and fixed the next day (undifferentiated cells) or differentiated for 4 days with 20 µM retinoic acid (differentiated cells). Mitochondria are stained with MitoTracker Red. Images are representative and were taken from a deconvoluted Z- stack by a DeltaVision deconvolution microscope (Applied Precision). Bars correspond to 10 μm.

140

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

As earlier shown in Chapter 3., transfected PLCη2 in COS-7 cells has a high co- localisation rate with mitochondria. To ascertain whether this occurs in an endogenous PLCη2 model such as Neuro-2a cells, non-differentiated and 4-day differentiated cells were subjected to MitoTracker Red treatment that was followed by the fluorescent labelling of endogenous PLCη2 (Figure 4.11.). Deconvoluted images show a partial co-localisation of PLCη2 with mitochondrial membranes although the co-localisation coefficient was just above 0.5 (Pearson coefficient). Although this could be due to the presence of PLCη2 in the nucleus as mitochondrial staining should never occur inside these organelle.

4.2.4. The physiological level of PLCη2 is essential for the retinoic acid-induced differentiation of Neuro-2a cells

The finding that PLCη2 expression is upregulated during the differentiation of Neuro-2a cells (Figure 4.8.) strongly supports the idea that PLCη2 is a key molecule for neuritogenesis. Therefore, the next aim was to investigate of its importance for Neuro-2a cell differentiation. PLCη2 knock-down cell lines were generated by the stable transfection of shRNA targeting the expression of PLCη2. Four shRNA constructs in psi-H1 vector (Genecopoeia) were transfected by electroporation and stable clones were selected using puromycin. Single-cell-derived clones were tested with Western blot to select two cell lines with the highest knock-down efficiency (shRNAPLCη2-1 and shRNAPLCη2-2, Figure 4.12.a). A control cell line was produced by the stable transfection of scrambled control shRNA. The protein level of PLCη2 21a/22/23 variant was calculated based on the densitometry of two western blots and was normalised to β-actin levels (Figure 4.12.). According to this, PLCη2 expression was decreased to 33% and 61% in shRNAPLCη2-1 and shRNAPLCη2-2 cell lines, respectively.

Previously, a role for PLCη1 was proposed in the enhancement of GPCR-induced PLC activity in Neuro-2a cells. In this study, PLCη1 and PLCη2 knock-down cells were treated with the Ca2+ ionophore, ionomycin, followed by the measurement of

141

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY inositol-turnover. Interestingly, only the downregulation of PLCη1 level was effective in reducing the Ca2+-induced phospholipase activity in this experiment. Unfortunately, Kim et al. did not demonstrate the level of PLCη2 reduction in knock- down cells and therefore it might not be appropriate to compare the participation of the two enzymes in this Ca2+-dependent process (Kim et al. 2011).

Figure 4.12.: PLCη2 protein level was downregulated by the stable transfection of shRNA in Neuro-2a cells. A) Representative immunoblot showing the expression of PLCη2 splice variants 21a/23 (164.3 kDa) and the 21a/22/23 (119.2 kDa). The whole cell lysate from Neuro-2a cells was separated on a 4-12% SDS-polyacrylamide gel. B) Calculation of PLCη2 (21a/22/23) knock-down efficiency by densitometry on 2 western blots. PLCη2 levels were normalised to β-actin levels and the level of shRNAcontrol was determined as 100%.

To ascertain whether PLCη2 is involved in this process, an inositol phosphate (IP) release assay was employed with the three cell lines to determine phospholipid turnover in response to Ca2+. The only Ca2+-mobilising agent which effectively increased the activity of PLCη2 in transfected COS-7 cells was the Na+ ionophore monensin (Chapter 3) although this might have been due to the specific cellular localisation of the enzyme in these cells. In Neuro-2a cells, the distribution of PLCη2 is certainly altered and our aim was to find an agent which is more likely to increase the overall PLC activity in these cells. Although, the treatment of the Ca2+ ionophore A23187 was unsuccessful to increase inositol phosphate level above the control level in PLCη2-expressing COS-7 cells, it stimulated the endogenous PLC activity 3-fold in the control cells. As this agent was found to be a potent stimulator of PLC activity,

142

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY it was used to treat the Neuro-2a cell lines in the IP release assays. Unfortunately, monensin treatments were not efficient in the elevation of endogenous PLC activity in these cells (data not shown) therefore this agent was not used in the following experiments. Preliminary experiments implied that the cell content of a 12-well plate that was previously used with the COS-7 cells would not provide enough material to effectively count IP-levels in Neuro-2a cells and therefore experimental conditions have been altered. Cells were grown and treated with 1 μCi/ml myo-D-[3H]inositol in 14 cm diameter dishes. On the day of the IP assay cells were trypsinised, sorted into tubes (500.000 cells/tube) and were pre-incubated with 10 mM LiCl containing buffer A for 1 hour. Subsequently, 5 µM A23187 was introduced to the cells for 3 hours and the stimulation was stopped by adding formic acid to a final concentration of 10 mM. This was followed by the conduction of the IP assay. Interestingly, the Ca2+ induced release of IPs was significantly reduced in both knock-down cell lines from 154% (shRNAcontrol) to 110% (shRNAPLCη2-1, one-way ANOVA, p<0.01) and 118% (shRNAPLCη2-2, one-way ANOVA, p<0.01, Figure 4.13.). This supports the idea that PLCη2 is activated by Ca2+-transients in Neuro-2a cells and might be also crucial for the synergistic activation of other PLCs similarly to PLCη1 (Kim et al. 2011).

Next the effect of PLCη2 knock-down on the differentiation process of Neuro-2a cells was investigated. Control and knock-down cells were differentiated as described previously for 4 days and bright images were taken with a x10 objective on each day to follow the morphological changes during the whole process. Representative images of 4-day differentiated cells from each cell line are shown in Figure 4.14a. Differentiation was significantly inhibited in the knock-down cells to an extent which largely mirrored the reduction in PLCη2 levels of the two cell lines.

143

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

Figure 4.13: Calcium induced phospholipid turnover is significantly reduced in PLCη2 knock-down cells. ShRNAcontrol and PLCη2 knock-down Neuro-2a cells were incubated with myo-D-[3H]inositol for 24 hours. Subsequently cells (500.000 cells/tube) were pretreated with 10 mM LiCl for an hour, treated with 5 µM A23187 (Ca2+ -ionophore) in 10 mM LiCl containing buffer A for 3 hours and were subjected to inositol -phosphate release assay. The activity of the untreated shRNA control was defined as 1. Error bars represent ± S.E.M. (one-way ANOVA, p<0.01, n=3).

The shRNAPLCη2-1 cells showed very little neurite outgrowth whereas the shRNAPLCη2-2 cells possessed longer neurites. To quantitatively evaluate these results the rate of differentiation for each day was calculated from three independent experiments (Figure 4.14.b). A cell was defined differentiated upon possessing at least one neurite that was longer than twice the diameter of the cell body. Two images were analysed in each experimental group (at least 75 cells in each group). ImageJ software was used to accurately measure cell body and neurite lengths. Significant differences between the differentiation rates of shRNAcontrol and knock- down cell lines were detected already on day 1 of treatment (one-way ANOVA, P<0.05) which difference gradually increased during the 4 days (Figure 4.14b). On day 4 the differentiation rate of shRNAcontrol cells was 73%, whereas only 8% and 22% of the shRNAPLCη2-1 and shRNAPLCη2-2 cells were defined as differentiated, respectively (one-way ANOVA, p<0.001 and p<0.01, respectively).

144

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

Figure 4.14.: The retinoic acid-induced differentiation of Neuro-2a cells is reduced in PLCη2 knock-down cells. A) Bright field images showing control and PLCη2 knock-down cells (shRNAPLCη2-1, shRNAPLCη2-2) after 4 days of retinoic acid treatment. Bars correspond to 100 µm. B) Differentiation rate of control and PLCη2 knock-down cells. A cell was defined differentiated when it possessed at least one neurite with a length longer than twice the cell body diameter. The results are calculated from 3 independent experiments and at least 75 cells were measured in each group. Error bars represent ± S.E.M. (one-way ANOVA, p<0.05: *, p<0.01: **, p<0.001: ***, n=3).

Total neurite outgrowth was calculated based on a previously described stereological approach (description is provided in Chapter 2) (Ronn et al. 2000; Lucocq 2008). Results were calculated from three independent experiments and four micrographs were analysed in each experimental group (at least 180 cells in each group, Figure 4.15.). As it was expected, the control cells showed gradual increase in neuronal outgrowth expanding the basal 14.7 µm to 243.3 µm (total neurite length/cell) on day 4. In contrast, neuronal outgrowth was largely deteriorated in PLCη2 knock-down

145

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY cells. From the basal 18.4 µm, the shRNAPLCη2-1 cells reached 34.9 µm on the first day, but remained on this level throughout the whole differentiation process (35.7 µm on day 4, one-way ANOVA, p<0.001 compared to control). Although the spontaneous neuritogenesis (basal level) was lower in shRNAPLCη2-2 cells (8.8 µm), it was increased to 39.6 µm on the first day. On day 4, the total length of neurites/cell in these cells was 51.5 µm on average (one-way ANOVA, p<0.001 compared to control, Figure 4.15.).

Figure 4.13.: Retinoic acid-induced neuronal outgrowth is significantly reduced in PLCη2 knock-down cells. The rate of neuronal outgrowth is determined as the total length of neurites/cell and was determined by using a stereological approach (Ronn et al. 2000; Lucocq 2008). The results are calculated from 3 independent experiments. Four images and at least 180 cells were measured in each group. Error bars represent ± S.E.M. (one-way ANOVA, p<0.01: **, p<0.001: ***, n=3).

4.2.5. PLCη2 is involved in the regulation of retinoic acid receptor signalling

As suggested by the previous experiments, it appears that PLCη2 plays an important role in the differentiation of Neuro-2a cells. However, the exact mechanism is far from being understood. PLCη2 is mainly found in the nuclei of Neuro-2a cells. This raises the possibility that it might be involved in the regulation of the local Ca2+

146

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY dynamics in the nucleus which might consequently affect transcriptional activity. Although transcriptional regulation as a function of PLCη2 has not been proposed before, its participation in this process would not be unique across the PLC family (Cocco et al. 2006). As the differentiation of Neuro-2a cells was induced by retinoic acid in previous experiments and its downstream signalling highly affects the expression of various genes, the detection of changes in RA-dependent changes in control and PLCη2 knock-down cells seems to be a good tool for further investigating the physiological role of PLCη2.

Retinoic acid signalling is processed by its nuclear receptor (RAR), which is able to regulate the expression of various genes upon ligand-induced stimulation. In addition, RAR undergoes heterodimerisation with the retinoid X receptor (RXR) after ligand-binding which enables the efficient binding to gene promoters (Lane and Bailey 2005). A short DNA sequence on the promoter, which is the target of the receptor-ligand complex, is termed as retinoic acid responsive element (RARE) (Lane and Bailey 2005). Three variants of retinoid receptors are known to exist such as RARα, RARβ and RARγ (Chambon 1996). The expressions of all three RAR variants were tested by RT-PCR (Figure 4.16). As several splice variants of these receptors exist, the primers used in this experiment were designed to be non-specific for variations. Mouse brain cDNA was used as a positive control and RPLP0 level as a loading control. After 35 PCR cycles, high levels of RARα mRNA were detected in both Neuro-2a and brain samples whereas the RARβ gene was only expressed in brain. The latter result was expected because RARβ gene expression is often downregulated in tumours and the Neuro-2a cell line was developed from a spontaneous tumour of the mice (Xu 2001). RARγ mRNA was undetectable in both Neuro-2a cells and the mouse brain. The expression of this gene in developing neural structures is almost completely absent and therefore it possibly has little significance for neuronal physiology (Ruberte et al. 1993).

147

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

Figure 4.16.: RT-PCR showing the expression of retinoid receptors in Neuro-2a cells and in mouse brain. PCR products corresponding to RARα, RARβ and the standard gene mRNA, RPLP0 are detected at their expected sizes (159 bp, 173 bp and 163 bp, respectively). RARγ transcript (169bp) couldn`t be detected possible due to the lack of its expression in Neuro-2a cells and in mouse brain. Amplification was stopped after 25 cycles for RPLP0 and after 35 cycles for receptor transcripts. In the marker lane the band corresponding to 200 bp is shown.

As the only detected receptor variant in Neuro-2a cells was RARα, this was further investigated in the control and PLCη2 knock-down cells. RARα mRNA levels were measured by quantitative PCR (qPCR) and were calculated relative to RPLP0 levels (Figure 4.175). The results showed no significant difference in the mRNA level of RARα among these groups (one-way ANOVA). As the next purpose of the study was to investigate the rate of retinoid signalling downstream from this receptor, it was essential to determine if there was any difference in its basal levels in these cells. With the detection of equal retinoid receptor level in control and knock-down cells, differences measured in the retinoid signalling in further experiments will not be the causative of altered receptor numbers.

148

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

Figure 4.17.: Quantitative PCR showing the relative levels of RARα mRNA in control and knock-down Neuro-2a cell lines. The unstimulated stable cell lines did not show significant difference in the mRNA levels of RARα. Results were normalised to the expression of the ribosomal mRNA, RPLP0. The RARα level of the shRNAcontrol was defined 1. Error bars represent ± S.E.M. (one-way ANOVA, n=3).

Figure 4.18.: Simple representation of luciferase reporter constructs designed for the detection of retinoic acid induced gene transcription. The retinoic acid (RA)-RAR- RXR complex binds to the RARE sequence which induces the expression of the firefly luciferase gene. A control vector is co-transfected with the reporter construct which contains a Renilla luciferase under the control of a CMV (Cytomegalovirus) promoter. This provides an endogenous control to measure transfection efficiency and to normalise the expression of the firefly luciferase. Figure was produced by the author of this thesis.

149

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

A dual-luciferase reporter assay was employed to measure retinoic acid-induced transcriptional activity in the control and PLCη2 knock-down Neuro-2a cells as described in Chapter 2. Briefly, 2 vectors were co-transfected into Neuro-2a cells; one containing a firefly luciferase gene which is under the regulation of a RARE sequence (AGGTCACCAGGAGGTCA) and one vector encoding Renilla luciferase preceded by a constitutively active promoter (Figure 4.18., designed and synthetized at SABiosciences, Qiagen). This latter construct provided an internal reporter of the transfection efficiency.

Control and PLCη2 knock-down cells were seeded into a 96-well plate (50.000 cells/well) and the plasmid mixture (100ng/well) was transfected the next day by Lipofectamine. Six hours after the transfection complexes were added, the culture medium was replaced with serum-free medium. The following day, transfected cells were either treated with 20 µM retinoic acid or with vehicle (DMSO) in serum-free medium for 4 hours. Cells were then lysed and the activities of both firefly and renilla luciferases were measured sequentially. In Figure 4.19., the activity of vehicle-treated cells represents the basal activity of retinoid signalling. Interestingly, both knock-down cell lines had reduced levels of basal retinoic acid signalling compared to the control cells. The luciferase activity was decreased to 40% in shRNAPLCη2-1 (one-way ANOVA, p<0.001) whereas it was reduced to 70% in shRNAPLCη2-2 cells (one-way ANOVA, p<0.01) when the luciferase activity of the control was defined as 100%. Interestingly, there was also a significant difference between shRNAPLCη2-1 and shRNAPLCη2-2 (one-way ANOVA, p<0.01) which might be the causative of different levels of PLCη2 in these cells.

The luciferase activity of the retinoic acid stimulated cells was expressed as the fold change of the basal activity in each group (Figure 4.20.). Large elevation in luciferase activity was detected in the control cells in response to the retinoic acid- stimulation (101-fold). In contrast, the shRNAPLCη2-1 and shRNAPLCη2-2 cells exhibited only 35- and 44-fold increase in activity, respectively (one-way ANOVA,

150

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY p<0.05). This indicates that PLCη2 possesses a high impact on both basal and stimulated retinoid signalling.

Figure 4.19.: Basal retinoid receptor activity in control and PLCη2 knock-down cell lines as determined by a dual-luciferase assay. The basal activity was measured 1 day following the transfection of the reporter constructs. The results were normalised to the constitutively produced Renilla luciferase activity. The luciferase activity of the shRNAcontrol was defined as one. Error bars represent ± S.E.M. (one-way ANOVA, **: p<0.01, ***p<0.001, n=3).

Figure 4.20.: Retinoic acid-stimulated signalling is suppressed in PLCη2 knock- down cell lines as detected by dual-luciferase assay. Cells were treated with 20 µM retinoic acid for 4 hours one day after the transfection of the reporter constructs. The results were normalised to the constitutively produced Renilla luciferase activity which was transfected simultaneously with the firefly luciferase-encoding construct. The basal luciferase activities for all 3 cell lines were defined as one. Error bars represent ± S.E.M. (one-way ANOVA, p<0.05:*, n=3).

151

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

4.2.6. The interaction of PLCη2 and Lim domain kinase-1 and its consequences for neuronal outgrowth

A bacterial two-hybrid (B2H) screen was previously carried out in our lab by Deana Finelli (B.Sc. honours student, 2011) to identify possible interacting partners of the C-terminal domains from different PLCη2 splice variants. The B2H screen consists of the co-transfection of the bait vector (pBT) containing the PLCη2 C-terminus and a cDNA library encoded by the target vector (pTRG) into a reporter E. coli strain (BacterioMatch II Two Hybrid System, Agilent Technologies, Figure 4.21.). The bait (PLCη2 C-terminus) is fused to a full-length bacteriophage λ lambda repressor protein whereas the target (pancreas cDNA library) is connected to the α-subunit of RNA polymerase (RNAP). When the proteins encoded by the co-transfected plasmids interact in one cell, the RNAP is tethered to the promoter region of the reporter gene which allows the transcription of two selection markers. The reporter strain is defective in the endogen synthesis of histidine. Although cells are able to grow on minimal medium due to the basal transcription activity of the reporter gene encoding an enzyme which catalyses an essential step of the histidine synthesis pathway (HIS3). The compound 3-AT is a competitive inhibitor of the HIS3 gene product and when it is added to the medium the bacteria may grow only upon possessing an interacting protein pair encoded by the pBT and pTRG vectors. The reporter gene also encodes a streptomycin resistance gene to provide a second selection for verifying interactions.

152

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

Figure 4.21.: Simplified description of the 2-hybrid system. The cDNA encoding a protein which is screened for interaction partners is inserted into the bait vector (pBT) and this provides its fusion to the full-length bacteriophage λ lambda repressor protein (λcI). A cDNA library is also produced in the pTRG plasmid. This generates a fusion of the transcripts to the α-subunit of RNA polymerase (RNAP). The two vectors are co-transfected into the E. coli reporter strain. Upon interaction of the encoded proteins in one cell, the RNAP is tethered to the promoter region of the reporter gene which enables the transcription of genes important for histamine synthesis and streptomycin resistance. Figure is taken from the BacterioMatch II Two Hybrid System manual (Agilent Technologies).

By using the B2H screen, Lim domain kinase-1 (LIMK1) was identified as a putative interaction partner of the C-terminus of the 21a/22/23 PLCη2 variant. This variant encodes the splice variant exhibiting the shortest amino acid sequence which is integral to all the other variants except for the last three amino acids (VRD; derived from the exon 22). This suggests that the putative interaction between PLCη2 and LIMK1 would not likely to be splice variant specific. LIMK1 is a Ser/Thr/Tyr kinase, which phosphorylates downstream substrates such as cofilin with its c- terminal kinase domain (Arber et al. 1998). The other domains serve to regulate this

153

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY function (Manetti 2011). The target vector from the B2H screen only contained a partial sequence of LIMK1 (519-647, red line in Figure 4.22.). This encodes a fragment of the kinase domain which excludes the substrate binding site and the kinase active region.

Figure 4.22.: Domain structure of the human LIMK1. The main domains are as follows: LIM1 (5-77 residues) and LIM2 (84-138 residues) constituted of zinc finger pairs, PDZ (163-255 residues) and the protein interaction site/protein kinase domain possessing the catalytic activity of the enzyme (345-624). Residues involved in substrate binding and phosphorylation activity are supplied by the kinase domain between 345-514 residues. An important phosphorylation site (Tyr508) is indicated by an arrow which is involved in the regulation of kinase activity. The sequence identified by the B2H screen is highlighted with a red line (519-647 residues). The structure was created based on an alignment in the Conserved Domain Database (NCBI) by the author of this thesis.

In the study conducted by Deana Finelli, the interaction of PLCη2-LIMK1 was only verified with the 3-AT selection. To confirm this interaction, purified plasmids were separated by agarose gel electrophoresis and purified by gel extraction (Qiagen). Equal amounts (100ng-100ng) of the plasmids were retransformed to the reporter E. coli strain by heat-shock and after incubating the bacteria in minimal medium they were spread onto 3-AT plates containing selective antibiotics for the plasmids (tetracyclin and chloramphenicol). Next day, a mixture of the growing colonies was spread onto 3-AT, streptomycin, tetracyclin and chloramphenicol containing plates. Colonies were detected after an overnight incubation indicating the interaction of the bait and the target (Figure 4. 23).

154

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

Figure 4.23.: E. coli reporter strain encompassing both PLCη2-pBT and LIMK1- pTRG plasmids grow on selective plate containing streptomycin, 3-AT, tetracyclin and chloramphenicol. A mixture of colonies was transferred from the 3-AT selection plate and was incubated for 24 hours at 37oC.

The B2H system is a useful method for identifying protein-protein interactions (Joung et al. 2000). However, to verify an interaction, an alternative method is often required. To provide more evidence on PLCη2-LIMK1 interaction, a pull-down experiment was carried out by using the c-Myc Tag IP/Co-IP Kit (Thermo Scientific). This consisted of the overexpression of the full-length LIMK1 labelled with a c-myc tag on its c-terminus (kind gift from Prof. Michael Olson, Beatson Institute, Glasgow) and PLCη2 in COS-7 cells. As controls, cells transfected with only one of the constructs were used. Two days following transfection, cells were lysed and 500 µg total protein containing lysates were incubated with 5 µg anti-c- myc agarose overnight. Agarose beads were washed with TBST five times and the proteins were eluted. Both the input (cell lysates) and the output (eluted samples) of the pull-down were run on 4-12% polyacrylamide gels and proteins were identified by western blot with specific antibodies against the c-myc tag and PLCη2. The analysis of the cell lysates showed that PLCη2 and LIMK1 were efficiently transfected and expressed in COS-7 cells (Figure 4.24a). After conducting the pull- down experiment, LIMK1 was bound onto the myc-tagged beads with high efficiency. However, the co-immunoprecipitation of PLCη2 was not detected (4.24b). Unfortunately, the pull-down experiment failed to confirm the interaction

155

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY between LIMK1 and PLCη2. This might be due to their weak or temporary interaction under the conditions employed. Unfortunately, the constitution of the lysis and the elution buffers is not provided by the supplier of the immunoprecipitation kit, therefore it is hard to estimate what contents should be adjusted for getting better results. Nevertheless, failure of the pull-down experiment to show interaction between these proteins does not provide definitive proof that these proteins do not interact in vivo.

Figure 4.24.: Co-immunoprecipitation experiment failed to show the interaction between LIM kinase 1 and PLCη2. C-myc-LIMK1, PLCη2 or both constructs were transfected into COS-7 cells. The transfection efficiency was assessed by western blot for which β-actin level was used as a loading control (A). The output of the co- immunoprecipitation experiment shows no interaction between LIMK1 and PLCη2 (B). Samples were separated on a 4-12% SDS-polyacrylamide gel, electroblotted onto PVDF membrane which was probed with anti-PLCη2, anti-c-myc and anti-β- actin antibodies, respectively.

As previously discussed, the hypothetical PLCη2 binding site on LIMK1 was mapped based on the partial nature of the LIMK1 sequence identified from the B2H screen. According to this, PLCη2 binds to the C-terminal portion of the kinase domain not including the substrate binding site or the enzyme catalytic region (Figure 4.25.). The kinase domain of LIMK1 has been recently crystallised in complex with its inhibitor, staurosporine (Beltrami et al., unpublished, PDB: 3s95). Based on this structure, the possible interaction sites were modelled (Figure 4.25.). The main phosphorylation site of the kinase domain is the Thr508 residue targeted by two kinases, PAK (p21-activated kinase) and ROCK (rho-associated protein kinase) (Edwards et al. 1999; Ohashi et al. 2000). In addition, two other residues are

156

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY phosphorylated by protein kinase A (Ser 323 and Ser 596) (Nadella et al. 2009). The Thr508 residue is found next to the substrate binding site and it has been suggested to regulate the ability of the domain to target proteins for phosphorylation (Edwards and Gill 1999). In contrast, it is unknown how the phosphorylation of the Ser596 residue might contribute to this process as it is found relatively far from the residues involved in kinase activity. Nevertheless, this C-terminal portion of the kinase domain has important regulatory function and it is likely that the binding of the C- terminal of PLCη2 is involved in this process.

Figure 4.25.: Structure of the kinase domain of human LIMK1. The partial sequence found in the target vector is shown in yellow. The substrate binding site and the 2 phosphorylation sites (Thr508 and Ser596 in red) are indicated by arrows. The role of Ser596 residue in regulating kinase activity is not fully understood particularly as it is located relatively far from the functional region. The structure was modelled with Accelrys Discovery Studio 3.1 by the author of this thesis.

For an actual interaction of proteins, the similar pattern of their cellular distribution is required. Thus, to provide more evidence on the interaction of PLCη2 and LIMK1 it is essential to investigate their localisation in a cell model that contains these proteins endogenously. Both PLCη2 and LIMK1 is highly expressed in the brain and therefore the Neuro-2a cells are certainly good models for this purpose. Both undifferentiated and differentiated cells were stained for endogenous PLCη2 and LIMK1 by co-immunostaining (Figure 4.26.).

157

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

Figure 4.26.: Endogenous PLCη2 and LIMK1 co-localise in the nucleus of undifferentiated (A) and differentiated (B) Neuro-2a cells. Cells were seeded onto coverslips and fixed the next day (undifferentiated cells) or differentiated for 4 days with 20 µM retinoic acid (differentiated cells). PLCη2 possessed a high co- localisation rate with LIMK1 in both undifferentiated (0.6762, S.E.M: 0.0305, Pearson coefficient) and differentiated cells (0.6794, S.E.M.: 0.0619, n=3, Pearson coefficient). Images are representative and were taken from a deconvoluted Z-stack. Bars correspond to 10 µm.

Both LIMK1 and PLCη appeared to be abundant in the nucleus (Figure 4.26.). This is consistent with the previous finding that LIMK1 localised mainly to the nucleus in certain types of neurons (Bernard et al. 1994).

The localisation of LIMK1 was also compared to transfected GFP-PLCη2 in undifferentiated and differentiated cells (Figure 4.27.). This provided similar results to the endogenous PLCη2-staining. LIMK1 and GFP-PLCη2 possessed a high co- localisation rate in the nucleus of both differentiated and undifferentiated cells. Moreover, they were both detected in the neurites of the differentiated cells and exhibited similar patterns.

158

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

Figure 4.27.: GFP-tagged PLCη2 and LIMK1 co-localise in the nucleus of undifferentiated (A) and differentiated (B) Neuro-2a cells. Cells were seeded onto coverslips and transfected by GFP- PLCη2 the next day. Fixation was carried out the following day (undifferentiated cells) or cells were differentiated for 2 days with 20 µM retinoic acid (differentiated cells) and fixed. A neurite is shown with a higher magnification to reveal co-localisation of PLCη2 and LIMK1. GFP-PLCη2 possessed a high co-localisation rate with LIMK1 in both undifferentiated (0.7663, S.E.M: 0.0619, Pearson coefficient) and differentiated cells (0.8866, S.E.M.: 0.0300, n=3, Pearson coefficient). Images are representative and were taken from a deconvoluted Z-stack. Bars correspond to 10 µm.

The consequences of the potential interaction between LIMK1 and PLCη2 are unclear. However the assessment of the overall effect of decreased PLCη2 levels in knock-down cells on LIMK1 activity might provide a better insight into whether these proteins cooperate during Neuro-2a cell differentiation. It is necessary to emphasize that the interaction of these proteins might be only important for their close position and PLCη2 might not regulate LIMK1 enzyme activity directly. LIMK1 activity is highly sensitive to changes in Ca2+-dynamics. During neuronal differentiation of Neuro-2a cells induced by a Ca2+-ionophore (ionomycin), LIMK1

159

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY is phosphorylated by Ca2+/Calmodulin kinase IV at Thr508 (Takemura et al. 2009). Earlier in this Chapter, it was shown that Ca2+ activated PLC activity is reduced in PLCη2 knock-down cells, consequently leading to decreased release of Ca2+ from

I(1,4,5)P3 sensitive stores. To assess whether LIMK1 activity is affected by the PLCη2 knock-down, the level of its phosphorylation level at Thr508 was detected at different stages of retinoic acid-induced differentiation. The shRNAcontrol and shRNAPLCη2 cell lines were selected for this experiment to be treated with retinoic acid for 4 hours, 2 days and 4 days (Figure 4.28.).

Figure 4.28.: Retinoic acid stimulated LIMK1 and CREB phosphorylation levels are reduced in PLCη2 knock-down cells. A) Immunoblot showing the phosphorylation levels for LIMK1 (at Thr508) and CREB (at Ser133) at different time points during retinoic acid induced differentiation of shRNAcontrol (Cont.) and shRNAPLCη2-1 (PLCη2) cells (basal, 4 hours, 2 days, 4 days). The levels of total LIMK1 and CREB as well as β-actin loading control were also measured. The blot shown is representative of 3 independent experiments. The whole cell lysates from Neuro-2a cells were separated on a 4-12% SDS-polyacrylamide gel. B) Phosphorylation level of LIMK1 and CREB relative to total LIMK1 and CREB levels. The pLIMK1/LIMK1 and pCREB/CREB levels were calculated relative to the shRNAcontrol level in each treatment group. White bars: control level, black bars: shRNAPLCη2-1 levels. Error bars represent ± S.E.M. (t-test, p<0.05:*, p<0.001:*** n=3).

160

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

The retinoic acid–induced phosphorylation of LIMK1 was greatly reduced in the shRNAPLCη2-1 cells (Figure 4.28.). Phosphorylation of LIMK1-Thr508 (pT508) was undetectable in the untreated cells and therefore its level was not calculated. After 4 hour stimulation, the level of pThr508 was downregulated by 75.3 % (t-test, p<0.001) and remained low at Day 2 and 4 (89% and 46.7% reduction, t-test, p<0.001 and p<0.05, respectively) compared to the shRNAcontrol. The phosphorylation rate of a LIMK1 downstream effector was also tested. The transcription factor, CREB is mainly phosphorylated and consequently activated by the cAMP pathway (Montminy and Bilezikjian 1987). Although in differentiating neurons, CREB is also directly phosphorylated by LIMK1 to direct the transcription of essential genes of neuronal outgrowth (Yang et al. 2004). Basal level of the phosphorylation of CREB at Ser133 was detectable but was similar in the shRNAPLCη2-1 and shRNAcontrol cells (t-test). After 4 hours, CREB phosphorylation was reduced by 30% in knock-down cells compared to the control cells although this was not a significant effect (t-test). At two and four days of differentiation, pCREB levels were 69.4% and 41.7% lower in shRNAPLCη2-1 than in the control cells, respectively (t-test, p<0.001 and p<0.05). In conclusion, the lack of PLCη2 in Neuro-2a cells disrupted certain signalling pathways that led to the alterations in the phosphorylation of LIMK1 and CREB, however, the exact mechanism is not quite clear at this point. Either the consequently reduced PLC activity-releated Ca2+-release or a direct action of PLCη2 on LIMK1 may lead to reduced phosphorylation of these proteins. Nevertheless, these results highlight the importance of PLCη2 in LIMK1 signalling.LIMK1 is also involved in the control of actin dynamics through the phosphorylation and the subsequent inactivation of cofillin and the actin depolymerising factor (ADF). When activated, cofillin and ADH depolymerise F-actin (polymerised form of actin) and release G-actin (depolymerised form). Hence, upon LIMK1 stimulation, the level of F-actin elevates (Arber et al. 1998; Yang et al. 1998). In the growth cone of the neuron, the cooperation of LIM kinases (1 and 2) and phosphatases of cofillin and ADH (slingshot proteins, SSHs) is required to maintain a balance in actin dynamics.

161

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

Neurite outgrowth is inhibited when either LIMK1 or the SSHs levels are disturbed by the addition of siRNA targeting the expression of these proteins (Endo et al. 2007).

The distribution of F-actin was detected in control and PLCη2 knock-down cells by tetramethylrhodamine isothiocyanate (TRITC)-linked phalloidin in 4-day differentiated cells (Figure 4.29.) (Barden et al. 1987). The shRNAcontrol cells contained elongated actin filaments along the neurites. In contrast, both shRNAPLCη2-1 and shRNAPLCη2-1 cells possessed multiple processes and punctate distribution of actin. This suggests a role for PLCη2 in regulating actin dynamics possibly via controlling LIMK1 activity. Multiple growth cones in knock- down cells might be the result of imbalanced F-actin/G-actin ratio. The downregulation of LIM kinase activity in PC12 cells significantly reduced neuronal outgrowth. Although the authors did not mention, in these cells the number of processes was higher compared to the control cells as suggested by the presented images (Endo et al. 2007). Potentially, the phenotype of PLCη2 knock-down cells in our experiment is originated from the upregulation of cofillin activity and the consequently reduced polymerisation rate of actin.

Figure 4.29.: The actin dynamics is disturbed in PLCη2 knock-down cells. The shRNAcontrol cells showed the morphology of differentiated neurons with elongated actin fibers. In shRNAPLCη2-1 and shRNAPLCη2-2 cells multiple projections were detected with limited outgrowth and the actin staining showed punctate distribution. Cells were permeabilised after fixation and incubated with TRITC-phalloidin for F- actin staining. Slides were examined on a Zeiss Axioplan fluorescent microscope system. Bars correspond to 10 μm.

162

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

4.2.7. The localisation of PLCη2 in differentiated Neuro-2a cells is controlled by PI(3,4,5)P3 levels

In Chapter 3, the PH domain of PLCη2 (and PLCη1) was shown to bind preferentially to PI(3,4,5)P3, however its association with PI(4,5)P2 was also observed. PI(3,4,5)P3 is involved in neuronal polarisation by its accumulation in growth cones which triggers the local activation of Akt and PKCζ activity (Menager et al. 2004; Khodosevich and Monyer 2010). As PI(3,4,5)P3 might be a critical factor in defining the cellular distribution of PLCη2, it might direct PLCη2 to growth cones 2+ to regulate local Ca dynamics. Here, the co-localisation of PLCη2 and PI(3,4,5)P3 was assessed in differentiated Neuro-2a cells (Figure 4.30.). As it was seen in undifferentiated cells in Chapter 3, PI(3,4,5)P3 accumulates in the nucleus and there it possessed high co-localisation rate with the endogenous PLCη2 or the transfected

GFP-PLCη2 proteins. PI(3,4,5)P3 was also present on the cell membrane throughout the cell. Moreover, local elevations of PI(3,4,5)P3 at the nerve tip of the growing neurite were detected with similar distribution to PLCη2 (indicated by arrows in Figure 4.30a).

As mentioned above, PLCη2 PH domain also associates with PI(4,5)P2 in vitro. In

Chapter 3, PI(4,5)P2 was visualised in undifferentiated Neuro-2a cells by the transfection of a construct encoding the PH domain of PLCδ1 fused to an mCherry- tag (Watt et al. 2002). Here, this plasmid was transfected similarly but the cells were differentiated for two days afterwards (Figure 4.31.).

163

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

Figure 4.30.: PI(3,4,5)P3 is co-localised with endogenous PLCη2 (A) and transfected GFP-PLCη2 (B) in differentiated Neuro-2a cells. Cells were seeded onto coverslips and were differentiated for 4 days (A) or GFP-PLCη2 was delivered by lipotransfection and then differentiation was conducted for 2 days with 20 µM retinoic acid. Fixed cells were immunostained with PLCη2 antibody (green) and PI(3,4,5)P3 antibody (red) or alternatively in GFP-PLCη2 transfected cells only PI(3,4,5)P3 antibody was used. Nuclei were stained with DAPI. Both endogenous PLCη2 and transfected GFP-tagged construct colocalised with PI(3,4,5)P3 (0,8415, S.E.M.: 0,0379 and 0.8032, S.E.M.: 0.313, respectively, n=3, Pearson coefficient). Images are representative and were taken from a deconvoluted Z-stack. Bars correspond to 10 µm.

164

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

Figure 4.31.: Localisation of endogenous PLCη2 (A) and GFP-PLCη2 (B) relative to the PI(4,5)P2 marker, mCherry-PH-PLCδ1 in differentiated Neuro-2a cells. PLCη2 only co-localises with mCherry-PH-PLCδ1 on the plasma membrane and in the neurite in both experimental conditions. Neuro-2a cells were seeded onto coverslips and mCherry-PH-PLCδ1 alone or together with GFP-PLCη2 was delivered by lipotransfection. Cells were differentiated for 2 days with 20 µM retinoic acid. Fixed cells were immunostained with PLCη2 antibody (green) in the first experimental condition. Nuclei were stained with DAPI. Pictures were taken with a Leica confocal microscope. Bars correspond to 10 µm.

165

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

In differentiated Neuro-2a cells, PH-PLCδ1 was detected mainly on the plasma membrane where it showed limited co-localisation with the endogenous PLCη2 or the overexpressed GFP-PLCη2 proteins. The presence of PH-PLCδ1 was hardly detectable in the nucleus. This would suggest that when PLCη2 enters the nucleus it becomes inactive due to the lack of its substrate. However, it was previously shown that the nucleus also contains substantial amount of PI(4,5)P2 (Watt et al. 2002). In summary, the driving force of PLCη2 localisation is PI(3,4,5)P3 whereas local elevations in PI(4,5)P2 may also regulate the cellular pattern of PLCη2 in neurons.

4.3. Discussion 4.3.1. Implications of the specific pattern of PLCη2 in the brain

In the first part of this Chapter, the tissue specific localisation of PLCη2 was described (Figure 4.1–4.4) based on the data provided by the Allen Brain Atlas (Lein et al. 2007). Apart from its known presence in the cerebral cortex, cerebellum, hippocampus, medial habenula and olfactory bulb, new structures have been identified to contain PLCη2 mRNA. High PLCη2 mRNA levels were found in the suprachiasmatic nucleus, pineal gland, pyriform cortex and the olfactory tubercule.

In contrast to the CA1-CA3 region, the dentate gyrus of the hippocampal formation only contained a few PLCη2 positive cells. This region contains a subgranular zone which produces new granular cells throughout life (Eriksson et al. 1998). The small number of PLCη2-positive cells in the dentate gyrus suggests that these cells might represent neurons in a particular maturation state. As there are inconsistencies in the literature of PLCη2 being present in the DG or not (Nakahara et al. 2005; Kanemaru et al. 2010), it is important to assess whether it is due to the limitation of methods used or it is caused by altered ages and/or environment of the mice. Nevertheless, the presence of PLCη2 in the CA1-CA3 region of the hippocampal formation is unquestionable and implicates a possible role for PLCη2 in processes relating to learning and memory formation. Long-term potentiation (LTP) is the process of “learning” on the level of the cells, which is manifested as alterations in the

166

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY efficiency of synaptic transmission. It requires local elevations in Ca2+ levels in dendritic spines (Guthrie et al. 1991). High abundance of PLCη2 in hippocampal pyramidal cells suggests its role in regulating their Ca2+ dynamics and participating in the process of LTP. The role of PLCη2 in memory formation should be assessed for which the generation of a knock-out mouse-line would be crucial. By using this model a series of learning tests could be performed as well as it would allow the local measurement of synaptic transmission in the hippocampus

The piriform cortex and the olfactory tubercule receive projections from the olfactory bulb and process olfactory information and olfaction-associated learning (Wilson 2003; Carriero et al. 2009). The piriform cortex is the centre of odour-related associative learning (Roman et al. 1993). In addition, LTP was also registered in the level of the main olfactory bulb mitral cell-granule cell synapse (Zhang et al. 2010). As PLCη2 is abundant in the above mentioned regions, it might possess a special role in the cellular physiology in these regions.

Interestingly, PLCη2 is also specifically elevated in cerebellar granule cells. The traditional theory is that cerebellar learning is processed at the level of the granule cell parallel fiber-Purkinje cell synapse (Ito and Kano 1982). The stimulation of the parallel fiber induces a prolonged decrease in synaptic excitability of the Purkinje cell (long term depression, LTD). Hence the first theory of cerebellar learning was that it is operated by the inhibition of neuron firing. Since then, plasticity has been recorded on other synaptic levels of the cerebellum. High frequency stimulation of the mossy fiber induces the development of LTP on the level of the mossy fiber - granule cell contact that is directed by NMDA and metabotropic glutamate receptors. PLCη2 might participate in the subsequent development of Ca2+ signal and the activation of PKC (D'Angelo et al. 1999).

PLCη2 is also abundant in regions associated with the circadian clock system. The retina expresses high amounts of PLCη2 and its levels gradually elevate after birth (Kanemaru et al. 2010). Another structure involved in the circadian system is the

167

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY pineal gland which is often photoreceptive in non-vertebrates and its melatonin secretion is regulated by the daily light/dark cycles (Oksche 1965). In vertebrates, the melatonin secretion is regulated by light-sensing neurons in the retina (Moore et al. 1967). This group of neurons also innervate the main intrinsic clock generator, the suprachiasmatic nucleus and the habenula (Hattar et al. 2006). The connections of these regions can be complicated further yet they represent a functional cooperation to regulate the circadian clock. The special appearance of PLCη2 in these regions might indicate a role in the cellular physiology of the generation of the intrinsic rhythmicity. In the SCN, the efficacy of external stimuli on spontaneous firing of the neurons is inhibited by an IP3R antagonist (2-APB) (Hamada et al. 1999). It is generally thought that PLCβ4 directs PLC activity in these cells and contributes to the synchronisation of neurons (Ikeda et al. 2000). However, the cholinergic stimulation induced Ca2+ rise was unaffected by the absence of PLCβ4 hence the role of other PLCs have been indicated in this process (Ikeda et al. 2000). Consequently, PLCη2 might have a major role in directing Ca2+ dynamics in these cells. Hopefully, the characterisation of PLCη2 tissue distribution in the brain provides a basis for further experimental approaches in the future.

4.3.2. The gradual elevation in PLCη2 protein levels is essential for neuronal differentiation of Neuro-2a cells

The role of PLCη2 in neuronal physiology and differentiation was assessed in Neuro- 2a cells. Interestingly, different splice variants were detected in these cells compared to whole mouse brain lysates (Figure 4.6., Table 4.2.) although currently we are unaware of the importance of the alternative splicing of the C-terminus in PLCη2. In the weakly expressed PLCη2 variant of Neuro-2a cells (21a/23), but not in any of the brain variants, a putative class II PDZ motif was identified (Zhou et al. 2005). A similar motif on the PLCβ3 C-terminus directs its binding to post-synaptic density proteins (Shank2 and Homer1b complex) to regulate mGlu receptor mediated Ca2+- signals (Hwang et al. 2005b). Alternative splicing of the PLCη2 C-terminus might alter its cellular distribution via changing its ability to bind PDZ-domain containing

168

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY proteins. Further investigation is required to assess the specific roles of the spliceforms and to detect regional variations in their tissue distribution.

The Neuro-2a cells represent an excellent tool to study neuronal differentiation and it has been used to describe the function of many proteins in this process (Pignatelli et al. 1999; Bryan et al. 2006; Zeng and Zhou 2008). Results of this Chapter revealed that PLCη2 protein expression substantially increases during the retinoic-acid induced differentiation of Neuro-2a cells. The role of PLCη2 in neuronal outgrowth was further indicated by the insufficiency of this process in PLCη2 knock-down cells. Both the rate of differentiation and the total neurite outgrowth was decreased by the downregulation of PLCη2 levels. This might be simply the causative of 2+ 2+ altered Ca -induced Ca release of the IP3Rs suggested by the inositol phosphate response to a Ca2+-ionophore in our experiments. Differentiating neurons show spontaneous Ca2+ spikes and waves (Gu and Spitzer 1995) and due to the unique Ca2+ sensitivity of PLCη2 it might contribute to the generation of these signals and to their translation into downstream effects.

The cellular localisation of PLCη2 was assessed in undifferentiated and differentiated Neuro-2a cells. Earlier studies demonstrated that PLCη2 was found mainly on the plasma membrane in COS-7 and HEK-293 cells (Chapter 3) (Nakahara et al. 2005). Moreover, the protein had little presence in the cytoplasm in these cells. Based on this, it was concluded earlier that PLCη2 might direct the early responses to external stimuli as it does not require activation-induced translocation to the membrane (Popovics et al. 2011). However in Neuro-2a cells, PLCη2 mainly appeared in the nucleus and in the perinuclear area and only a small proportion of the protein was present on the plasma membrane. Additionally, it also localised to nerve terminals suggesting its role in neurotransmission. Further studies are required to evaluate the distribution of PLCη2 in other cellular models or primary cells. Possibly, the hippocampal pyramidal cells or cerebellar granule cells would provide a better tool for this analysis based on their high PLCη2 content.

169

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

4.3.4. The role of PLCη2 in nuclear signalling

Although it was unexpected to detect PLCη2 in the nucleus of Neuro-2a cells, it is not a unique feature across the PLC family. PLCβ isozymes are also abundant in the nucleus although their local role is just beginning to come to light. They are suggested to appear in nuclear speckles which are the locations of several transcription-regulating molecules (Martelli et al. 2005). PLCβ1 is directly involved in the regulation of promoter-binding of the transcription factors, c-Jun and AP1 in differentiating myogenic cells (Ramazzotti et al. 2008). PLCβ1 also stimulates protein kinase Cα- activity (PKCα) via the production of diacylglycerol (DAG). The activated PKCα phosphorylates lamin B1 and triggers the breakdown of the nuclear lamina preceding mitotic division in murine erythroleukemia cells (Fiume et al.

2009). Interestingly, the presence of IP3Rs on the inner nuclear membrane as well as inside the nucleus was shown by immunocytochemistry and immunoelectron 2+ microscopy. In addition, IP3R-dependent Ca transients in the nucleus have been observed and are implicated in the regulation of CREB-mediated signalling

(Cardenas et al. 2005). The production of I(1,4,5)P3 is consequently associated with the decrease in PI(4,5)P2 levels. PI(4,5)P2 is involved in chromatin remodelling by linking the BAF (Brahma-related gene Associated Factors) protein complex to the chromatin (Zhao et al. 1998). PLCη2 might participate in the regulation of these processes in neurons based on its high level in the nuclei of these cells.

In our experiment, PLCη2 knock-down had a substantial effect on basal and retinoic acid stimulated retinoid signalling. This effect might not be restricted to the RARE containing promoters and might appear as a general effect on phosphoinositide- regulated gene expression. This might contribute to the decreased differentiation rate detected in PLCη2 knock-down cells as neuronal differentiation is always associated with a substantial change in gene expression profiles (Gurok et al. 2004). This might be further investigated by using luciferase reporters of different DNA regulatory elements in PLCη2 knock-down or overexpressing cells.

170

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

4.3.5. The role of PLCη2-LIMK1 interaction for neuronal outgrowth of Neuro-2a cells

The interaction of PLCη2 with LIMK1 was identified with a bacterial two-hybrid screen, whereas the myc-tag co-immunoprecipitation experiment was unable to verify this. The latter experiment might have been unsuccessful due to the experimental conditions (ionic composition etc.) suggested in the description of the results. Moreover other limitation might be that the C-terminal tag on LIMK1 altered the binding properties of the protein. This interaction should be further investigated in the future by using differently tagged LIMK1-PLCη2 pairs in the pull-down experiments.

Nevertheless, LIMK1 and PLCη2 have a high co-localisation rate in both undifferentiated and differentiated Neuro-2a cells suggesting that their distribution allows the interaction of the proteins. Western blot analysis revealed that retinoic- acid stimulated LIMK1 and CREB phosphorylation is highly reduced in the PLCη2 knock-down cells. This effect may originate from two possible biological roles of PLCη2. As both LIMK1 and CREB phosphorylation is affected by Ca2+ transients via the phosphorylation activity of Ca2+/Calmodulin kinase IV (Matthews et al. 1994; Takemura et al. 2009) PLCη2 may affect these proteins by modulating Ca2+ dynamics. Additionally, PLCη2 might exhibit a direct effect on LIMK1, which consequently influences CREB phosphorylation (Yang et al. 2004).

The GFP-PLCη2 and the endogenous LIMK1 possessed similar localisation in the neurite of Neuro-2a cells. Their local interaction might affect the phosphorylation state of the LIMK1 substrate, cofillin and thereby increases the F-actin/G-actin ratio (Yang et al. 1998). This theory is supported by the disturbed organisation of actin skeleton seen in PLCη2 knock-down cells.

171

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

4.3.6. PLCη2 localisation is regulated by PI(3,4,5)P3

In Chapter 3, the PH domains of both PLCη1 and PLCη2 were shown to possess a preference toward PI(3,4,5)P3 binding and the co-localisation of PLCη2 with

PI(3,4,5)P3 was confirmed in undifferentiated Neuro-2a cells. In this Chapter, the localisation of endogenous PLCη2 and its transfected GFP-tagged variant was assessed in differentiated Neuro-2a cells relative to PI(3,4,5)P3 and PI(4,5)P2. PLCη2 and PI(3,4,5)P3 co-localised mainly in the nucleus and also on the cellular membrane to some extent. PI(3,4,5)P3 is likely to be the main phospholipid binding partner of PLCη2 PH domain and may enhance the accumulation of PLCη2 in the nucleus.

However, PLCη2 is not functional without its substrate, PI(4,5)P2 being present. For the detection of PI(4,5)P2 distribution in differentiated Neuro-2a cells, the mCherry- tagged PH domain of PLCδ1 was transfected. PLCη2 and the mCherry-PH-PLCδ1 co-localised partially on the plasma membrane. In addition, PI(4,5)P2 staining seemed to be lacking from the cell nucleus. The presence of PI(4,5)P2 in the nucleus was confirmed earlier by electron microscopy in different cell types (Watt et al.

2002). Thus, the amount of PI(4,5)P2 might be low in the nucleus as it is only detectable by very sensitive methods. In conclusion, the localisation of PLCη2 is mainly dependent on PI(3,4,5)P3 distribution and its enzyme activity might be regulated by the amount of the accessible PI(4,5)P2 levels.

4.3.7. Final conclusion

In this Chapter, a series of experiments were conducted to acquire evidence on the involvement of PLCη2 in neuronal differentiation and physiology. PLCη2 is expressed in special regions of the brain which are implicated in learning, memory formation, hormonal secretion or the maintenance of the intrinsic clock. Hypothetical functions of PLCη2 were also suggested based on tissue distribution. PLCη2 was shown to behave uniquely in neuronal cells based on its different cellular distribution in neuroblastoma cells compared to previous cellular models. PLCη2 was essential for the retinoic acid-induced differentiation of Neuro-2a cells. This might be the

172

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY result of the multiple roles of PLCη2 in these cells such as provoking Ca2+ signals, interacting with LIMK1 and regulating gene transcription patterns as indicated in Figure 4.32.

Figure 4.32.: PLCη2 activity contributes to neuronal differentiation on multiple levels. Upon activation of PLCη2, the level of PI(4,5)P2 decreases whereas the concentrations of the enzymatic products, I(1,4,5)P3 and DAG elevate. I(1,4,5)P3 triggers Ca2+-release from the endoplasmic reticulum which activates LIMK1 and CREB through their phosphorylation by CamKIV. Alternatively, CREB might also be phosphorylated directly by LIMK. PLCη2 may also directly activate LIMK1 by an unknown mechanism. LIMK1 inhibits actin depolymerising activity of cofilin resulting in the assembly of actin filaments required for neuronal outgrowth. Activated CREB initiates the transcription of essential genes for differentiation. Changes in the nuclear DAG and PI(4,5)P2 levels may also affect the transcriptional activity through the activation of PKC or altering PI(4,5)P2-chromatin interactions. Abbreviations: PLCη2: Phospholipase C-η2, I(1,4,5)P3: Inositol 1,4,5-trisphosphate, PI(4,5)P2: Phosphatidylinositol 4,5-bisphosphate, DAG: diacylglycerol, CamKIV: Ca2+/Calmodulin-dependent protein kinase IV, LIMK1: Lim-domain kinase 1, CREB: cAMP response element-binding protein, PKC: Protein kinase C. Figure was produced by the author of this thesis.

173

CHAPTER 4. THE ROLE OF PLCη2 IN NEURONAL PHYSIOLOGY

174

Chapter 5.

Future experiments

175

176

CHAPTER 5. FUTURE EXPERIMENTS

The work described in this thesis reveals that PLCη enzymes represent a unique class of PLCs. The importance and properties of several of their structural domains (PH-, EF- and C2) were ascertained. Moreover, a physiological role for PLCη2 in neuronal differentiation was identified. Scientific findings often give rise to more questions than they answer, which is a key driving force for Science. This Chapter contains suggestions for future experiments inspired by the key findings of the thesis.

5.1.: How does Ca2+ activate PLCη2?

It was successfully demonstrated in this thesis that Ca2+-activation of PLCη2 is operated by the EF-hand domain which triggers an allosteric switch to regulate the catalytic domain. More work is required to determine the structure of this unique EF- hand and to describe the structural changes of the whole protein upon Ca2+-binding. Purification of the EF-hand domain would provide the opportunity to determine the Ca2+-binding properties of this domain. This might be tested by Isothermal Titration Calorimetry (ITC) which is a quantitative technique that is used to detect the interactions between proteins and small molecules. The ITC detects interactions based on the very sensitive measurement of temperature changes generated by chemical interactions (Bounds et al. 2002). This way, we might gain a better understanding on how the non-canonical second EF-loop coordinates Ca2+ or what concentrations of the metal are required for this interaction. The extension of the study would be particularly challenging and include the purification and crystallisation of the whole PLCη2 protein with or without Ca2+ or its analogues Ba2+ and La3+ as in (Essen et al. 1997a). These structural studies are required to acquire a greater insight into the regulation of PLCη enzymes.

5.2.: What are the characteristics of PLCη2 C2 domain phospholipid-binding?

An interesting new feature of PLCη2 provided by the findings of the thesis is that association of the PLCη2 C2 domain with membranes is required for the

177

CHAPTER 5. FUTURE EXPERIMENTS physiological activity of the enzyme. Moreover, one mutant of the domain (D861N) was suggested to alter the lipid-binding selectivity of the domain (Ananthanarayanan et al. 2002). C2 domains in various proteins exhibit a wide range of phospholipid specificity (Stahelin 2009). Implications of this process in PLCηs are hard to estimate as the membrane targeting mechanism is dictated by the PH domain. However, considering that the lipid specificity of the PLCη2 C2 domain is important for activity, further investigation of this feature might provide useful information. Lipid-specificity of the C2 domain may be determined by various techniques such as a slight modification of the FRET-assay or PIP-strip method employed in the thesis. In addition to the mutations generated in the C2 domain, a new mutant lacking the whole domain might be a relevant tool to assess the role of this structure in cellular localisation or membrane affinity. Determination of the structure of the whole protein (suggested in the previous section) would also provide valuable information as to how Ca2+ directs the association of the domain to the membrane.

5.3. Cellular localisation – still in doubts

The cellular distribution of PLCη2 is cell type-specific. Neuro-2a cells were excellent tool for the initial characterisation of cellular distribution in neurons and determine the role of PLCη2 in neuronal differentiation. However, further studies are required to assess PLCη2 cellular distribution in more accepted models such as in primary neurons and in tissue sections derived from different brain regions. A technique which provides the highest resolution of the cellular distribution for proteins is immuno-electron microscopy. By quantifying the organelle-specific distribution of the protein in a range of cells an overall picture may be provided about its abundance on different membranes (Lucocq 2008). Moreover, the presence of PLCη2 in nerve endings and in dendrites should be further assessed to evaluate its role in pre- and postsynaptic events.

Phospholipid content of membranes undergoes a substantial change upon agonist induced stimulation suggested by the high number of molecules which are linked to

178

CHAPTER 5. FUTURE EXPERIMENTS lipid metabolism. Though PLCη2 is predominantly membrane-bound in COS-7 cells, a limited rate of cytosolic staining was detected in Neuro-2a cells. This might represent a cytosolic pool of the protein which is translocated to the membrane upon extracellular stimulation. Moreover, the redistribution of the membrane-bound forms might be also provoked by this process. To investigate these possibilities, live cell imaging using GFP-tagged PLCη2 could be conducted during treatment with Ca2+- ionophores or other drugs that influence intracellular Ca2+ dynamics. As the thesis provides evidence for the regulation of PLCη2 localisation by PI(3,4,5)P3 levels, cells might be treated by stimulators or inhibitors of the PI3K-pathway, such as insulin and wortmannin, respectively (Powis et al. 1994). The localisation of

PI(4,5)P2 and PI(3,4,5)P3 may be simultaneously detected by fluorescently tagged PH domains of PLCδ1 and GRP1 (general receptor for phosphoinositides-1) (Venkateswarlu et al. 1998; Varnai and Balla 2008). With these experiments, a better understanding about how PLCη2 is shuttled between membranes or in and out the nucleus may be provided.

5.4. PLCη2 levels throughout life and in disease

A key question that remains to be answered is how PLCη2 levels changes during pre- and postnatal development or during ageing. According to the data presented in this thesis, PLCη2 level increases during neuronal differentiation of Neuro-2a cells. However, further assessment of this phenomenon is required to be conducted in a mammalian model organism. PLCη2-levels should be followed during embryogenesis to allow us to determine which stage of the embryo is when PLCη2 gains an essential role for development. There might also be differences in its regional distribution during embryogenesis whose investigation might provide a better insight into its physiological role. Apart from its developmental role, PLCη2 function has been linked to ageing and the progression of Alzheimer`s disease (AD) (Popovics and Stewart 2012a). However, further research is required to support this hypothesis. It has been shown that PLCη2 levels gradually increase after birth in the brain and in the retina (Nakahara et al. 2005; Kanemaru et al. 2010). Moreover, data

179

CHAPTER 5. FUTURE EXPERIMENTS provided by the Allen Brain Atlas highlights the same tendency of PLCη2 expression in man (http://developinghumanbrain.org). Protein levels of PLCη2 should be further assessed throughout life in mouse and in man to gain more evidence on this process. Elevated PLCη2 levels may disturb the physiological Ca2+ responses at later stages of life but may also severe the progression of AD that is known to be connected to distorted Ca2+ homeostasis (Berridge 2010; Popovics and Stewart 2012a). Accordingly, it might sound sensible to study PLCη2 levels in animal models of Alzheimer`s disease and in post mortem human brain with diagnosed disease. In addition, Neuro-2a cells might serve as good cellular tools to study the effect of elevated PLCη2 levels on Ca2+-dynamics or to evaluate how these cells cope with stress (oxidative stress or amyloid treatment).

5.5. Splice variants of PLCη2

My results and the study conducted by Zhou et al. revealed that two of the five splice variants of PLCη2 are dominantly expressed in mouse brain (21b/22/23 and 21c/22/23). Interestingly, neither of these variants contains the PDZ-binding motif found on exon 23, due to a stop codon on the preceding exon (Zhou et al. 2005). In contrast, in the retina almost all spliceforms were detected including one with a PDZ sequence (21a/23). Further investigation is required to ascertain whether there are regional differences in the distribution of splice variants in the brain. Moreover, if the presence of multiple forms of PLCη2 is required for the processing of visual information in the retina, it would be interesting to investigate the participation of each spliceform in this process. In Chapter 4, splice variants were determined by RT- PCR using spliceform-specific primers and by western blot. A possibly more accurate technique would be provided by the Northern blot analysis of RNA expression which may be used to determine the tissue-specific distribution of splice variants in the brain (Alwine et al. 1977). In addition, cloning of the different forms and their expression as GFP-fused proteins might reveal alterations in their cellular distribution. These suggested experiments are likely to reveal the importance of having 5 different spliceforms of PLCη2.

180

CHAPTER 5. FUTURE EXPERIMENTS

5.6. LIMK1-PLCη2 interaction: more evidence is required

A B2H-screen provided some evidence for a putative interaction between PLCη2 and LIMK1. However, subsequent pull-down experiments failed to confirm the interaction. It is essential to further investigate this hypothetical interaction and uncover the mechanism by which PLCη2 regulates LIMK1 function. A sensitive technique which readily detects the interaction of proteins in a cellular context is conducted by the utilisation of FRET. The two proteins of interest are fused to two different fluorescent proteins which are capable of transferring energy upon excitation from one to another (from the donor to the acceptor protein). This only occurs if the fluorescent tags exhibit an overlap in their emission and absorption spectra. In addition, the FRET signal is generated if the distance between the two fusion proteins are in the range of 0.002 to 0.01 µm (Day et al. 2001). Interacting proteins are expected to reach a close proximity and therefore this technique may be employed to very efficiently and sensitively detect protein-protein interactions. FRET-analysis may be conducted to detect the interaction of PLCη2 and LIMK1 in live cells in basal and agonist-stimulated conditions. This may aid in determining whether this interaction occurs in the cell.

5.7. What is the role of PLCη2 in nuclear signalling?

The thesis provides evidence that PLCη2 has roles in regulating nuclear signalling. Its presence in the nucleus strongly suggests that PLCη2 responds to or triggers 2+ nuclear Ca signals. In a study conducted by Zima et al., IP3R-dependent nuclear Ca2+ transients were detected in isolated cardiac nuclei by using the Ca2+ sensitive dye, fluo-4 dextran (Zima et al. 2007). A similar experiment may be employed by the isolation of nuclei from control and PLCη2 knock-down Neuro-2a cells. This would serve as a good tool to investigate the role of PLCη2 in nuclear Ca2+ signalling. It is also intriguing, how PLCη2 is involved in the regulation of transcriptional activity. This may be through regulation of intranuclear Ca2+ dynamics. Phospholipids, which have the potential to determine the subnuclear localisation of PLCη2, are known to

181

CHAPTER 5. FUTURE EXPERIMENTS be present in the nucleus but it is not known how they are linked to the nuclear matrix. One theory is that they reside on the inner nuclear membrane and on invaginations of the nuclear envelope (Malhas et al. 2011; Martelli et al. 2011). Although, this would suggest that the nuclear phosphoinositide-binding proteins should translocate onto these membranes upon activation. Also, this requires these invaginations to frequently occur in the nucleus, however in primary neurons these structures were not even detected (Bezin et al. 2008). In contrast, when the localisation of PI(4,5)P2 and PI(3,4,5)P3 was detected by monoclonal antibodies their nuclear distribution greatly differed from what would have been indicated by their presence on only membraneous structures. Whereas PI(4,5)P2 specifically accumulates in dense structures, PI(3,4,5)P3 exhibits a more diffused appearance (Mazzotti et al. 1995; Kwon et al. 2010). These two lipids seem to regulate PLCη2 localisation and activity according to the results of the thesis. Interestingly, PLCβ nuclear distribution is similar to that of PI(4,5)P2, although its PH domain was shown to bind phospholipids in a non-specific way (Evangelisti et al. 2006; McCullar et al. 2007). As suggested before, immuno-electron microscopy would provide a much greater resolution which should be employed to map PLCη2 distribution in the nucleus. It has been suggested before, that proteins of the nuclear matrix exist in a highly complexed organisation (Martelli et al. 2011). This may reinforce the search for new interacting partners for PLCη2 in the nucleus.

An additional question is how PLCη2 is transferred to the nucleus. Molecules that are larger than >50 kDa require a nuclear transport mechanism initiated by a nuclear localisation signal (NLS) sequence in the molecule (Poon and Jans 2005). Such signal was identified in PLCβ sequence as clustered basic residues on the C-terminus (Kim et al. 1996). It would be intriguing to ascertain where the NLS is mapped on PLCη2 and whether it is represented in both isoforms and all spliceforms. In addition, it also has to be highlighted, that PLCη2 is only transported to the nucleus in neurons (as detected so far). This strongly suggests that it has a specific role in neuronal nuclear signalling.

182

CHAPTER 5. FUTURE EXPERIMENTS

Since its initial description in 2005, PLCη2 research gave limited amount of information on its contribution to cellular and physiological processes. The work presented in this thesis has provided a fuller understanding of its physiological function and how its activity is regulated. The work suggested in this Chapter would provide even greater insight into the importance of PLCη2 and its various properties.

183

184

Chapter 6.

Bibliography

185

CHAPTER 6. BIBLIOGRAPHY

Abdel-Latif, A. A., Green, K., Smith, J. P., McPherson, J. C. and Matheny, J. L. (1978), 'Norepinephrine-stimulated breakdown of triphosphoinositide of rabbit iris smooth muscle: effects of surgical sympathetic denervation and in vivo electrical stimulation of the sympathetic nerve of the eye', Journal of Neurochemistry, 30 (3), 517-25. Adebanjo, O. A., Biswas, G., Moonga, B. S., Anandatheerthavarada, H. K., Sun, L., Bevis, P. J., Sodam, B. R., Lai, F. A., Avadhani, N. G. and Zaidi, M. (2000), 'Novel biochemical and functional insights into nuclear Ca2+ transport through IP3Rs and RyRs in osteoblasts', Am J Physiol Renal Physiol, 278 (5), F784-91. Agranoff, B. W., Murthy, P. and Seguin, E. B. (1983), 'Thrombin-induced phosphodiesteratic cleavage of phosphatidylinositol bisphosphate in human platelets', J Biol Chem, 258 (4), 2076-8.

Akasu, T., Hasuo, H. and Tokimasa, T. (1987), 'Activation of 5-HT3 receptor subtypes causes rapid excitation of rabbit parasympathetic neurones', Br J Pharmacol, 91 (3), 453-5. Alicia, S., Angelica, Z., Carlos, S., Alfonso, S. and Vaca, L. (2008), 'STIM1 converts TRPC1 from a receptor-operated to a store-operated channel: moving TRPC1 in and out of lipid rafts', Cell Calcium, 44 (5), 479-91. Allan, D. and Michell, R. H. (1978) 'A calcium-activated polyphosphoinositide phosphodiesterase in the plasma membrane of human and rabbit erythrocytes' Biochim Biophys Acta, 508 (2), 277-86. Allbritton, N. L. and Meyer, T. (1993), 'Localized calcium spikes and propagating calcium waves', Cell Calcium, 14 (10), 691-7. Alousi, A. A., Jasper, J. R., Insel, P. A. and Motulsky, H. J. (1991), 'Stoichiometry of receptor-Gs-adenylate cyclase interactions', FASEB J, 5 (9), 2300-3. Alwine, J. C., Kemp, D. J. and Stark, G. R. (1977), 'Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes', Proc Natl Acad Sci U S A, 74 (12), 5350-4. Ananthanarayanan, B., Das, S., Rhee, S. G., Murray, D. and Cho, W. (2002), 'Membrane targeting of C2 domains of phospholipase Cδ isoforms', J Biol Chem, 277 (5), 3568-75. Anderson, D., Koch, C. A., Grey, L., Ellis, C., Moran, M. F. and Pawson, T. (1990), 'Binding of SH2 domains of phospholipase Cγ1, GAP, and Src to activated growth factor receptors', Science, 250 (4983), 979-82. Aoki, J., Inoue, A., Makide, K., Saiki, N. and Arai, H. (2007), 'Structure and function of extracellular phospholipase A1 belonging to the pancreatic lipase gene family', Biochimie, 89 (2), 197-204.

186

CHAPTER 6. BIBLIOGRAPHY

Arber, S., Barbayannis, F. A., Hanser, H., Schneider, C., Stanyon, C. A., Bernard, O. and Caroni, P. (1998), 'Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase', Nature, 393 (6687), 805-9. Baimbridge, K. G., Celio, M. R. and Rogers, J. H. (1992), 'Calcium-binding proteins in the nervous system', Trends Neurosci, 15 (8), 303-8. Bairoch, A. and Cox, J. A. (1990), 'EF-hand motifs in inositol phospholipid-specific phospholipase C', FEBS Lett, 269 (2), 454-6. Banno, Y., Yada, Y. and Nozawa, Y. (1988), 'Purification and characterization of membrane-bound phospholipase C specific for phosphoinositides from human platelets', J Biol Chem, 263 (23), 11459-65. Barden, J. A., Miki, M., Hambly, B. D. and Dos Remedios, C. G. (1987), 'Localization of the phalloidin and nucleotide-binding sites on actin', Eur J Biochem, 162 (3), 583-8. Basavappa, S., Mangel, A. W., Scott, L. and Liddle, R. A. (1999), 'Activation of calcium channels by cAMP in STC-1 cells is dependent upon Ca2+ calmodulin-dependent protein kinase II', Biochem Biophys Res Commun, 254 (3), 699-702. Batra-Safferling, R., Granzin, J., Modder, S., Hoffmann, S. and Willbold, D. (2010), 'Structural studies of the phosphatidylinositol 3-kinase (PI3K) SH3 domain in complex with a peptide ligand: role of the anchor residue in ligand binding', Biol Chem, 391 (1), 33-42. Baughman, J. M., Perocchi, F., Girgis, H. S., Plovanich, M., Belcher-Timme, C. A., Sancak, Y., Bao, X. R., Strittmatter, L., Goldberger, O., Bogorad, R. L., Koteliansky, V. and Mootha, V. K. (2011), 'Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter', Nature, 476 (7360), 341-5. Baukal, A. J., Guillemette, G., Rubin, R., Spat, A. and Catt, K. J. (1985), 'Binding sites for inositol trisphosphate in the bovine adrenal cortex', Biochem Biophys Res Commun, 133 (2), 532-8. Belmaker, R. H., Bersudsky, Y., Agam, G., Levine, J. and Kofman, O. (1996), 'How does lithium work on manic depression? Clinical and psychological correlates of the inositol theory', Annu Rev Med, 47, 47-56. Bennett, C. F., Balcarek, J. M., Varrichio, A. and Crooke, S. T. (1988), 'Molecular cloning and complete amino-acid sequence of form-I phosphoinositide- specific phospholipase C', Nature, 334 (6179), 268-70. Bennett, C. F. and Crooke, S. T. (1987), 'Purification and characterization of a phosphoinositide-specific phospholipase C from guinea pig uterus. Phosphorylation by protein kinase C in vivo', J Biol Chem, 262 (28), 13789- 97.

187

CHAPTER 6. BIBLIOGRAPHY

Bernard, O., Ganiatsas, S., Kannourakis, G. and Dringen, R. (1994), 'Kiz-1, a protein with LIM zinc finger and kinase domains, is expressed mainly in neurons', Cell Growth Differ, 5 (11), 1159-71. Berridge, M. J. (2010), 'Calcium hypothesis of Alzheimer's disease', Pflugers Arch, 459 (3), 441-9. Berridge, M. J. (2012b), 'Cell Signalling Biology' doi:10.1042/csb0001001. Berridge, M. J. (2012a), 'Cell Signalling Biology' doi:10.1042/csb0001002. Berridge, M. J. (2012c), 'Cell Signalling Biology' doi:10.1042/csb0001003. Berridge, M. J., Bootman, M. D. and Lipp, P. (1998), 'Calcium- a life and death signal', Nature, 395 (6703), 645-8. Berridge, M. J., Bootman, M. D. and Roderick, H. L. (2003), 'Calcium signalling: dynamics, homeostasis and remodelling', Nat Rev Mol Cell Biol, 4 (7), 517- 29. Berridge, M. J., Dawson, R. M., Downes, C. P., Heslop, J. P. and Irvine, R. F. (1983), 'Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides', Biochem J, 212 (2), 473-82. Berridge, M. J., Lipp, P. and Bootman, M. D. (2000), 'The versatility and universality of calcium signalling', Nat Rev Mol Cell Biol, 1 (1), 11-21. Bezin, S., Fossier, P. and Cancela, J. M. (2008), 'Nucleoplasmic reticulum is not essential in nuclear calcium signalling mediated by cyclic ADP-ribose in primary neurons', Pflugers Arch, 456 (3), 581-6. Boittin, F. X., Macrez, N., Halet, G. and Mironneau, J. (1999) 'Norepinephrine- 2+ induced Ca waves depend on InsP3 and ryanodine receptor activation in vascular myocytes', Am J Physiol, 277, C139-51. Bootman, M. D. and Lipp, P. (2001), 'Calcium Signalling and Regulation of Cell Function', eLS (John Wiley & Sons, Ltd). Bounds, P. L., Sutter, B. and Koppenol, W. H. (2002), 'Studies of metal-binding properties of Cu, Zn superoxide dismutase by isothermal titration calorimetry', Methods Enzymol, 349, 115-23. Boyer, J. L., Waldo, G. L. and Harden, T. K. (1992), 'Beta gamma-subunit activation of G-protein-regulated phospholipase C', J Biol Chem, 267 (35), 25451-6. BrainSpan: Atlas of the Developing Human Brain [Internet]. Funded by ARRA Awards 1RC2MH089921-01, 1RC2MH090047-01, and 1RC2MH089929-01. © 2011. Available from: http://developinghumanbrain.org. Brini, M. and Carafoli, E. (2011), 'The plasma membrane Ca2+ ATPase and the plasma membrane sodium calcium exchanger cooperate in the regulation of cell calcium', Cold Spring Harb Perspect Biol, 3 (2).

188

CHAPTER 6. BIBLIOGRAPHY

Brown, R. A., Domin, J., Arcaro, A., Waterfield, M. D. and Shepherd, P. R. (1999), 'Insulin activates the alpha isoform of class II phosphoinositide 3-kinase', J Biol Chem, 274 (21), 14529-32. Bryan, B. A., Cai, Y. and Liu, M. (2006), 'The Rho-family guanine nucleotide exchange factor GEFT enhances retinoic acid- and cAMP-induced neurite outgrowth', J Neurosci Res, 83 (7), 1151-9. Burgess, W. H., Dionne, C. A., Kaplow, J., Mudd, R., Friesel, R., Zilberstein, A., Schlessinger, J. and Jaye, M. (1990), 'Characterization and cDNA cloning of phospholipase C-γ, a major substrate for heparin-binding growth factor 1 (acidic fibroblast growth factor)-activated tyrosine kinase', Mol Cell Biol, 10 (9), 4770-7. Burgoyne, R. D. (2007), 'Neuronal calcium sensor proteins: generating diversity in neuronal Ca2+ signalling', Nat Rev Neurosci, 8 (3), 182-93. Burke, J. E. and Dennis, E. A. (2009), 'Phospholipase A2 structure/function, mechanism, and signalling', Journal of Lipid Research, 50 (Supplement), S237-S42. Busillo, J. M. and Benovic, J. L. (2007), 'Regulation of CXCR4 signalling', Biochim Biophys Acta, 1768 (4), 952-63. Bygrave, F. L. and Benedetti, A. (1996), 'What is the concentration of calcium ions in the endoplasmic reticulum?', Cell Calcium, 19 (6), 547-51. Calcraft, P. J., Ruas, M., Pan, Z., Cheng, X., Arredouani, A., Hao, X., Tang, J., Rietdorf, K., Teboul, L., Chuang, K. T., Lin, P., Xiao, R., Wang, C., Zhu, Y., Lin, Y., Wyatt, C. N., Parrington, J., Ma, J., Evans, A. M., Galione, A. and Zhu, M. X. (2009), 'NAADP mobilizes calcium from acidic organelles through two-pore channels', Nature, 459 (7246), 596-600. Camacho, M., Machado, J. D., Alvarez, J. and Borges, R. (2008), 'Intravesicular calcium release mediates the motion and exocytosis of secretory organelles: a study with adrenal chromaffin cells', J Biol Chem, 283 (33), 22383-9. Cardenas, C., Liberona, J. L., Molgo, J., Colasante, C., Mignery, G. A. and Jaimovich, E. (2005), 'Nuclear inositol 1,4,5-trisphosphate receptors regulate local Ca2+ transients and modulate cAMP response element binding protein phosphorylation', J Cell Sci, 118 (Pt 14), 3131-40. Carriero, G., Uva, L., Gnatkovsky, V. and de Curtis, M. (2009), 'Distribution of the olfactory fiber input into the olfactory tubercle of the in vitro isolated guinea pig brain', Journal of Neurophysiology, 101 (3), 1613-19. Catterall, W. A., Perez-Reyes, E., Snutch, T. P. and Striessnig, J. (2005), 'International Union of Pharmacology. XLVIII. Nomenclature and structure- function relationships of voltage-gated calcium channels', Pharmacol Rev, 57 (4), 411-25.

189

CHAPTER 6. BIBLIOGRAPHY

Chambon, P. (1996), 'A decade of molecular biology of retinoic acid receptors', FASEB J, 10 (9), 940-54. Chapman, E. R., An, S., Edwardson, J. M. and Jahn, R. (1996), 'A novel function for the second C2 domain of synaptotagmin. Ca2+-triggered dimerization', J Biol Chem, 271 (10), 5844-9. Cheng, H. F., Jiang, M. J., Chen, C. L., Liu, S. M., Wong, L. P., Lomasney, J. W. and King, K. (1995), 'Cloning and identification of amino acid residues of human phospholipase C δ 1 essential for catalysis', J Biol Chem, 270 (10), 5495-505. Cheng, Q. and Yeh, H. H. (2005), 'PLCγ signalling underlies BDNF potentiation of Purkinje cell responses to GABA', J Neurosci Res, 79 (5), 616-27. Cho, W. and Stahelin, R. V. (2006), 'Membrane binding and subcellular targeting of C2 domains', Biochim Biophys Acta, 1761 (8), 838-49. Churchill, G. C., Okada, Y., Thomas, J. M., Genazzani, A. A., Patel, S. and Galione, A. (2002), 'NAADP mobilizes Ca2+ from reserve granules, lysosome-related organelles, in sea urchin eggs', Cell, 111 (5), 703-8. Cocco, L., Faenza, I., Fiume, R., Maria Billi, A., Gilmour, R. S., and Manzoli, F. A. (2006), 'Phosphoinositide-specific phospholipase C (PI-PLC) β1 and nuclear lipid-dependent signalling', Biochim Biophys Acta, 1761 (5-6), 509-21. Cockcroft, S. (2006), 'The latest phospholipase C, PLCη, is implicated in neuronal function', Trends Biochem Sci, 31 (1), 4-7. Cohen, G. B., Ren, R. and Baltimore, D. (1995), 'Modular binding domains in signal transduction proteins', Cell, 80 (2), 237-48. Colegrove, S. L., Albrecht, M. A. and Friel, D. D. (2000), 'Quantitative analysis of mitochondrial Ca2+ uptake and release pathways in sympathetic neurons. Reconstruction of the recovery after depolarization-evoked [Ca2+]i elevations', J Gen Physiol, 115 (3), 371-88. Collo, G., North, R. A., Kawashima, E., Merlo-Pich, E., Neidhart, S., Surprenant, A. and Buell, G. (1996), 'Cloning OF P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gated ion channels', J Neurosci, 16 (8), 2495-507. Cox, D. A., Conforti, L., Sperelakis, N., and Matlib, M. A. (1993), 'Selectivity of inhibition of Na+-Ca2+ exchange of heart mitochondria by benzothiazepine CGP-37157', J Cardiovasc Pharmacol, 21 (4), 595-9. Crompton, M. (1999), 'The mitochondrial permeability transition pore and its role in cell death', Biochem J, 341 (Pt 2), 233-49. Cunha, C., Brambilla, R. and Thomas, K. L. (2010), 'A simple role for BDNF in learning and memory?', Front Mol Neurosci, 3, 1.

190

CHAPTER 6. BIBLIOGRAPHY

D'Angelo, E., Rossi, P., Armano, S. and Taglietti, V. (1999), 'Evidence for NMDA and mGlu receptor-dependent long-term potentiation of mossy fiber-granule cell transmission in rat cerebellum', J Neurophysiol, 81 (1), 277-87. Davletov, B. A. and Sudhof, T. C. (1993), 'A single C2 domain from synaptotagmin I is sufficient for high affinity Ca2+/phospholipid binding', J Biol Chem, 268 (35), 26386-90. Day, R. N., Periasamy, A. and Schaufele, F. (2001), 'Fluorescence resonance energy transfer microscopy of localized protein interactions in the living cell nucleus', Methods, 25 (1), 4-18. del Rio, E., McLaughlin, M., Downes, C. P. and Nicholls, D. G. (1999), 'Differential coupling of G-protein-linked receptors to Ca2+ mobilization through inositol(1,4,5)trisphosphate or ryanodine receptors in cerebellar granule cells in primary culture', Eur J Neurosci, 11 (9), 3015-22. Denton, R. M. and McCormack, J. G. (1980), 'The role of calcium in the regulation of mitochondrial metabolism', Biochem Soc Trans, 8 (3), 266-8. Deterre, P., Bigay, J., Forquet, F., Robert, M. and Chabre, M. (1988), 'cGMP phosphodiesterase of retinal rods is regulated by two inhibitory subunits', Proc Natl Acad Sci U S A, 85 (8), 2424-8. Dode, L., Andersen, J. P., Raeymaekers, L., Missiaen, L., Vilsen, B. and Wuytack, F. (2005), 'Functional comparison between secretory pathway Ca2+/Mn2+- ATPase (SPCA) 1 and sarcoplasmic reticulum Ca2+-ATPase (SERCA) 1 isoforms by steady-state and transient kinetic analyses', J Biol Chem, 280 (47), 39124-34. Dolmetsch, R. E., Xu, K., and Lewis, R. S. (1998), 'Calcium oscillations increase the efficiency and specificity of gene expression', Nature, 392 (6679), 933-6. Drin, G., Douguet, D. and Scarlata, S. (2006), 'The pleckstrin homology domain of phospholipase Cβ transmits enzymatic activation through modulation of the membrane-domain orientation', Biochemistry, 45 (18), 5712-24. Dubyak, G. R. (2004), 'Ion homeostasis, channels, and transporters: an update on cellular mechanisms', Adv Physiol Educ, 28 (1-4), 143-54. Eder, A. and Bading, H. (2007), 'Calcium signals can freely cross the nuclear envelope in hippocampal neurons: somatic calcium increases generate nuclear calcium transients', BMC Neurosci, 8, 57. Edwards, D. C. and Gill, G. N. (1999), 'Structural features of LIM kinase that control effects on the actin cytoskeleton', J Biol Chem, 274 (16), 11352-61. Edwards, D. C., Sanders, L. C., Bokoch, G. M. and Gill, G. N. (1999), 'Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics', Nat Cell Biol, 1 (5), 253-9.

191

CHAPTER 6. BIBLIOGRAPHY

Ehninger, D. and Kempermann, G. (2008), 'Neurogenesis in the adult hippocampus', Cell Tissue Res, 331 (1), 243-50. Eliot, L. S., Dudai, Y., Kandel, E. R. and Abrams, T. W. (1989), 'Ca2+/calmodulin sensitivity may be common to all forms of neural adenylate cyclase', Proc Natl Acad Sci U S A, 86 (23), 9564-8. Ellis, M. V., James, S. R., Perisic, O., Downes, C. P., Williams, R. L. and Katan, M. (1998), 'Catalytic domain of phosphoinositide-specific phospholipase C (PLC). Mutational analysis of residues within the active site and hydrophobic ridge of PLCδ1', J Biol Chem, 273 (19), 11650-9. Endo, M., Ohashi, K. and Mizuno, K. (2007), 'LIM kinase and slingshot are critical for neurite extension', J Biol Chem, 282 (18), 13692-702. Endo, M., Tanaka, M. and Ogawa Y. (1970), 'Calcium induced release of calcium from the sarcoplasmic reticulum of skinned skeletal muscle fibres', Nature, 228, 34-36. Erickson, E. S., Mooren, O. L., Moore-Nichols, D. and Dunn, R. C. (2004), 'Activation of ryanodine receptors in the nuclear envelope alters the conformation of the nuclear pore complex', Biophys Chem, 112 (1), 1-7. Eriksson, P. S., Perfilieva, E., Bjork-Eriksson, T., Alborn, A. M., Nordborg, C., Peterson, D. A. and Gage, F. H. (1998), 'Neurogenesis in the adult human hippocampus', Nat Med, 4 (11), 1313-7. Ertel, E. A., Campbell, K. P., Harpold, M. M., Hofmann, F., Mori, Y., Perez-Reyes, E., Schwartz, A., Snutch, T. P., Tanabe, T., Birnbaumer, L., Tsien, R. W. and Catterall, W. A. (2000), 'Nomenclature of voltage-gated calcium channels', Neuron, 25 (3), 533-5. Essen, L. O., Perisic, O., Cheung, R., Katan, M. and Williams, R. L. (1996), 'Crystal structure of a mammalian phosphoinositide-specific phospholipase C δ', Nature, 380 (6575), 595-602. Essen, L. O., Perisic, O., Katan, M., Wu, Y., Roberts, M. F. and Williams, R. L. (1997b), 'Structural mapping of the catalytic mechanism for a mammalian phosphoinositide-specific phospholipase C', Biochemistry, 36 (7), 1704-18. Essen, L. O., Perisic, O., Lynch, D. E., Katan, M. and Williams, R. L. (1997a), 'A ternary metal binding site in the C2 domain of phosphoinositide-specific phospholipase C-δ1', Biochemistry, 36 (10), 2753-62. Evangelisti, C., Riccio, M., Faenza, I., Zini, N., Hozumi, Y., Goto, K., Cocco, L. and Martelli, A. M. (2006), 'Subnuclear localization and differentiation-dependent increased expression of DGK-ζ in C2C12 mouse myoblasts', J Cell Physiol, 209 (2), 370-8. Faber, E. S. and Sah, P. (2003), 'Calcium-activated potassium channels: multiple contributions to neuronal function', Neuroscientist, 9 (3), 181-94.

192

CHAPTER 6. BIBLIOGRAPHY

Falasca, M., Logan, S. K., Lehto, V. P., Baccante, G., Lemmon, M. A., and Schlessinger, J. (1998), 'Activation of phospholipase C γ by PI 3-kinase- induced PH domain-mediated membrane targeting', EMBO J, 17 (2), 414-22. Fatt, P. and Ginsborg, B.L. (1958), 'The ionic requirements for the production of action potentials in crustacean muscle fibres', J Physiol-London, 142 (3), 516–543. Ferguson, K. M., Lemmon, M. A., Schlessinger, J., and Sigler, P. B. (1995), 'Structure of the high affinity complex of inositol trisphosphate with a phospholipase C pleckstrin homology domain', Cell, 83 (6), 1037-46. Fesenko, E. E., Kolesnikov, S. S., and Lyubarsky, A. L. (1985), 'Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment', Nature, 313 (6000), 310-3. Filomatori, C. V. and Rega, A. F. (2003), 'On the mechanism of activation of the plasma membrane Ca2+-ATPase by ATP and acidic phospholipids', J Biol Chem, 278 (25), 22265-71. Fiume, R., Ramazzotti, G., Teti, G., Chiarini, F., Faenza, I., Mazzotti, G., Billi, A. M. and Cocco, L. (2009), 'Involvement of nuclear PLCβ1 in lamin B1 phosphorylation and G2/M cell cycle progression', FASEB J, 23 (3), 957-66. Flucher, B. E., Andrews, S. B., Fleischer, S., Marks, A. R., Caswell, A. and Powell, J. A. (1993), 'Triad formation: organization and function of the sarcoplasmic reticulum calcium release channel and triadin in normal and dysgenic muscle in vitro', J Cell Biol, 123 (5), 1161-74. Forstermann, U., Pollock, J. S., Schmidt, H. H., Heller, M. and Murad, F. (1991), 'Calmodulin-dependent endothelium-derived relaxing factor/nitric oxide synthase activity is present in the particulate and cytosolic fractions of bovine aortic endothelial cells', Proc Natl Acad Sci U S A, 88 (5), 1788-92. Frieden, M., James, D., Castelbou, C., Danckaert, A., Martinou, J. C. and Demaurex, N. (2004) 'Ca2+ homeostasis during mitochondrial fragmentation and perinuclear clustering induced by hFis1' J Biol Chem, 279 (21), 22704-14. Fujii, M., Yi, K. S., Kim, M. J., Ha, S. H., Ryu, S. H., Suh, P. G. and Yagisawa, H. (2009), 'Phosphorylation of phospholipase C-δ 1 regulates its enzymatic activity', J Cell Biochem, 108 (3), 638-50. Fukami, Kiyoko (2002), 'Structure, Regulation, and Function of Phospholipase C Isozymes', Journal of Biochemistry, 131 (3), 293-99. Fukui, T., Lutz, R. J. and Lowenstein, J. M. (1988), 'Purification of a phospholipase C from rat liver cytosol that acts on phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate', J Biol Chem, 263 (33), 17730-7. Furuichi, T., Yoshikawa, S. and Mikoshiba, K. (1989a), 'Nucleotide sequence of cDNA encoding P400 protein in the mouse cerebellum', Nucleic Acids Res, 17 (13), 5385-6.

193

CHAPTER 6. BIBLIOGRAPHY

Furuichi, T., Yoshikawa, S., Miyawaki, A., Wada, K., Maeda, N. and Mikoshiba, K. (1989b), 'Primary structure and functional expression of the inositol 1,4,5- trisphosphate-binding protein P400', Nature, 342 (6245), 32-8. Gadsby, D. C. (2004), 'Ion transport: spot the difference', Nature, 427 (6977), 795-7. Gartner, A., Polnau, D. G., Staiger, V., Sciarretta, C., Minichiello, L., Thoenen, H., Bonhoeffer, T. and Korte, M. (2006), 'Hippocampal long-term potentiation is supported by presynaptic and postsynaptic tyrosine receptor kinase B- mediated phospholipase Cγ signalling', J Neurosci, 26 (13), 3496-504. Gassama-Diagne, A, Rogalle, P, Fauvel, J, Willson, M, Klaébé, A and Chap, H (1992), 'Substrate specificity of phospholipase B from guinea pig intestine. A glycerol ester lipase with broad specificity', Journal of Biological Chemistry, 267 (19), 13418-24. Gerasimenko, O. V., Gerasimenko, J. V., Tepikin, A. V. and Petersen, O. H. (1995), 'ATP-dependent accumulation and inositol trisphosphate- or cyclic ADP- ribose-mediated release of Ca2+ from the nuclear envelope', Cell, 80 (3), 439- 44. Gerstner, A., Zong, X., Hofmann, F. and Biel, M. (2000), 'Molecular cloning and functional characterization of a new modulatory cyclic nucleotide-gated channel subunit from mouse retina', J Neurosci, 20 (4), 1324-32. Gether, U. (2000), 'Uncovering molecular mechanisms involved in activation of G protein-coupled receptors', Endocr Rev, 21 (1), 90-113. Ghosh, S., Pawelczyk, T. and Lowenstein, J. M. (1997), 'Phospholipase C isoforms δ 1 and δ 3 from human fibroblasts. High-yield expression in Escherichia coli, simple purification, and properties', Protein Expr Purif, 9 (2), 262-78. Gifford, J. L., Walsh, M. P. and Vogel, H. J. (2007), 'Structures and metal-ion- binding properties of the Ca2+-binding helix-loop-helix EF-hand motifs', Biochem J, 405 (2), 199-221. Gilbertson, T. A., Scobey, R. and Wilson, M. (1991), 'Permeation of calcium ions through non-NMDA glutamate channels in retinal bipolar cells', Science, 251 (5001), 1613-5. Gilkey, J. C., Jaffe, L. F., Ridgway, E. B. and Reynolds, G. T. (1978), 'A free calcium wave traverses the activating egg of the medaka, Oryzias latipes', J Cell Biol, 76 (2), 448-66. Gincel, D., Zaid, H. and Shoshan-Barmatz, V. (2001), 'Calcium binding and translocation by the voltage-dependent anion channel: a possible regulatory mechanism in mitochondrial function', Biochem J, 358 (Pt 1), 147-55. Ginsborg, B. L., House, C. R. and Mitchell, M. R. (1980), 'On the role of calcium in the electrical responses of cockroach salivary gland cells to dopamine', J Physiol, 303, 325-35.

194

CHAPTER 6. BIBLIOGRAPHY

Glitsch, M. D., Bakowski, D. and Parekh, A. B. (2002), 'Store-operated Ca2+ entry depends on mitochondrial Ca2+ uptake', EMBO J, 21 (24), 6744-54. Gluzman, Y. (1981), 'SV40-transformed simian cells support the replication of early SV40 mutants', Cell, 23 (1), 175-82. Godi, A., Di Campli, A., Konstantakopoulos, A., Di Tullio, G., Alessi, D. R., Kular, G. S., Daniele, T., Marra, P., Lucocq, J. M. and De Matteis, M. A. (2004), 'FAPPs control Golgi-to-cell-surface membrane traffic by binding to ARF and PtdIns(4)P', Nat Cell Biol, 6 (5), 393-404. Gordienko, D. V. and Bolton, T. B. (2002), 'Crosstalk between ryanodine receptors 2+ and IP3 receptors as a factor shaping spontaneous Ca -release events in rabbit portal vein myocytes', J Physiol, 542 (Pt 3), 743-62. Grabarek, Z. (2005), 'Structure of a trapped intermediate of calmodulin: calcium regulation of EF-hand proteins from a new perspective', J Mol Biol, 346 (5), 1351-66. Grabarek, Z. (2006), 'Structural basis for diversity of the EF-hand calcium-binding proteins', J Mol Biol, 359 (3), 509-25. Gray, A., Olsson, H., Batty, I. H., Priganica, L. and Peter Downes, C. (2003), 'Nonradioactive methods for the assay of phosphoinositide 3-kinases and phosphoinositide phosphatases and selective detection of signalling lipids in cell and tissue extracts', Anal Biochem, 313 (2), 234-45. Griffith, O. H., Volwerk, J. J. and Kuppe, A. (1991), 'Phosphatidylinositol-specific phospholipases C from Bacillus cereus and Bacillus thuringiensis', Methods Enzymol, 197, 493-502. Grobler, J. A., Essen, L. O., Williams, R. L. and Hurley, J. H. (1996), 'C2 domain conformational changes in phospholipase C-δ 1', Nat Struct Biol, 3 (9), 788- 95. Gu, X. and Spitzer, N. C. (1995), 'Distinct aspects of neuronal differentiation encoded by frequency of spontaneous Ca2+ transients', Nature, 375 (6534), 784-7. Guild, S. (1991), 'Effects of adenosine 3':5'-cyclic monophosphate and guanine nucleotides on calcium-evoked ACTH release from electrically permeabilized AtT-20 cells', Br J Pharmacol, 104 (1), 117-22. Guillemette, G., Balla, T., Baukal, A. J. and Catt, K. J. (1987), 'Inositol 1,4,5- trisphosphate binds to a specific receptor and releases microsomal calcium in the anterior pituitary gland', Proc Natl Acad Sci U S A, 84 (23), 8195-9. Gunteski-Hamblin, A. M., Clarke, D. M. and Shull, G. E. (1992), 'Molecular cloning and tissue distribution of alternatively spliced mRNAs encoding possible mammalian homologues of the yeast secretory pathway calcium pump', Biochemistry, 31 (33), 7600-8.

195

CHAPTER 6. BIBLIOGRAPHY

Gurok, U., Steinhoff, C., Lipkowitz, B., Ropers, H. H., Scharff, C. and Nuber, U. A. (2004), 'Gene expression changes in the course of neural progenitor cell differentiation', J Neurosci, 24 (26), 5982-6002. Guthrie, P. B., Segal, M. and Kater, S. B. (1991), 'Independent regulation of calcium revealed by imaging dendritic spines', Nature, 354 (6348), 76-80. Gyorke, I. and Gyorke, S. (1998), 'Regulation of the cardiac ryanodine receptor channel by luminal Ca2+ involves luminal Ca2+ sensing sites', Biophys J, 75 (6), 2801-10. Haak, L. L., Song, L. S., Molinski, T. F., Pessah, I. N., Cheng, H. and Russell, J. T. (2001), 'Sparks and puffs in oligodendrocyte progenitors: cross talk between ryanodine receptors and inositol trisphosphate receptors', J Neurosci, 21 (11), 3860-70. Hajnoczky, G., Hager, R. and Thomas, A. P. (1999), 'Mitochondria suppress local feedback activation of inositol 1,4, 5-trisphosphate receptors by Ca2+', J Biol Chem, 274 (20), 14157-62. Hamada, T., Liou, S. Y., Fukushima, T., Maruyama, T., Watanabe, S., Mikoshiba, K. and Ishida, N. (1999), 'The role of inositol trisphosphate-induced Ca2+ release from IP3-receptor in the rat suprachiasmatic nucleus on circadian entrainment mechanism', Neurosci Lett, 263 (2-3), 125-8. Hamm, H. E. and Gilchrist, A. (1996), 'Heterotrimeric G proteins', Curr Opin Cell Biol, 8 (2), 189-96. Hargreaves, A. C., Lummis, S. C. and Taylor, C. W. (1994), 'Ca2+ permeability of cloned and native 5-hydroxytryptamine type 3 receptors', Mol Pharmacol, 46 (6), 1120-8. Harper, C., Wootton, L., Michelangeli, F., Lefievre, L., Barratt, C. and Publicover, S. (2005), 'Secretory pathway Ca2+-ATPase (SPCA1) Ca2+ pumps, not SERCAs, regulate complex [Ca2+]i signals in human spermatozoa', J Cell Sci, 118 (Pt 8), 1673-85. Hartzell, C., Putzier, I. and Arreola, J. (2005), 'Calcium-activated chloride channels', Annu Rev Physiol, 67, 719-58. Haslam, Richard J., Koide, Hiroshi B. and Hemmings, Brian A. (1993), 'Pleckstrin domain homology', Nature, 363 (6427), 309-10. Hathaway, D. R. and Adelstein, R. S. (1979), 'Human platelet myosin light chain kinase requires the calcium-binding protein calmodulin for activity', Proc Natl Acad Sci U S A, 76 (4), 1653-7. Hattar, S., Kumar, M., Park, A., Tong, P., Tung, J., Yau, K. W. and Berson, D. M. (2006), 'Central projections of melanopsin-expressing retinal ganglion cells in the mouse', J Comp Neurol, 497 (3), 326-49.

196

CHAPTER 6. BIBLIOGRAPHY

He, L. and Lemasters, J. J. (2002), 'Regulated and unregulated mitochondrial permeability transition pores: a new paradigm of pore structure and function?', FEBS Lett, 512 (1-3), 1-7. Hicks, S. N., Jezyk, M. R., Gershburg, S., Seifert, J. P., Harden, T. K. and Sondek, J. (2008), 'General and versatile autoinhibition of PLC isozymes', Mol Cell, 31 (3), 383-94. Hiles, I. D., Otsu, M., Volinia, S., Fry, M. J., Gout, I., Dhand, R., Panayotou, G., Ruiz-Larrea, F., Thompson, A., Totty, N. F. and et al. (1992), 'Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit', Cell, 70 (3), 419-29. Hirano, N., Shibasaki, F., Kato, H., Sakai, R., Tanaka, T., Nishida, J., Yazaki, Y., Takenawa, T. and Hirai, H. (1994), 'Molecular cloning and characterization of a cDNA for bovine phospholipase C-α: proposal of redesignation of phospholipase C-α', Biochem Biophys Res Commun, 204 (1), 375-82. Hirose, K., Kadowaki, S., Tanabe, M., Takeshima, H., and Iino, M. (1999), 'Spatiotemporal dynamics of inositol 1,4,5-trisphosphate that underlies complex Ca2+ mobilization patterns', Science, 284 (5419), 1527-30. Hodge, T. and Colombini, M. (1997), 'Regulation of metabolite flux through voltage- gating of VDAC channels', J Membr Biol, 157 (3), 271-9. Hofmann, S. L. and Majerus, P. W. (1982), 'Identification and properties of two distinct phosphatidylinositol-specific phospholipase C enzymes from sheep seminal vesicular glands', J Biol Chem, 257 (11), 6461-9. Hofmann, T., Obukhov, A. G., Schaefer, M., Harteneck, C., Gudermann, T. and Schultz, G. (1999), 'Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol', Nature, 397 (6716), 259-63. Hohenester, E., Maurer, P., Hohenadl, C., Timpl, R., Jansonius, J. N., and Engel, J. (1996), 'Structure of a novel extracellular Ca2+-binding module in BM-40', Nat Struct Biol, 3 (1), 67-73. Hokin, M. R. and Hokin, L. E. (1953), 'Enzyme secretion and the incorporation of p32 into phospholipides of pancreas slices', Journal of Biological Chemistry, 203 (2), 967-77. Hollenbeck, P. J. and Bray, D. (1987), 'Rapidly transported organelles containing membrane and cytoskeletal components: their relation to axonal growth', J Cell Biol, 105 (6 Pt 1), 2827-35. Homma, Y., Imaki, J., Nakanishi, O. and Takenawa, T. (1988), 'Isolation and characterization of two different forms of inositol phospholipid-specific phospholipase C from rat brain', J Biol Chem, 263 (14), 6592-8. Horch, H. L. and Sargent, P. B. (1995), 'Perisynaptic surface distribution of multiple classes of nicotinic acetylcholine receptors on neurons in the chicken ciliary ganglion', J Neurosci, 15 (12), 7778-95.

197

CHAPTER 6. BIBLIOGRAPHY

Horne, E. A. and Dell'Acqua, M. L. (2007), 'Phospholipase C is required for changes in postsynaptic structure and function associated with NMDA receptor- dependent long-term depression', J Neurosci, 27 (13), 3523-34. Huang, C. Y., Chau, V., Chock, P. B., Wang, J. H. and Sharma, R. K. (1981), 'Mechanism of activation of cyclic nucleotide phosphodiesterase: requirement of the binding of four Ca2+ to calmodulin for activation', Proc Natl Acad Sci U S A, 78 (2), 871-4. Hubbard, S. R., Mohammadi, M. and Schlessinger, J. (1998), 'Autoregulatory mechanisms in protein-tyrosine kinases', J Biol Chem, 273 (20), 11987-90. Humbert, J. P., Matter, N., Artault, J. C., Koppler, P. and Malviya, A. N. (1996), 'Inositol 1,4,5-trisphosphate receptor is located to the inner nuclear membrane vindicating regulation of nuclear calcium signalling by inositol 1,4,5- trisphosphate. Discrete distribution of inositol phosphate receptors to inner and outer nuclear membranes', J Biol Chem, 271 (1), 478-85. Humphries, L. A., Dangelmaier, C., Sommer, K., Kipp, K., Kato, R. M., Griffith, N., Bakman, I., Turk, C. W., Daniel, J. L. and Rawlings, D. J. (2004), 'Tec kinases mediate sustained calcium influx via site-specific tyrosine phosphorylation of the phospholipase Cgamma Src homology 2-Src homology 3 linker', J Biol Chem, 279 (36), 37651-61. Hwang, J. I., Kim, H. S., Lee, J. R., Kim, E., Ryu, S. H. and Suh, P. G. (2005b), 'The interaction of phospholipase C-β3 with Shank2 regulates mGluR-mediated calcium signal', J Biol Chem, 280 (13), 12467-73. Hwang, J. I., Oh, Y. S., Shin, K. J., Kim, H., Ryu, S. H. and Suh, P. G. (2005a), 'Molecular cloning and characterization of a novel phospholipase C, PLC-η', Biochem J, 389 (Pt 1), 181-6. Ichas, F. and Mazat, J. P. (1998), 'From calcium signalling to cell death: two conformations for the mitochondrial permeability transition pore. Switching from low- to high-conductance state', Biochim Biophys Acta, 1366 (1-2), 33- 50. Igwe, O. J. and Filla, M. B. (1995), 'Regulation of phosphatidylinositide transduction system in the rat spinal cord during aging', Neuroscience, 69 (4), 1239-51. Igwe, O. J. and Filla, M. B. (1997), 'Aging-related regulation of myo-inositol 1,4,5- trisphosphate signal transduction pathway in the rat striatum', Brain Res Mol Brain Res, 46 (1-2), 39-53. Iino, M. (1990), 'Biphasic Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca release in smooth muscle cells of the guinea pig taenia caeci', J Gen Physiol, 95 (6), 1103-22. Ikeda, M., Sugiyama, T., Suzuki, K., Moriya, T., Shibata, S., Katsuki, M., Allen, C. N. and Yoshioka, T. (2000), 'PLC β 4-independent Ca2+ rise via muscarinic receptors in the mouse suprachiasmatic nucleus', Neuroreport, 11 (5), 907-12.

198

CHAPTER 6. BIBLIOGRAPHY

Illenberger, D., Walliser, C., Strobel, J., Gutman, O., Niv, H., Gaidzik, V., Kloog, Y., Gierschik, P. and Henis, Y. I. (2003), 'Rac2 regulation of phospholipase C-β 2 activity and mode of membrane interactions in intact cells', J Biol Chem, 278 (10), 8645-52. Imagawa, T., Smith, J. S., Coronado, R. and Campbell, K. P. (1987), 'Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the Ca2+- permeable pore of the calcium release channel', J Biol Chem, 262 (34), 16636-43. Imai, T., Kasai, K., Kurita, J., Fukami, K., Tashiro, M. and Shimotakahara, S. (2007), 'Expression and characterization of a pleckstrin homology domain in phospholipase C, PLC-η1', Protein Expr Purif, 56 (2), 247-52. Ito, M. and Kano, M. (1982), 'Long-lasting depression of parallel fiber-Purkinje cell transmission induced by conjunctive stimulation of parallel fibers and climbing fibers in the cerebellar cortex', Neurosci Lett, 33 (3), 253-8. James, P., Maeda, M., Fischer, R., Verma, A. K., Krebs, J., Penniston, J. T., and Carafoli, E. (1988), 'Identification and primary structure of a calmodulin binding domain of the Ca2+ pump of human erythrocytes', J Biol Chem, 263 (6), 2905-10. Jean-Quartier, C., Bondarenko, A. I., Alam, M. R., Trenker, M., Waldeck- Weiermair, M., Malli, R. and Graier, W. F. (2012), 'Studying mitochondrial Ca2+ uptake - a revisit', Mol Cell Endocrinol, 353 (1-2), 114-27. Jhon, D. Y., Lee, H. H., Park, D., Lee, C. W., Lee, K. H., Yoo, O. J. and Rhee, S. G. (1993), 'Cloning, sequencing, purification, and Gq-dependent activation of phospholipase C-β 3', J Biol Chem, 268 (9), 6654-61. Jia, Y., Subramanian, K. K., Erneux, C., Pouillon, V., Hattori, H., Jo, H., You, J., Zhu, D., Schurmans, S. and Luo, H. R. (2007), 'Inositol 1,3,4,5- tetrakisphosphate negatively regulates phosphatidylinositol-3,4,5- trisphosphate signalling in neutrophils', Immunity, 27 (3), 453-67. Jiang, D., Zhao, L. and Clapham, D. E. (2009), 'Genome-wide RNAi screen identifies Letm1 as a mitochondrial Ca2+/H+ antiporter', Science, 326 (5949), 144-7. Jin, T. G., Satoh, T., Liao, Y., Song, C., Gao, X., Kariya, K., Hu, C. D. and Kataoka, T. (2001), 'Role of the CDC25 homology domain of phospholipase Cε in amplification of Rap1-dependent signalling', J Biol Chem, 276 (32), 30301-7. Jones, D., Morgan, C. and Cockcroft, S. (1999), 'Phospholipase D and membrane traffic. Potential roles in regulated exocytosis, membrane delivery and vesicle budding', Biochim Biophys Acta, 1439 (2), 229-44. Jouaville, L. S., Pinton, P., Bastianutto, C., Rutter, G. A. and Rizzuto, R. (1999), 'Regulation of mitochondrial ATP synthesis by calcium: evidence for a long- term metabolic priming', Proc Natl Acad Sci U S A, 96 (24), 13807-12.

199

CHAPTER 6. BIBLIOGRAPHY

Joung, J. K., Ramm, E. I. and Pabo, C. O. (2000), 'A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions', Proc Natl Acad Sci U S A, 97 (13), 7382-7. Juhaszova, M., Church, P., Blaustein, M. P. and Stanley, E. F. (2000), 'Location of calcium transporters at presynaptic terminals', Eur J Neurosci, 12 (3), 839-46. Kaja, S., van de Ven, R. C., Broos, L. A., Frants, R. R., Ferrari, M. D., van den Maagdenberg, A. M., and Plomp, J. J. (2007), 'Characterization of acetylcholine release and the compensatory contribution of non-Cav2.1 channels at motor nerve terminals of leaner Cav2.1-mutant mice', Neuroscience, 144 (4), 1278-87. Kaneko, N. and Sawamoto, K. (2009), 'Adult neurogenesis and its alteration under pathological conditions', Neurosci Res, 63 (3), 155-64. Kanemaru, K., Nakahara, M., Nakamura, Y., Hashiguchi, Y., Kouchi, Z., Yamaguchi, H., Oshima, N., Kiyonari, H. and Fukami, K. (2010), 'Phospholipase C-η2 is highly expressed in the habenula and retina', Gene Expr Patterns, 10 (2-3), 119-26. Karten, Y. J., Olariu, A. and Cameron, H. A. (2005), 'Stress in early life inhibits neurogenesis in adulthood', Trends Neurosci, 28 (4), 171-2. Katan, M., Kriz, R. W., Totty, N., Philp, R., Meldrum, E., Aldape, R. A., Knopf, J. and Parker, P. J. (1988), 'Primary structure of PLC-154', Cold Spring Harb Symp Quant Biol, 53 Pt 2, 921-6. Katan, M. and Parker, P. J. (1987), 'Purification of phosphoinositide-specific phospholipase C from a particulate fraction of bovine brain', Eur J Biochem, 168 (2), 413-8. Kavalali, E. T., Hwang, K. S. and Plummer, M. R. (1997), 'cAMP-dependent enhancement of dihydropyridine-sensitive calcium channel availability in hippocampal neurons', J Neurosci, 17 (14), 5334-48. Kennerly, D. A., Sullivan, T. J., Sylwester, P. and Parker, C. W. (1979), 'Diacylglycerol metabolism in mast cells: a potential role in membrane fusion and arachidonic acid release', J Exp Med, 150 (4), 1039-44. Kent, C. (1995), 'Eukaryotic phospholipid biosynthesis', Annu Rev Biochem, 64, 315- 43. Khodosevich, K. and Monyer, H. (2010), 'Signalling involved in neurite outgrowth of postnatally born subventricular zone neurons in vitro', BMC Neurosci, 11, 18. Kim, C. G., Park, D. and Rhee, S. G. (1996), 'The role of carboxyl-terminal basic amino acids in Gqα-dependent activation, particulate association, and nuclear localization of phospholipase C-β1', J Biol Chem, 271 (35), 21187-92.

200

CHAPTER 6. BIBLIOGRAPHY

Kim, D. H., Ohnishi, S. T. and Ikemoto, N. (1983), 'Kinetic studies of calcium release from sarcoplasmic reticulum in vitro', J Biol Chem, 258 (16), 9662-8. Kim, J. K., Choi, J. W., Lim, S., Kwon, O., Seo, J. K., Ryu, S. H. and Suh, P. G. (2011), 'Phospholipase C-η1 is activated by intracellular Ca2+ mobilization and enhances GPCRs/PLC/Ca2+ signalling', Cell Signal, 23 (6), 1022-9. Kirischuk, S., Pronchuk, N. and Verkhratsky, A. (1992), 'Measurements of intracellular calcium in sensory neurons of adult and old rats', Neuroscience, 50 (4), 947-51. Klebe, R. J. and Ruddle, F. H. (1969) 'Neuroblastoma: Cell culture analysis of a differentiating stem cell system', J Cell Biol,43, 69A (abstract). Koch, G. L. (1990), 'The endoplasmic reticulum and calcium storage', Bioessays, 12 (11), 527-31. Koch, K. W., Duda, T. and Sharma, R. K. (2002), 'Photoreceptor specific guanylate cyclases in vertebrate phototransduction', Mol Cell Biochem, 230 (1-2), 97- 106. Koenig, E., Kinsman, S., Repasky, E. and Sultz, L. (1985), 'Rapid mobility of motile varicosities and inclusions containing alpha-spectrin, actin, and calmodulin in regenerating axons in vitro', J Neurosci, 5 (3), 715-29. Kohda, D., Hatanaka, H., Odaka, M., Mandiyan, V., Ullrich, A., Schlessinger, J. and Inagaki, F. (1993), 'Solution structure of the SH3 domain of phospholipase C- γ', Cell, 72 (6), 953-60. Kordel, J., Skelton, N. J., Akke, M. and Chazin, W. J. (1993), 'High-resolution structure of calcium-loaded calbindin D9k', J Mol Biol, 231 (3), 711-34. Kort, A. A., Capogrossi, M. C. and Lakatta, E. G. (1985), 'Frequency, amplitude, and propagation velocity of spontaneous Ca++-dependent contractile waves in intact adult rat cardiac muscle and isolated myocytes', Circ Res, 57 (6), 844- 55. Kouchi, Z., Shikano, T., Nakamura, Y., Shirakawa, H., Fukami, K. and Miyazaki, S. (2005), 'The role of EF-hand domains and C2 domain in regulation of enzymatic activity of phospholipase Cζ', J Biol Chem, 280 (22), 21015-21. Kouchi, Z., Igarashi, T., Shibayama, N., Inanobe, S., Sakurai, K., Yamaguchi, H., Fukuda, T., Yanagi, S., Nakamura, Y. and Fukami, K. (2011), 'Phospholipase Cδ3 regulates RhoA/Rho kinase signalling and neurite outgrowth', J Biol Chem, 286 (10), 8459-71. Kretsinger, R. H. and Nockolds, C. E. (1973), 'Carp muscle calcium-binding protein. II. Structure determination and general description', J Biol Chem, 248 (9), 3313-26. Kurose, H. (2003), 'Gα12 and Gα13 as key regulatory mediator in signal transduction', Life Sci, 74 (2-3), 155-61.

201

CHAPTER 6. BIBLIOGRAPHY

Kwon, I. S., Lee, K. H., Choi, J. W. and Ahn, J. Y. (2010), 'PI(3,4,5)P3 regulates the interaction between Akt and B23 in the nucleus', BMB Rep, 43 (2), 127-32. Lai, F. A., Erickson, H. P., Rousseau, E., Liu, Q. Y. and Meissner, G. (1988), 'Purification and reconstitution of the calcium release channel from skeletal muscle', Nature, 331 (6154), 315-9. Lally, G., Faull, R. L., Waldvogel, H. J., Ferrari, S. and Emson, P. C. (1997), 'Calcium homeostasis in ageing: studies on the calcium binding protein calbindin D28K', J Neural Transm, 104 (10), 1107-12. Lane, M. A. and Bailey, S. J. (2005), 'Role of retinoid signalling in the adult brain', Prog Neurobiol, 75 (4), 275-93. Lee, S. B. and Rhee, S. G. (1996), 'Molecular cloning, splice variants, expression, and purification of phospholipase C-δ 4', J Biol Chem, 271 (1), 25-31. Lee, W. K., Kim, J. K., Seo, M. S., Cha, J. H., Lee, K. J., Rha, H. K., Min, D. S., Jo, Y. H. and Lee, K. H. (1999), 'Molecular cloning and expression analysis of a mouse phospholipase C-δ1', Biochem Biophys Res Commun, 261 (2), 393-9. Lein, E. S. Hawrylycz, M. J. Ao, N. Ayres, M. Bensinger, A. Bernard, A. Boe, A. F. Boguski, M. S. Brockway, K. S. Byrnes and et al. (2007), 'Genome-wide atlas of gene expression in the adult mouse brain', Nature, 445 (7124), 168-76. Lewit-Bentley, A. and Rety, S. (2000), 'EF-hand calcium-binding proteins', Curr Opin Struct Biol, 10 (6), 637-43. Li, H. S., Xu, X. Z. and Montell, C. (1999), 'Activation of a TRPC3-dependent cation current through the neurotrophin BDNF', Neuron, 24 (1), 261-73. Li, X., Rainnie, D. G., McCarley, R. W. and Greene, R. W. (1998), 'Presynaptic nicotinic receptors facilitate monoaminergic transmission', J Neurosci, 18 (5), 1904-12. Liou, J., Kim, M. L., Heo, W. D., Jones, J. T., Myers, J. W., Ferrell, J. E., Jr. and Meyer, T. (2005), 'STIM is a Ca2+ sensor essential for Ca2+-store-depletion- triggered Ca2+ influx', Curr Biol, 15 (13), 1235-41. Liscovitch, M., Czarny, M., Fiucci, G. and Tang, X. (2000), 'Phospholipase D: molecular and cell biology of a novel gene family', Biochem J, 345 Pt 3, 401- 15. Liu, J., Blin, N., Conklin, B. R. and Wess, J. (1996), 'Molecular mechanisms involved in muscarinic acetylcholine receptor-mediated G protein activation studied by insertion mutagenesis', J Biol Chem, 271 (11), 6172-8. Livak, K. J. and Schmittgen, T. D. (2001), 'Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT Method', Methods, 25 (4), 402-8.

202

CHAPTER 6. BIBLIOGRAPHY

Lodish, H. F., Kong, N. and Wikstrom, L. (1992), 'Calcium is required for folding of newly made subunits of the asialoglycoprotein receptor within the endoplasmic reticulum', J Biol Chem, 267 (18), 12753-60. Lomasney, J. W., Cheng, H. F., Wang, L. P., Kuan, Y., Liu, S., Fesik, S. W. and King, K. (1996), 'Phosphatidylinositol 4,5-bisphosphate binding to the pleckstrin homology domain of phospholipase C-δ1 enhances enzyme activity', J Biol Chem, 271 (41), 25316-26. Lopez, I., Mak, E. C., Ding, J., Hamm, H. E. and Lomasney, J. W. (2001), 'A novel bifunctional phospholipase c that is regulated by Gα 12 and stimulates the Ras/mitogen-activated protein kinase pathway', J Biol Chem, 276 (4), 2758- 65. Lopez, J. J., Camello-Almaraz, C., Pariente, J. A., Salido, G. M. and Rosado, J. A. (2005) 'Ca2+ accumulation into acidic organelles mediated by Ca2+- and vacuolar H+-ATPases in human platelets', Biochem J,;390, 243–252. Lucas, N. and Cho, W. (2011), 'Phosphatidylserine binding is essential for plasma membrane recruitment and signalling function of 3-phosphoinositide- dependent kinase-1', J Biol Chem, 286 (48), 41265-72. Lucocq, J. (2008), 'Quantification of structures and gold labeling in transmission electron microscopy', Methods Cell Biol, 88, 59-82. Lyon, A. M., Tesmer, V. M., Dhamsania, V. D., Thal, D. M., Gutierrez, J., Chowdhury, S., Suddala, K. C., Northup, J. K. and Tesmer, J. J. (2011), 'An autoinhibitory helix in the C-terminal region of phospholipase C-β mediates Galphaq activation', Nat Struct Mol Biol, 18 (9), 999-1005. Macho, A., Decaudin, D., Castedo, M., Hirsch, T., Susin, S. A., Zamzami, N. and Kroemer, G. (1996), 'Chloromethyl-X-Rosamine is an aldehyde-fixable potential-sensitive fluorochrome for the detection of early apoptosis', Cytometry, 25 (4), 333-40. Mahapatra, N. R., Mahata, M., Hazra, P. P., McDonough, P. M., O’Connor, D. T. and Mahata, S. K. (2004) 'A dynamic pool of calcium in catecholamine storage vesicles. Exploration in living cells by a novel vesicle-targeted chromogranin A-aequorin chimeric photoprotein', J Biol Chem, 279 (49), 51107–51121. Majumder, P., Trujillo, C. A., Lopes, C. G., Resende, R. R., Gomes, K. N., Yuahasi, K. K., Britto, L. R. and Ulrich, H. (2007), 'New insights into purinergic receptor signalling in neuronal differentiation, neuroprotection, and brain disorders', Purinergic Signal, 3 (4), 317-31. Malhas, A., Goulbourne, C. and Vaux, D. J. (2011), 'The nucleoplasmic reticulum: form and function', Trends Cell Biol, 21 (6), 362-73.

203

CHAPTER 6. BIBLIOGRAPHY

Malmendal, A., Linse, S., Evenas, J., Forsen, S. and Drakenberg, T. (1999), 'Battle for the EF-hands: magnesium-calcium interference in calmodulin', Biochemistry, 38 (36), 11844-50. Mandal, M. and Yan, Z. (2009), 'Phosphatidylinositol (4,5)-bisphosphate regulation of N-methyl-D-aspartate receptor channels in cortical neurons', Mol Pharmacol, 76 (6), 1349-59. Manetti, F. (2011), 'LIM kinases are attractive targets with many macromolecular partners and only a few small molecule regulators', Med Res Rev. Manning, E. E., Ransome, M. I., Burrows, E. L. and Hannan, A. J. (2012), 'Increased adult hippocampal neurogenesis and abnormal migration of adult-born granule neurons is associated with hippocampal-specific cognitive deficits in phospholipase C-β1 knockout mice', Hippocampus, 22 (2), 309-19. Martelli, A. M., Fiume, R., Faenza, I., Tabellini, G., Evangelista, C., Bortul, R., Follo, M. Y., Fala, F. and Cocco, L. (2005), 'Nuclear phosphoinositide specific phospholipase C (PI-PLC)-β 1: a central intermediary in nuclear lipid-dependent signal transduction', Histol Histopathol, 20 (4), 1251-60. Martelli, A. M., Ognibene, A., Buontempo, F., Fini, M., Bressanin, D., Goto, K., McCubrey, J. A., Cocco, L. and Evangelisti, C. (2011), 'Nuclear phosphoinositides and their roles in cell biology and disease', Crit Rev Biochem Mol Biol, 46 (5), 436-57. Marty, I., Robert, M., Villaz, M., De Jongh, K., Lai, Y., Catterall, W. A. and Ronjat, M. (1994), 'Biochemical evidence for a complex involving dihydropyridine receptor and ryanodine receptor in triad junctions of skeletal muscle', Proc Natl Acad Sci U S A, 91 (6), 2270-4. Matsuyama, S., Matsumoto, A., Enomoto, T. and Nishizaki, T. (2000), 'Activation of nicotinic acetylcholine receptors induces long-term potentiation in vivo in the intact mouse dentate gyrus', Eur J Neurosci, 12 (10), 3741-7. Matthews, R. P., Guthrie, C. R., Wailes, L. M., Zhao, X., Means, A. R. and McKnight, G. S. (1994), 'Calcium/calmodulin-dependent protein kinase types II and IV differentially regulate CREB-dependent gene expression', Mol Cell Biol, 14 (9), 6107-16. Mayer, M. L. and Westbrook, G. L. (1987), 'Permeation and block of N-methyl-D- aspartic acid receptor channels by divalent cations in mouse cultured central neurones', J Physiol, 394, 501-27. Mazzotti, G., Zini, N., Rizzi, E., Rizzoli, R., Galanzi, A., Ognibene, A., Santi, S., Matteucci, A., Martelli, A. M. and Maraldi, N. M. (1995), 'Immunocytochemical detection of phosphatidylinositol 4,5-bisphosphate localization sites within the nucleus', J Histochem Cytochem, 43 (2), 181-91. McCullar, J. S., Larsen, S. A., Millimaki, R. A. and Filtz, T. M. (2003), 'Calmodulin is a phospholipase C-β interacting protein', J Biol Chem, 278 (36), 33708-13.

204

CHAPTER 6. BIBLIOGRAPHY

McCullar, J. S., Malencik, D. A., Vogel, W. K., Crofoot, K. M., Anderson, S. R. and Filtz, T. M. (2007), 'Calmodulin potentiates Gβγ activation of phospholipase C-β3', Biochem Pharmacol, 73 (2), 270-8. McPherson, P. S., Kim, Y. K., Valdivia, H., Knudson, C. M., Takekura, H., Franzini- Armstrong, C., Coronado, R. and Campbell, K. P. (1991), 'The brain ryanodine receptor: a caffeine-sensitive calcium release channel', Neuron, 7 (1), 17-25. Meier, R. and Hemmings, B. A. (1999), 'Regulation of protein kinase B', J Recept Signal Transduct Res, 19 (1-4), 121-8. Meissner, G., Darling, E. and Eveleth, J. (1986), 'Kinetics of rapid Ca2+ release by sarcoplasmic reticulum. Effects of Ca2+, Mg2+, and adenine nucleotides', Biochemistry, 25 (1), 236-44. Meissner, G. and Lu, X. (1995), 'Dihydropyridine receptor-ryanodine receptor interactions in skeletal muscle excitation-contraction coupling', Biosci Rep, 15 (5), 399-408.

Menager, C., Arimura, N., Fukata, Y. and Kaibuchi, K. (2004), 'PIP3 is involved in neuronal polarization and axon formation', J Neurochem, 89 (1), 109-18. Merritt, J. E., Armstrong, W. P., Benham, C. D., Hallam, T. J., Jacob, R., Jaxa- Chamiec, A., Leigh, B. K., McCarthy, S. A., Moores, K. E. and Rink, T. J. (1990), 'SK&F 96365, a novel inhibitor of receptor-mediated calcium entry', Biochem J, 271 (2), 515-22. Mery, L., Mesaeli, N., Michalak, M., Opas, M., Lew, D. P. and Krause, K. H. (1996), 'Overexpression of calreticulin increases intracellular Ca2+ storage and decreases store-operated Ca2+ influx', J Biol Chem, 271 (16), 9332-9.

Mignen, O. and Shuttleworth, T. J. (2000), 'IARC, a novel arachidonate-regulated, noncapacitative Ca2+ entry channel', J Biol Chem, 275 (13), 9114-9. Mignen, O., Thompson, J. L. and Shuttleworth, T. J. (2009), 'The molecular architecture of the arachidonate-regulated Ca2+-selective ARC channel is a pentameric assembly of Orai1 and Orai3 subunits', J Physiol, 587 (Pt 17), 4181-97. Mignen, O., Thompson, J. L., Yule, D. I. and Shuttleworth, T. J. (2005), 'Agonist activation of arachidonate-regulated Ca2+-selective ARC channels in murine parotid and pancreatic acinar cells', J Physiol, 564 (Pt 3), 791-801. Miledi, R. (1973), 'Transmitter release induced by injection of calcium ions into nerve terminals', Proc R Soc Lond B Biol Sci, 183 (73), 421-5. Millhouse, O. E. (1987), 'Granule cells of the olfactory tubercle and the question of the islands of Calleja', J Comp Neurol, 265 (1), 1-24. Mitchell, K. J., Lai, F. A. and Rutter, G. A. (2003), 'Ryanodine receptor type I and nicotinic acid adenine dinucleotide phosphate receptors mediate Ca2+ release

205

CHAPTER 6. BIBLIOGRAPHY

from insulin-containing vesicles in living pancreatic β-cells (MIN6)', J Biol Chem, 278 (13), 11057-64. Mitchell, K. J., Pinton, P., Varadi, A., Tacchetti, C., Ainscow, E. K., Pozzan, T., Rizzuto, R. and Rutter, G. A. (2001), 'Dense core secretory vesicles revealed as a dynamic Ca2+ store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera', J Cell Biol, 155 (1), 41-51. Miyawaki, A., Furuichi, T., Maeda, N. and Mikoshiba, K. (1990), 'Expressed cerebellar-type inositol 1,4,5-trisphosphate receptor, P400, has calcium release activity in a fibroblast L cell line', Neuron, 5 (1), 11-8. Montero, M., Alonso, M. T., Carnicero, E., Cuchillo-Ibanez, I., Albillos, A., Garcia, A. G., Garcia-Sancho, J. and Alvarez, J. (2000), 'Chromaffin-cell stimulation triggers fast millimolar mitochondrial Ca2+ transients that modulate secretion', Nat Cell Biol, 2 (2), 57-61. Montminy, M. R. and Bilezikjian, L. M. (1987), 'Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene', Nature, 328 (6126), 175-8. Moore, R. Y., Heller, A., Wurtman, R. J. and Axelrod, J. (1967), 'Visual pathway mediating pineal response to environmental light', Science, 155 (3759), 220- 3. Moreau, B., Nelson, C. and Parekh, A. B. (2006), 'Biphasic regulation of mitochondrial Ca2+ uptake by cytosolic Ca2+ concentration', Curr Biol, 16 (16), 1672-7. Morris, A. J. and Malbon, C. C. (1999), 'Physiological regulation of G protein-linked signalling', Physiol Rev, 79 (4), 1373-430. Mundy, W., Tandon, P., Ali, S. and Tilson, H. (1991), 'Age-related changes in receptor-mediated phosphoinositide hydrolysis in various regions of rat brain', Life Sci, 49 (14), PL97-102. Muntener, M., Kaser, L., Weber, J. and Berchtold, M. W. (1995), 'Increase of skeletal muscle relaxation speed by direct injection of parvalbumin cDNA', Proc Natl Acad Sci U S A, 92 (14), 6504-8. Murakami, M. and Kudo, I. (2002), 'Phospholipase A2', J Biochem, 131 (3), 285-92. Murga, C., Laguinge, L., Wetzker, R., Cuadrado, A. and Gutkind, J. S. (1998), 'Activation of Akt/protein kinase B by G protein-coupled receptors. A role for α and β γ subunits of heterotrimeric G proteins acting through phosphatidylinositol-3-OH kinasegamma', J Biol Chem, 273 (30), 19080-5. Murphy, S. N. and Miller, R. J. (1989), 'Regulation of Ca++ influx into striatal neurons by kainic acid', J Pharmacol Exp Ther, 249 (1), 184-93.

206

CHAPTER 6. BIBLIOGRAPHY

Murthy, S. N., Lomasney, J. W., Mak, E. C. and Lorand, L. (1999), 'Interactions of Gh/transglutaminase with phospholipase Cδ1 and with GTP', Proc Natl Acad Sci U S A, 96 (21), 11815-9. Nadella, K. S., Saji, M., Jacob, N. K., Pavel, E., Ringel, M. D. and Kirschner, L. S. (2009), 'Regulation of actin function by protein kinase A-mediated phosphorylation of Limk1', EMBO Rep, 10 (6), 599-605. Nakahara, M., Shimozawa, M., Nakamura, Y., Irino, Y., Morita, M., Kudo, Y. and Fukami, K. (2005), 'A novel phospholipase C, PLCη2, is a neuron-specific isozyme', J Biol Chem, 280 (32), 29128-34. Nakanishi, S. (1992), 'Molecular diversity of glutamate receptors and implications for brain function', Science, 258 (5082), 597-603. Nakashima, S., Banno, Y., Watanabe, T., Nakamura, Y., Mizutani, T., Sakai, H., Zhao, Y., Sugimoto, Y. and Nozawa, Y. (1995), 'Deletion and site-directed mutagenesis of EF-hand domain of phospholipase C-δ 1: effects on its activity', Biochem Biophys Res Commun, 211 (2), 365-9. Nemoto, W., Fukui, K. and Toh, H. (2011), 'GRIPDB - G protein coupled Receptor Interaction Partners DataBase', J Recept Signal Transduct Res, 31 (3), 199- 205. Neves, S. R., Ram, P. T. and Iyengar, R. (2002), 'G protein pathways', Science, 296 (5573), 1636-9. Nguyen Tle, X., Ye, K., Cho, S. W. and Ahn, J. Y. (2007), 'Overexpression of phospholipase C-γ1 inhibits NGF-induced neuronal differentiation by proliferative activity of SH3 domain', Int J Biochem Cell Biol, 39 (11), 2083- 92. Nishizuka, Y. (1995), 'Protein kinase C and lipid signalling for sustained cellular responses', FASEB J, 9 (7), 484-96. Nieuwenhuys, R., Voogd, J. and van Huijzen, C. (2008), 'The human central nervous system' (4th edn.: New York: Springer) 967. Nomikos, M., Blayney, L. M., Larman, M. G., Campbell, K., Rossbach, A., Saunders, C. M., Swann, K. and Lai, F. A. (2005), 'Role of phospholipase C-ζ domains in Ca2+-dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca2+ oscillations', J Biol Chem, 280 (35), 31011-8. Nomikos, M., Elgmati, K., Theodoridou, M., Calver, B. L., Nounesis, G., Swann, K. and Lai, F. A. (2011a), 'Phospholipase Cζ binding to PtdIns(4,5)P2 requires the XY-linker region', J Cell Sci, 124 (Pt 15), 2582-90. Nomikos, M., Elgmati, K., Theodoridou, M., Georgilis, A., Gonzalez-Garcia, J. R., Nounesis, G., Swann, K. and Lai, F. A. (2011b), 'Novel regulation of PLCζ activity via its XY-linker', Biochem J, 438 (3), 427-32.

207

CHAPTER 6. BIBLIOGRAPHY

North, R. A. (2004), 'P2X3 receptors and peripheral pain mechanisms', J Physiol, 554 (Pt 2), 301-8. Offermanns, S. and Simon, M. I. (1995), 'G α 15 and G α 16 couple a wide variety of receptors to phospholipase C', J Biol Chem, 270 (25), 15175-80. Ogawa, Y. (1985), 'Calcium binding to troponin C and troponin: effects of Mg2+, ionic strength and pH', J Biochem, 97 (4), 1011-23. Ogunbayo, O. A., Zhu, Y., Rossi, D., Sorrentino, V., Ma, J., Zhu, M. X. and Evans, A. M. (2011), 'Cyclic adenosine diphosphate ribose activates ryanodine receptors, whereas NAADP activates two-pore domain channels', J Biol Chem, 286 (11), 9136-40. Ohashi, K., Nagata, K., Maekawa, M., Ishizaki, T., Narumiya, S. and Mizuno, K. (2000), 'Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop', J Biol Chem, 275 (5), 3577-82. Oksche, A. (1965), 'Survey of the Development and Comparative Morphology of the Pineal Organ', Prog Brain Res, 10, 3-29. Ozdener, F., Dangelmaier, C., Ashby, B., Kunapuli, S. P. and Daniel, J. L. (2002), 'Activation of phospholipase Cγ2 by tyrosine phosphorylation', Mol Pharmacol, 62 (3), 672-9. Palty, R., Silverman, W. F., Hershfinkel, M., Caporale, T., Sensi, S. L., Parnis, J., Nolte, C., Fishman, D., Shoshan-Barmatz, V., Herrmann, S., Khananshvili, D. and Sekler, I. (2010), 'NCLX is an essential component of mitochondrial Na+/Ca2+ exchange', Proc Natl Acad Sci U S A, 107 (1), 436-41. Pamonsinlapatham, P., Hadj-Slimane, R., Lepelletier, Y., Allain, B., Toccafondi, M., Garbay, C. and Raynaud, F. (2009), 'p120-Ras GTPase activating protein (RasGAP): a multi-interacting protein in downstream signalling', Biochimie, 91 (3), 320-8. Pannese, E. (1994), 'Neurocytology: Fine Structure of Neurons, Nerve Processes and Neuroglial Cells' (1th edn.: New York and Stuttgart: Georg Thieme) 264. Parham, J. H., Iannone, M. A., Overton, L. K. and Hutchins, J. T. (1998), 'Optimization of transient gene expression in mammalian cells and potential for scale-up using flow electroporation', Cytotechnology, 28 (1-3), 147-55. Park, D., Jhon, D. Y., Lee, C. W., Lee, K. H. and Rhee, S. G. (1993), 'Activation of phospholipase C isozymes by G protein β γ subunits', J Biol Chem, 268 (7), 4573-6. Patel, S. and Docampo, R. (2010), 'Acidic calcium stores open for business: expanding the potential for intracellular Ca2+ signalling', Trends Cell Biol, 20 (5), 277-86.

208

CHAPTER 6. BIBLIOGRAPHY

Paulus, H. and Kennedy, E. P. (1960), 'The enzymatic synthesis of inositol monophosphatide', J Biol Chem, 235, 1303-11. Perez-Chacon, G., Astudillo, A. M., Balgoma, D., Balboa, M. A. and Balsinde, J. (2009), 'Control of free arachidonic acid levels by phospholipases A2 and lysophospholipid acyltransferases', Biochim Biophys Acta, 1791 (12), 1103- 13. Perez-Reyes, E. (2003), 'Molecular physiology of low-voltage-activated t-type calcium channels', Physiol Rev, 83 (1), 117-61. Perez-Reyes, E., Cribbs, L. L., Daud, A., Lacerda, A. E., Barclay, J., Williamson, M. P., Fox, M., Rees, M. and Lee, J. H. (1998), 'Molecular characterization of a neuronal low-voltage-activated T-type calcium channel', Nature, 391 (6670), 896-900. Perin, M. S., Fried, V. A., Mignery, G. A., Jahn, R. and Sudhof, T. C. (1990), 'Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C', Nature, 345 (6272), 260-3. Perisic, O., Fong, S., Lynch, D. E., Bycroft, M. and Williams, R. L. (1998), 'Crystal structure of a calcium-phospholipid binding domain from cytosolic phospholipase A2', J Biol Chem, 273 (3), 1596-604. Perocchi, F., Gohil, V. M., Girgis, H. S., Bao, X. R., McCombs, J. E., Palmer, A. E. and Mootha, V. K. (2010), 'MICU1 encodes a mitochondrial EF hand protein required for Ca2+ uptake', Nature, 467 (7313), 291-6. Pieske, B., Maier, L. S., Piacentino, V. 3rd, Weisser, J., Hasenfuss, G. and Houser, S. (2002) 'Rate dependence of [Na+]i and contractility in nonfailing and failing human myocardium', Circulation, 106 (4), 447-53. Pignatelli, M., Cortes-Canteli, M., Santos, A. and Perez-Castillo, A. (1999), 'Involvement of the NGFI-A gene in the differentiation of neuroblastoma cells', FEBS Lett, 461 (1-2), 37-42. Pinton, P., Pozzan, T. and Rizzuto, R. (1998), 'The Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca2+ store, with functional properties distinct from those of the endoplasmic reticulum', EMBO J, 17 (18), 5298-308. Pizzo, P., Lissandron, V. and Pozzan, T. (2010), 'The trans-golgi compartment: A new distinct intracellular Ca store', Commun Integr Biol, 3 (5), 462-4. Poon, I. K. and Jans, D. A. (2005), 'Regulation of nuclear transport: central role in development and transformation?', Traffic, 6 (3), 173-86. Popovics, P., Beswick, W., Guild, S. B., Cramb, G., Morgan, K., Millar, R. P. and Stewart, A. J. (2011), 'Phospholipase C-η2 is activated by elevated intracellular Ca2+ levels', Cell Signal, 23 (11), 1777-84. Popovics, P. and Stewart, A. J. (2012a), 'Phospholipase C-η Activity May Contribute to Alzheimer's Disease-Associated Calciumopathy', J Alzheimers Dis.

209

CHAPTER 6. BIBLIOGRAPHY

Popovics, P. and Stewart, A. J. (2012b), 'Putative roles for phospholipase Cη enzymes in neuronal Ca2+ signal modulation', Biochem Soc Trans, 40 (1), 282-6. Portzehl, H., Caldwell, P. C. and Rueegg, J. C. (1964), 'The Dependence of Contraction and Relaxation of Muscle Fibres from the Crab Maia Squinado on the Internal Concentration of Free Calcium Ions', Biochim Biophys Acta, 79, 581-91. Poulin, B., Sekiya, F. and Rhee, S. G. (2005), 'Intramolecular interaction between phosphorylated tyrosine-783 and the C-terminal Src homology 2 domain activates phospholipase C-γ1', Proc Natl Acad Sci U S A, 102 (12), 4276-81. Powis, G., Bonjouklian, R., Berggren, M. M., Gallegos, A., Abraham, R., Ashendel, C., Zalkow, L., Matter, W. F., Dodge, J., Grindey, G. and et al. (1994), 'Wortmannin, a potent and selective inhibitor of phosphatidylinositol-3- kinase', Cancer Res, 54 (9), 2419-23. Prakriya, M., Feske, S., Gwack, Y., Srikanth, S., Rao, A. and Hogan, P. G. (2006), 'Orai1 is an essential pore subunit of the CRAC channel', Nature, 443 (7108), 230-3. Putney, J. W., Jr. (1982), 'Inositol lipids and cell stimulation in mammalian salivary gland', Cell Calcium, 3 (4-5), 369-83. Putney, J. W., Jr. (1986), 'A model for receptor-regulated calcium entry', Cell Calcium, 7 (1), 1-12. Rae, M. G., Hilton, J. and Sharkey J.(2012) 'Putative TRP channel antagonists, SKF 96365, flufenamic acid and 2-APB, are non-competitive antagonists at recombinant human α1β2γ2 GABA(A) receptors' Neurochem Int, 60(6), 543- 54. Ramazzotti, G., Faenza, I., Gaboardi, G. C., Piazzi, M., Bavelloni, A., Fiume, R., Manzoli, L., Martelli, A. M. and Cocco, L. (2008), 'Catalytic activity of nuclear PLC-β1 is required for its signalling function during C2C12 differentiation', Cell Signal, 20 (11), 2013-21. Rao, V. R. and Finkbeiner, S. (2007), 'NMDA and AMPA receptors: old channels, new tricks', Trends Neurosci, 30 (6), 284-91. Rebecchi, M. J. and Pentyala, S. N. (2000), 'Structure, function, and control of phosphoinositide-specific phospholipase C', Physiol Rev, 80 (4), 1291-335. Rebecchi, M. J. and Rosen, O. M. (1987), 'Purification of a phosphoinositide-specific phospholipase C from bovine brain', J Biol Chem, 262 (26), 12526-32. Rhee, S. G. and Choi, K. D. (1992), 'Regulation of inositol phospholipid-specific phospholipase C isozymes', J Biol Chem, 267 (18), 12393-6. Rhee, S. G., Suh, P. G., Ryu, S. H. and Lee, S. Y. (1989), 'Studies of inositol phospholipid-specific phospholipase C', Science, 244 (4904), 546-50.

210

CHAPTER 6. BIBLIOGRAPHY

Ringer, S. (1882), 'Concerning the influence exerted by each of the constituents of the blood on the contraction of the ventricle', J Physiol, 3 (5-6), 380-93. Rizzuto, R. and Pozzan, T. (2006), 'Microdomains of intracellular Ca2+: molecular determinants and functional consequences', Physiol Rev, 86 (1), 369-408. Rizzuto, R., Pinton, P., Carrington, W., Fay, F. S., Fogarty, K. E., Lifshitz, L. M., Tuft, R. A. and Pozzan, T. (1998) 'Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses', Science, 280 (5370), 1763-6. Rizzuto, R., Simpson, A. W., Brini, M. and Pozzan, T. (1992), 'Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin', Nature, 358 (6384), 325-7. Robb-Gaspers, L. D., Rutter, G. A., Burnett, P., Hajnoczky, G., Denton, R. M. and Thomas, A. P. (1998), 'Coupling between cytosolic and mitochondrial calcium oscillations: role in the regulation of hepatic metabolism', Biochim Biophys Acta, 1366 (1-2), 17-32. Rodrigues, M. A., Gomes, D. A., Leite, M. F., Grant, W., Zhang, L., Lam, W., Cheng, Y. C., Bennett, A. M. and Nathanson, M. H. (2007), 'Nucleoplasmic calcium is required for cell proliferation', J Biol Chem, 282 (23), 17061-8. Roman, F. S., Chaillan, F. A. and Soumireu-Mourat, B. (1993), 'Long-term potentiation in rat piriform cortex following discrimination learning', Brain Res, 601 (1-2), 265-72. Ronn, L. C., Ralets, I., Hartz, B. P., Bech, M., Berezin, A., Berezin, V., Moller, A. and Bock, E. (2000), 'A simple procedure for quantification of neurite outgrowth based on stereological principles', J Neurosci Methods, 100 (1-2), 25-32. Ross, C. A., MacCumber, M. W., Glatt, C. E. and Snyder, S. H. (1989), 'Brain phospholipase C isozymes: differential mRNA localizations by in situ hybridization', Proc Natl Acad Sci U S A, 86 (8), 2923-7. Rotin, D., Honegger, A. M., Margolis, B. L., Ullrich, A. and Schlessinger, J. (1992), 'Presence of SH2 domains of phospholipase C γ 1 enhances substrate phosphorylation by increasing the affinity toward the epidermal growth factor receptor', J Biol Chem, 267 (14), 9678-83. Rotman, E. I., Murphy, B. J. and Catterall, W. A. (1995), 'Sites of selective cAMP- dependent phosphorylation of the L-type calcium channel α 1 subunit from intact rabbit skeletal muscle myotubes', J Biol Chem, 270 (27), 16371-7. Ruas, M., Rietdorf, K., Arredouani, A., Davis, L. C., Lloyd-Evans, E., Koegel, H., Funnell, T. M., Morgan, A. J., Ward, J. A., Watanabe, K., and et al. (2010), 'Purified TPC isoforms form NAADP receptors with distinct roles for Ca2+ signalling and endolysosomal trafficking', Curr Biol, 20 (8), 703-9.

211

CHAPTER 6. BIBLIOGRAPHY

Ruberte, E., Friederich, V., Chambon, P. and Morriss-Kay, G. (1993), 'Retinoic acid receptors and cellular retinoid binding proteins. III. Their differential transcript distribution during mouse nervous system development', Development, 118 (1), 267-82. Rutter, G. A. (2003), 'Calcium signalling: NAADP comes out of the shadows', Biochem J, 373 (Pt 2), e3-4. Rye, D. B., Wainer, B. H., Mesulam, M. M., Mufson, E. J. and Saper, C. B. (1984), 'Cortical projections arising from the basal forebrain: a study of cholinergic and noncholinergic components employing combined retrograde tracing and immunohistochemical localization of choline acetyltransferase', Neuroscience, 13 (3), 627-43. Ryu, S. H., Cho, K. S., Lee, K. Y., Suh, P. G. and Rhee, S. G. (1986), 'Two forms of phosphatidylinositol-specific phospholipase C from bovine brain', Biochem Biophys Res Commun, 141 (1), 137-44. Ryu, S. H., Suh, P. G., Cho, K. S., Lee, K. Y. and Rhee, S. G. (1987), 'Bovine brain cytosol contains three immunologically distinct forms of inositolphospholipid-specific phospholipase C', Proc Natl Acad Sci U S A, 84 (19), 6649-53. Sagara, Y. and Inesi, G. (1991), 'Inhibition of the sarcoplasmic reticulum Ca2+ transport ATPase by thapsigargin at subnanomolar concentrations', J Biol Chem, 266 (21), 13503-6. Saito, Y., Ihara, Y., Leach, M. R., Cohen-Doyle, M. F. and Williams, D. B. (1999), 'Calreticulin functions in vitro as a molecular chaperone for both glycosylated and non-glycosylated proteins', EMBO J, 18 (23), 6718-29.Santodomingo, J., Vay, L., Camacho, M., Hernandez-Sanmiguel, E., Fonteriz, R. I., Lobaton, C. D., Montero, M., Moreno, A. and Alvarez, J. (2008) 'Calcium dynamics in bovine adrenal medulla chromaffin cell secretory granules', Eur J Neurosci, 28 (7), 1265–1274. Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J., Blayney, L. M., Swann, K. and Lai, F. A. (2002), 'PLC ζ: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo development', Development, 129 (15), 3533- 44. Scheuber, A., Miles, R. and Poncer, J. C. (2004), 'Presynaptic Cav2.1 and Cav2.2 differentially influence release dynamics at hippocampal excitatory synapses', J Neurosci, 24 (46), 10402-9. Schmidt, M., Evellin, S., Weernink, P. A., von Dorp, F., Rehmann, H., Lomasney, J. W. and Jakobs, K. H. (2001), 'A new phospholipase-C-calcium signalling pathway mediated by cyclic AMP and a Rap GTPase', Nat Cell Biol, 3 (11), 1020-4.

212

CHAPTER 6. BIBLIOGRAPHY

Schumacher, M. A., Rivard, A. F., Bachinger, H. P. and Adelman, J. P. (2001), 'Structure of the gating domain of a Ca2+-activated K+ channel complexed with Ca2+/calmodulin', Nature, 410 (6832), 1120-4. Schurmans, S., Schiffmann, S. N., Gurden, H., Lemaire, M., Lipp, H. P., Schwam, V., Pochet, R., Imperato, A., Bohme, G. A. and Parmentier, M. (1997), 'Impaired long-term potentiation induction in dentate gyrus of calretinin- deficient mice', Proc Natl Acad Sci U S A, 94 (19), 10415-20. Sepulveda, M. R., Vanoevelen, J., Raeymaekers, L., Mata, A. M. and Wuytack, F. (2009), 'Silencing the SPCA1 (secretory pathway Ca2+-ATPase isoform 1) impairs Ca2+ homeostasis in the Golgi and disturbs neural polarity', J Neurosci, 29 (39), 12174-82. Shimohama, S., Sumida, Y., Fujimoto, S., Matsuoka, Y., Taniguchi, T., Takenawa, T. and Kimura, J. (1998), 'Differential expression of rat brain phospholipase C isozymes in development and aging', Biochem Biophys Res Commun, 243 (1), 210-6. Shinomura, T., Asaoka, Y., Oka, M., Yoshida, K. and Nishizuka, Y. (1991), 'Synergistic action of diacylglycerol and unsaturated fatty acid for protein kinase C activation: its possible implications', Proc Natl Acad Sci U S A, 88 (12), 5149-53. Singer, A. U., Waldo, G. L., Harden, T. K. and Sondek, J. (2002), 'A unique fold of phospholipase C-β mediates dimerization and interaction with Gαq', Nat Struct Biol, 9 (1), 32-6. Singh, A., Hildebrand, M. E., Garcia, E. and Snutch, T. P. (2010), 'The transient receptor potential channel antagonist SKF96365 is a potent blocker of low- voltage-activated T-type calcium channels', Br J Pharmacol, 160 (6), 1464- 75. Singh, S. M. and Murray, D. (2003), 'Molecular modeling of the membrane targeting of phospholipase C pleckstrin homology domains', Protein Sci, 12 (9), 1934- 53. Smrcka, A. V., Hepler, J. R., Brown, K. O. and Sternweis, P. C. (1991), 'Regulation of polyphosphoinositide-specific phospholipase C activity by purified Gq', Science, 251 (4995), 804-7. Song, C., Hu, C. D., Masago, M., Kariyai, K., Yamawaki-Kataoka, Y., Shibatohge, M., Wu, D., Satoh, T. and Kataoka, T. (2001), 'Regulation of a novel human phospholipase C, PLCε, through membrane targeting by Ras', J Biol Chem, 276 (4), 2752-7. Song, C., Satoh, T., Edamatsu, H., Wu, D., Tadano, M., Gao, X. and Kataoka, T. (2002), 'Differential roles of Ras and Rap1 in growth factor-dependent activation of phospholipase C ε', Oncogene, 21 (53), 8105-13.

213

CHAPTER 6. BIBLIOGRAPHY

Spat, A., Bradford, P. G., McKinney, J. S., Rubin, R. P. and Putney, J. W., Jr. (1986b), 'A saturable receptor for 32P-inositol-1,4,5-triphosphate in hepatocytes and neutrophils', Nature, 319 (6053), 514-6. Spat, A., Fabiato, A. and Rubin, R. P. (1986a), 'Binding of inositol trisphosphate by a liver microsomal fraction', Biochem J, 233 (3), 929-32. Stahelin, R. V. (2009), 'Lipid binding domains: more than simple lipid effectors', J Lipid Res, 50 Suppl, S299-304. Stathopulos, P. B., Li, G. Y., Plevin, M. J., Ames, J. B. and Ikura, M. (2006), 'Stored Ca2+ depletion-induced oligomerization of stromal interaction molecule 1 (STIM1) via the EF-SAM region: An initiation mechanism for capacitive Ca2+ entry', J Biol Chem, 281 (47), 35855-62. Steinhardt, R., Zucker, R. and Schatten, G. (1977), 'Intracellular calcium release at fertilization in the sea urchin egg', Dev Biol, 58 (1), 185-96. Stewart, A. J., Morgan, K., Farquharson, C. and Millar, R. P. (2007), 'Phospholipase C-η enzymes as putative protein kinase C and Ca2+ signalling components in neuronal and neuroendocrine tissues', Neuroendocrinology, 86 (4), 243-8. Stewart, A. J., Mukherjee, J., Roberts, S. J., Lester, D. and Farquharson, C. (2005), 'Identification of a novel class of mammalian phosphoinositol-specific phospholipase C enzymes', Int J Mol Med, 15 (1), 117-21. Streb, H., Irvine, R. F., Berridge, M. J. and Schulz, I. (1983), 'Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol- 1,4,5-trisphosphate', Nature, 306 (5938), 67-9. Suh, P. G., Park, J. I., Manzoli, L., Cocco, L., Peak, J. C., Katan, M., Fukami, K., Kataoka, T., Yun, S. and Ryu, S. H. (2008), 'Multiple roles of phosphoinositide-specific phospholipase C isozymes', BMB Rep, 41 (6), 415- 34. Suh, P. G., Ryu, S. H., Moon, K. H., Suh, H. W. and Rhee, S. G. (1988), 'Cloning and sequence of multiple forms of phospholipase C', Cell, 54 (2), 161-9. Supattapone, S., Worley, P. F., Baraban, J. M. and Snyder, S. H. (1988), 'Solubilization, purification, and characterization of an inositol trisphosphate receptor', J Biol Chem, 263 (3), 1530-4. Sutherland, M. K., Wong, L., Somerville, M. J., Yoong, L. K., Bergeron, C., Parmentier, M. and McLachlan, D. R. (1993), 'Reduction of calbindin-28k mRNA levels in Alzheimer as compared to Huntington hippocampus', Brain Res Mol Brain Res, 18 (1-2), 32-42. Sutton, R. B., Davletov, B. A., Berghuis, A. M., Sudhof, T. C. and Sprang, S. R. (1995), 'Structure of the first C2 domain of synaptotagmin I: a novel Ca2+/phospholipid-binding fold', Cell, 80 (6), 929-38.

214

CHAPTER 6. BIBLIOGRAPHY

Sutton, R. B. and Sprang, S. R. (1998), 'Structure of the protein kinase Cβ phospholipid-binding C2 domain complexed with Ca2+', Structure, 6 (11), 1395-405. Szabadkai, G., Bianchi, K., Várnai, P., De Stefani, D., Wieckowski, M. R., Cavagna, D., Nagy, A. I., Balla, T. and Rizzuto, R. (2006) 'Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels', J Cell Biol, 175 (6), 901-11. Takai, Y., Kishimoto, A., Kikkawa, U., Mori, T. and Nishizuka, Y. (1979), 'Unsaturated diacylglycerol as a possible messenger for the activation of calcium-activated, phospholipid-dependent protein kinase system', Biochem Biophys Res Commun, 91 (4), 1218-24. Takata, M. and Kurosaki, T. (1996), 'A role for Bruton's tyrosine kinase in B cell antigen receptor-mediated activation of phospholipase C-γ 2', J Exp Med, 184 (1), 31-40. Takemura, M., Mishima, T., Wang, Y., Kasahara, J., Fukunaga, K., Ohashi, K. and Mizuno, K. (2009), 'Ca2+/calmodulin-dependent protein kinase IV-mediated LIM kinase activation is critical for calcium signal-induced neurite outgrowth', J Biol Chem, 284 (42), 28554-62. Takenawa, T. and Nagai, Y. (1981), 'Purification of phosphatidylinositol-specific phospholipase C from rat liver', J Biol Chem, 256 (13), 6769-75. Tan, W. and Colombini, M. (2007), 'VDAC closure increases calcium ion flux', Biochim Biophys Acta, 1768 (10), 2510-5. Tanaka, O. and Kondo, H. (1994), 'Localization of mRNAs for three novel members (β3, β4 and γ2) of phospholipase C family in mature rat brain', Neurosci Lett, 182 (1), 17-20. Taussig, R., Iniguez-Lluhi, J. A. and Gilman, A. G. (1993), 'Inhibition of adenylyl cyclase by Gi α', Science, 261 (5118), 218-21. Taylor, R. S., Jones, S. M., Dahl, R. H., Nordeen, M. H. and Howell, K. E. (1997), 'Characterization of the Golgi complex cleared of proteins in transit and examination of calcium uptake activities', Mol Biol Cell, 8 (10), 1911-31. Taylor, S. J., Chae, H. Z., Rhee, S. G. and Exton, J. H. (1991), 'Activation of the β 1 isozyme of phospholipase C by α subunits of the Gq class of G proteins', Nature, 350 (6318), 516-8. Teclemariam-Mesbah, R., Ter Horst, G. J., Postema, F., Wortel, J. and Buijs, R. M. (1999), 'Anatomical demonstration of the suprachiasmatic nucleus-pineal pathway', J Comp Neurol, 406 (2), 171-82. Tecott, L. H., Maricq, A. V. and Julius, D. (1993), 'Nervous system distribution of the serotonin 5-HT3 receptor mRNA', Proc Natl Acad Sci U S A, 90 (4), 1430-4.

215

CHAPTER 6. BIBLIOGRAPHY

Thomas, A. P., Bird, G. S., Hajnoczky, G., Robb-Gaspers, L. D. and Putney, J. W., Jr. (1996), 'Spatial and temporal aspects of cellular calcium signalling', FASEB J, 10 (13), 1505-17. Tjoelker, L. W., Seyfried, C. E., Eddy, R. L., Jr., Byers, M. G., Shows, T. B., Calderon, J., Schreiber, R. B. and Gray, P. W. (1994), 'Human, mouse, and rat calnexin cDNA cloning: identification of potential calcium binding motifs and gene localization to human chromosome 5', Biochemistry, 33 (11), 3229- 36. Tsuneki, H., Dezaki, K. and Kimura, I. (1997), 'Neuronal nicotinic receptor operates slow Ca2+ mobilization at mouse muscle endplate', Neurosci Lett, 225 (3), 185-8. Turner, T. J., Mokler, D. J. and Luebke, J. I. (2004), 'Calcium influx through presynaptic 5-HT3 receptors facilitates GABA release in the hippocampus: in vitro slice and synaptosome studies', Neuroscience, 129 (3), 703-18. Tyers, M., Haslam, R. J., Rachubinski, R. A. and Harley, C. B. (1989), 'Molecular analysis of pleckstrin: the major protein kinase C substrate of platelets', J Cell Biochem, 40 (2), 133-45. Uchida, Y., Hasegawa, J., Chinnapen, D., Inoue, T., Okazaki, S., Kato, R., Wakatsuki, S., Misaki, R., Koike, M., Uchiyama, Y. and et al. (2011), 'Intracellular phosphatidylserine is essential for retrograde membrane traffic through endosomes', Proc Natl Acad Sci U S A, 108 (38), 15846-51. Uekama, N., Sugita, T., Okada, M., Yagisawa, H. and Tuzi, S. (2007), 'Phosphatidylserine induces functional and structural alterations of the membrane-associated pleckstrin homology domain of phospholipase C- delta1', FEBS J, 274 (1), 177-87. Unger, V. M., Hargrave, P. A., Baldwin, J. M. and Schertler, G. F. (1997), 'Arrangement of rhodopsin transmembrane α-helices', Nature, 389 (6647), 203-6. Unwin, N. (1993), 'Nicotinic acetylcholine receptor at 9 A resolution', J Mol Biol, 229 (4), 1101-24. Usachev, Y. M., Marchenko, S. M. and Sage, S. O. (1995), 'Cytosolic calcium concentration in resting and stimulated endothelium of excised intact rat aorta', J Physiol, 489 ( Pt 2), 309-17. Van Obberghen, E., Ballotti, R., Gazzano, H., Fehlmann, M., Rossi, B., Gammeltoft, S., Debant, A., Le Marchand-Brustel, Y. and Kowalski, A. (1985), 'The insulin receptor kinase', Biochimie, 67 (10-11), 1119-24. van Praag, H., Kempermann, G. and Gage, F. H. (1999), 'Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus', Nat Neurosci, 2 (3), 266-70.

216

CHAPTER 6. BIBLIOGRAPHY

Varnai, P. and Balla, T. (2008), 'Live cell imaging of phosphoinositides with expressed inositide binding protein domains', Methods, 46 (3), 167-76. Venkateswarlu, K., Gunn-Moore, F., Oatey, P. B., Tavare, J. M. and Cullen, P. J. (1998), 'Nerve growth factor- and epidermal growth factor-stimulated translocation of the ADP-ribosylation factor-exchange factor GRP1 to the plasma membrane of PC12 cells requires activation of phosphatidylinositol 3- kinase and the GRP1 pleckstrin homology domain', Biochem J, 335 ( Pt 1), 139-46. Verkhratsky, A. J. and Petersen, O. H. (1998), 'Neuronal calcium stores', Cell Calcium, 24 (5-6), 333-43. Wallace, M. A. and Claro, E. (1990), 'Comparison of serotoninergic to muscarinic cholinergic stimulation of phosphoinositide-specific phospholipase C in rat brain cortical membranes', J Pharmacol Exp Ther, 255 (3), 1296-300. Walliser, C., Retlich, M., Harris, R., Everett, K. L., Josephs, M. B., Vatter, P., Esposito, D., Driscoll, P. C., Katan, M., Gierschik, P. and Bunney, T. D. (2008), 'Rac regulates its effector phospholipase Cγ2 through interaction with a split pleckstrin homology domain', J Biol Chem, 283 (44), 30351-62. Walseth, T., F., Lin-Moshier, Y., Jain, P., Ruas, M., Parrington, J., Galione, A., Marchant, J. S. and Slama, J. T. (2012) 'Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg', J Biol Chem, 287 (4), 2308-15. Wang, D. S. and Shaw, G. (1995), 'The association of the C-terminal region of β I sigma II spectrin to brain membranes is mediated by a PH domain, does not require membrane proteins, and coincides with a inositol-1,4,5 triphosphate binding site', Biochem Biophys Res Commun, 217 (2), 608-15. Wang, T., Dowal, L., El-Maghrabi, M. R., Rebecchi, M. and Scarlata, S. (2000), 'The pleckstrin homology domain of phospholipase C-β(2) links the binding of Gβγ to activation of the catalytic core', J Biol Chem, 275 (11), 7466-9. Wang, X., Zhang, X., Dong, X. P., Samie, M., Li, X., Cheng, X., Goschka, A., Shen, D., Zhou, Y., Harlow, J., Zhu, M. X., Clapham, D. E., Ren, D. and Xu, H. (2012) 'TPC Proteins Are Phosphoinositide- Activated Sodium-Selective Ion Channels in Endosomes and Lysosomes', Cell, 151 (2), 372-83. Wang, X. D., Kiang, J. G., Scheibel, L. W. and Smallridge, R. C. (1999), 'Phospholipase C activation by Na+/Ca2+ exchange is essential for monensin- induced Ca2+ influx and arachidonic acid release in FRTL-5 thyroid cells', J Investig Med, 47 (8), 388-96. Wang, X., Su, B., Lee, H. G., Li, X., Perry, G., Smith, M. A. and Zhu, X. (2009) 'Impaired Balance of Mitochondrial Fission and Fusion in Alzheimer's Disease', J Neurosci, 29 (28), 9090-103.

217

CHAPTER 6. BIBLIOGRAPHY

Watanabe, M., Nakamura, M., Sato, K., Kano, M., Simon, M. I. and Inoue, Y. (1998), 'Patterns of expression for the mRNA corresponding to the four isoforms of phospholipase Cβ in mouse brain', Eur J Neurosci, 10 (6), 2016- 25. Watt, S. A., Kular, G., Fleming, I. N., Downes, C. P. and Lucocq, J. M. (2002), 'Subcellular localization of phosphatidylinositol 4,5-bisphosphate using the pleckstrin homology domain of phospholipase C δ1', Biochem J, 363 (Pt 3), 657-66. Weber C. R., Piacentino V., Ginsburg K. S., Houser S. R. and Bers D. M. (2002), 'Na+-Ca2+ exchange current and submembrane [Ca2+] during the cardiac action potential', Circ Res, 90 (2), 182–189. Weiss, S. J., McKinney, J. S. and Putney, J. W., Jr. (1982), 'Receptor-mediated net breakdown of phosphatidylinositol 4,5-bisphosphate in parotid acinar cells', Biochem J, 206 (3), 555-60. Wen, W., Yan, J. and Zhang, M. (2006), 'Structural characterization of the split pleckstrin homology domain in phospholipase C-γ1 and its interaction with TRPC3', J Biol Chem, 281 (17), 12060-8. Werth, J. L. and Thayer, S. A. (1994), 'Mitochondria buffer physiological calcium loads in cultured rat dorsal root ganglion neurons', J Neurosci, 14 (1), 348-56. Wetzker, R., Klinger, R., Haase, H., Vetter, R. and Bohmer, F. D. (1987), 'Fast activation of Ca2+-ATPases in plasma membranes from cardiac muscle and from ascites carcinoma cells: a possible function of endogenous calmodulin', Biomed Biochim Acta, 46 (8-9), S403-6. Whitman, M., Downes, C. P., Keeler, M., Keller, T. and Cantley, L. (1988), 'Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate', Nature, 332 (6165), 644-6. Wilson, D. A. (2003), 'Rapid, experience-induced enhancement in odorant discrimination by anterior piriform cortex neurons', J Neurophysiol, 90 (1), 65-72. Wilton, David C. (2005), 'Phospholipases A2: structure and function', European Journal of Lipid Science and Technology, 107 (3), 193-205. Wing, M. R., Houston, D., Kelley, G. G., Der, C. J., Siderovski, D. P. and Harden, T. K. (2001), 'Activation of phospholipase C-ε by heterotrimeric G protein βγ- subunits', J Biol Chem, 276 (51), 48257-61. Wing, M. R., Snyder, J. T., Sondek, J. and Harden, T. K. (2003), 'Direct activation of phospholipase C-ε by Rho', J Biol Chem, 278 (42), 41253-8. Woo, D. H., Jung, S. J., Zhu, M. H., Park, C. K., Kim, Y. H., Oh, S. B. and Lee, C. J. (2008), 'Direct activation of transient receptor potential vanilloid 1 (TRPV1) by diacylglycerol (DAG)', Mol Pain, 4, 42.

218

CHAPTER 6. BIBLIOGRAPHY

Wright, C. E. and Angus, J. A. (1996), 'Effects of N-, P- and Q-type neuronal calcium channel antagonists on mammalian peripheral neurotransmission', Br J Pharmacol, 119 (1), 49-56. Wu, D., Katz, A. and Simon, M. I. (1993), 'Activation of phospholipase C β 2 by the α and β γ subunits of trimeric GTP-binding protein', Proc Natl Acad Sci U S A, 90 (11), 5297-301. Wu, D., Katz, A., Lee, C. H. and Simon, M. I. (1992a), 'Activation of phospholipase C by alpha 1-adrenergic receptors is mediated by the α subunits of Gq family', J Biol Chem, 267 (36), 25798-802. Wu, D., Tadano, M., Edamatsu, H., Masago-Toda, M., Yamawaki-Kataoka, Y., Terashima, T., Mizoguchi, A., Minami, Y., Satoh, T. and Kataoka, T. (2003), 'Neuronal lineage-specific induction of phospholipase Cε expression in the developing mouse brain', Eur J Neurosci, 17 (8), 1571-80. Wu, D. Q., Lee, C. H., Rhee, S. G. and Simon, M. I. (1992b), 'Activation of phospholipase C by the α subunits of the Gq and G11 proteins in transfected COS-7 cells', J Biol Chem, 267 (3), 1811-7. Wu, X. and Bers, D. M. (2006), 'Sarcoplasmic reticulum and nuclear envelope are one highly interconnected Ca2+ store throughout cardiac myocyte', Circ Res, 99 (3), 283-91. Xie, W., Kaetzel, M. A., Bruzik, K. S., Dedman, J. R., Shears, S. B. and Nelson, D. J. (1996), 'Inositol 3,4,5,6-tetrakisphosphate inhibits the calmodulin- dependent protein kinase II-activated chloride conductance in T84 colonic epithelial cells', J Biol Chem, 271 (24), 14092-7. Xu, J. and Chuang, D. M. (1987), 'Serotonergic, adrenergic and histaminergic receptors coupled to phospholipase C in cultured cerebellar granule cells of rats', Biochem Pharmacol, 36 (14), 2353-8. Xu, L., Begum, S., Hearn, J. D. and Hynes, R. O. (2006), 'GPR56, an atypical G protein-coupled receptor, binds tissue transglutaminase, TG2, and inhibits melanoma tumor growth and metastasis', Proc Natl Acad Sci U S A, 103 (24), 9023-8. Xu, R. X., Pawelczyk, T., Xia, T. H. and Brown, S. C. (1997), 'NMR structure of a protein kinase C-γ phorbol-binding domain and study of protein-lipid micelle interactions', Biochemistry, 36 (35), 10709-17. Xu, S., Zhao, L., Larsson, A. and Venge, P. (2009), 'The identification of a phospholipase B precursor in human neutrophils', FEBS J, 276 (1), 175-86. Xu, X. C. (2001), 'Detection of altered retinoic acid receptor expression in tissue sections using in situ hybridization', Histol Histopathol, 16 (1), 205-12. Yamamoto, K., Hashimoto, K., Isomura, Y., Shimohama, S. and Kato, N. (2000), 2+ 2+ 'An IP3-assisted form of Ca -induced Ca release in neocortical neurons', Neuroreport, 11 (3), 535-9.

219

CHAPTER 6. BIBLIOGRAPHY

Yamamoto, T., Matsui, T., Nakafuku, M., Iwamatsu, A. and Kaibuchi, K. (1995), 'A novel GTPase-activating protein for R-Ras', J Biol Chem, 270 (51), 30557- 61. Yamamoto, T., Takeuchi, H., Kanematsu, T., Allen, V., Yagisawa, H., Kikkawa, U., Watanabe, Y., Nakasima, A., Katan, M. and Hirata, M. (1999), 'Involvement of EF hand motifs in the Ca2+-dependent binding of the pleckstrin homology domain to phosphoinositides', Eur J Biochem, 265 (1), 481-90. Yan, W., Sunavala, G., Rosenzweig, S., Dasso, M., Brand, J. G. and Spielman, A. I. (2001), 'Bitter taste transduced by PLC-β2-dependent rise in IP3 and α- gustducin-dependent fall in cyclic nucleotides', Am J Physiol Cell Physiol, 280 (4), C742-51. Yang, E. J., Yoon, J. H., Min, D. S. and Chung, K. C. (2004), 'LIM kinase 1 activates cAMP-responsive element-binding protein during the neuronal differentiation of immortalized hippocampal progenitor cells', J Biol Chem, 279 (10), 8903- 10. Yang, N., Higuchi, O., Ohashi, K., Nagata, K., Wada, A., Kangawa, K., Nishida, E. and Mizuno, K. (1998), 'Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization', Nature, 393 (6687), 809-12. Yeung, T., Gilbert, G. E., Shi, J., Silvius, J., Kapus, A. and Grinstein, S. (2008), 'Membrane phosphatidylserine regulates surface charge and protein localization', Science, 319 (5860), 210-3. Yin, L., Wang, J., Klein, P. S. and Lazar, M. A. (2006) 'Nuclear receptor Rev- erbalpha is a critical lithium-sensitive component of the circadian clock', Science, 311 (5763), 1002–5. Yoon, H. S., Hajduk, P. J., Petros, A. M., Olejniczak, E. T., Meadows, R. P. and Fesik, S. W. (1994), 'Solution structure of a pleckstrin-homology domain', Nature, 369 (6482), 672-5. Yue, C., Dodge, K. L., Weber, G. and Sanborn, B. M. (1998), 'Phosphorylation of serine 1105 by protein kinase A inhibits phospholipase Cβ3 stimulation by Galphaq', J Biol Chem, 273 (29), 18023-7. Yue, C., Ku, C. Y., Liu, M., Simon, M. I. and Sanborn, B. M. (2000), 'Molecular mechanism of the inhibition of phospholipase C β 3 by protein kinase C', J Biol Chem, 275 (39), 30220-5. Yuen, J. W., Poon, L. S., Chan, A. S., Yu, F. W., Lo, R. K. and Wong, Y. H. (2010), 'Activation of STAT3 by specific Gα subunits and multiple Gβγ dimers', Int J Biochem Cell Biol, 42 (6), 1052-9. Zaballos, M. A., Garcia, B. and Santisteban, P. (2008), 'Gβγ dimers released in response to thyrotropin activate phosphoinositide 3-kinase and regulate gene expression in thyroid cells', Mol Endocrinol, 22 (5), 1183-99.

220

CHAPTER 6. BIBLIOGRAPHY

Zeng, M. and Zhou, J. N. (2008), 'Roles of autophagy and mTOR signalling in neuronal differentiation of mouse neuroblastoma cells', Cell Signal, 20 (4), 659-65. Zeng, W., Xu, X. and Muallem, S. (1996), 'Gβγ transduces [Ca2+]i oscillations and Gαq a sustained response during stimulation of pancreatic acinar cells with [Ca2+]i-mobilizing agonists', J Biol Chem, 271 (31), 18520-6. Zhang, J. J., Okutani, F., Huang, G. Z., Taniguchi, M., Murata, Y. and Kaba, H. (2010), 'Common properties between synaptic plasticity in the main olfactory bulb and olfactory learning in young rats', Neuroscience, 170 (1), 259-67. Zhao, K., Wang, W., Rando, O. J., Xue, Y., Swiderek, K., Kuo, A. and Crabtree, G. R. (1998), 'Rapid and phosphoinositol-dependent binding of the SWI/SNF- like BAF complex to chromatin after T lymphocyte receptor signalling', Cell, 95 (5), 625-36. Zhou, Y., Sondek, J. and Harden, T. K. (2008), 'Activation of human phospholipase C-η2 by Gβγ', Biochemistry, 47 (15), 4410-7. Zhou, Y., Wing, M. R., Sondek, J. and Harden, T. K. (2005), 'Molecular cloning and characterization of PLC-η2', Biochem J, 391 (Pt 3), 667-76.

Zima, A. V., Bare, D. J., Mignery, G. A. and Blatter, L. A. (2007), 'IP3-dependent nuclear Ca2+ signalling in the mammalian heart', J Physiol, 584 (Pt 2), 601- 11.

221

222

Chapter 7.

Appendices

223

224

CHAPTER 7. APPENDICES

Appendix A - Sequences

DNA sequence of PLCη2 (223-3717 bp of NM_175556.4 ) inserted into pcDNA3.1. The construct was provided by Prof. Kiyoko Fukami.

223 ATGCCTGG TCCCCAGCCG 241 TCTGCTGCCA GCCAGACCAC AGGAGCCGTG GCTTGCCTGG CAGAGGTACT CCTCTGGGTT 301 GGAGGGAGCG TGGTAGTGTC ACCAAGATGG CAGCTCAGCC TTGTAGTGGA GCGATGCATG 361 AGTGCCATGC AAGAGGGGAC CCAGATGGTG AAGCTCCGTG GCAGCTCCAA GGGATTGGTC 421 CGCTTCTACT ACCTGGATGA GCACCGCTCC TGCCTCCGAT GGCGTCCCTC CCGCAAGAAC 481 GAGAAAGCCA AGATTTCCAT CGACTCCATC CAGGAGGTGA GTGAGGGCCG GCAGTCTGAA 541 ATCTTCCAGC GCTACCCGGA CAGCAGCTTT GACCCCAACT GCTGCTTCAG CATCTACCAT 601 GGCAGCCACA GAGAGTCGCT GGACTTGGTC TCACCCAGCA GTGAAGAGGC ACGTACCTGG 661 GTCACCGGCC TTCGCTACCT CATGGCTGGC ATCAGCGATG AAGACAGCCT AGCCCGTCGC 721 CAGCGTACCA GGGACCAGTG GCTGAAGCAG ACGTTTGATG AGGCTGACAA GAACGGGGAC 781 GGCAGCCTGA GCATCAGTGA GGTTCTGCAG CTGCTACATA AACTCAACGT GAACCTGCCC 841 CGGCAGAGGG TGAAACAGAT GTTCAGGGAG GCGGACACAG ATGACCATCA GGGGACATTA 901 GGCTTTGAGG AATTCTGCGC CTTCTACAAG ATGATGTCTA CCCGCCGAGA CCTCTACCTG 961 CTCATGCTGA CCTACAGCAA CCATAAGGAC CACTTGGATG CCTCCGACCT GCAGCGCTTT 1021 CTGGAGGTGG AGCAGAAGAT GAACGGTGTG ACCCTGGAGA GCTGTCAGAA CATCATTGAG 1081 CAGTTTGAGC CTTGCCTGGA AAATAAGAGC AAGGGGATGC TGGGGATTGA TGGCTTTACC 1141 AACTACACCC GGAGCCCCGC CGGTGACATC TTCAACCCTG AGCACAACAG AGTGCACCAA 1201 GACATGACGC AGCCGCTGAG TCACTACTTC ATCACCTCGT CCCACAACAC CTACCTCGTG 1261 GGTGACCAGC TCATGTCCCA GTCTCGGGTG GACATGTATG CCTGGGTCCT GCAGGCCGGC 1321 TGCCGCTGTG TGGAGGTGGA CTGCTGGGAC GGGCCCGATG GAGAACCCAT TGTCCATCAT 1381 GGCTACACTC TGACCTCCAA GATCCTTTTC AAAGATGTCA TCGAAACCAT CAACAAATAC 1441 GCCTTCATCA AGAATGAGTA CCCAGTGATC CTGTCCATTG AGAACCACTG TAGTGTTGTT 1501 CAGCAGAAGA AGATGGCCCA GTATCTGACT GACATCCTCG GGGACAAGCT GGACCTCTCC 1561 TCAGTGAGCA GCGAAGATGC CACCATGCTT CCATCTCCAC AGATGCTCAA GGGCAAGATC 1621 CTGGTGAAGG GGAAGAAGCT CCCTGCCAAT ATCAGTGAGG ATGCAGAAGA AGGCGAAGTA 1681 TCTGATGAGG ACAGTGCTGA TGAGATGGAG GATGACTGCA AGCTCCTCAA TGGGGATGCC 1741 TCCACCAATC GGAAGCGTGT GGAAAACATT GCAAAGAAGA AGCTGGATTC TCTGATCAAG 1801 GAGTCAAAGA TCCGTGACTG TGAAGACCCC AATGACTTCT CTGTGTCCAC ACTATCCCCC 1861 TCTGGAAAGC TTGGGCGCAA AGCAGAGGCC AAGAAGGGTC AGAGCAAGGT TGAGGAAGAC 1921 GTGGAGGCCG GGGAAGACAG CGGGGTGAGC AGGCAGAATA GCCGCCTCTT CATGAGCAGC 1981 TTCTCCAAGC GCAAGAAAAA AGGCAGCAAG ATAAAGAAGG TGGCCAGCGT GGAAGAGGGG 2041 GACGAGACCC TGGACTCCCC AGGAAGCCAG AGCCGAGGGA CTGCCCGGCA AAAGAAGACC 2101 ATGAAGCTGT CACGAGCCCT CTCGGACCTA GTGAAATATA CCAAGTCTGT GGGGACCCAT 2161 GACGTGGAGA TAGAGGTGGT ATCTAGCTGG CAGGTGTCGT CCTTCAGTGA GACCAAGGCC 2221 CATCAGATCC TGCAGCAGAA GCCCACCCAG TACCTGCGCT TCAACCAGCA CCAGCTCTCG 2281 CGCATATACC CCTCCTCCTA CCGTGTGGAC TCCAGCAACT ACAATCCACA ACCCTTCTGG 2341 AACGCTGGTT GCCAGATGGT TGCCCTGAAC TACCAGTCAG AGGGGCGGAT GCTACAGCTG 2401 AACAGGGCCA AGTTCAGCGC CAACGGTGAC TGTGGCTACG TGCTCAAACC CCAGTGCATG 2461 TGCCAGGGTG TCTTCAACCC CAACTCGGAG GATCCCCTGC CGGGGCAGCT CAAGAAGCAG 2521 CTGGCCCTGA GGATCATCAG TGGCCAGCAG CTGCCCAAAC CACGGGACTC GGTGCTGGGC 2581 GACCGTGGGG AGATCATCGA CCCCTTCGTG GAGGTGGAGG TCATTGGGCT CCCCGTGGAC 2641 TGCAGCAAGG AGCAGACCCG AGTGGTGGAC GACAACGGAT TCAACCCCAT GTGGGAGGAG

225

CHAPTER 7. APPENDICES

2701 ACACTGGTGT TCACCGTGCA CATGCCAGAG ATTGCGCTTG TACGCTTCCT GGTCTGGGAC 2761 CATGACCCCA TTGGACGTGA CTTCATCGGC CAGAGGACAC TAGCCTTCAG CAGCATAATG 2821 CCAGGCTACC GGCATGTGTA CCTAGAAGGG ATGGAAGAGG CCTCTATCTT TGTTCATGTG 2881 GCTGTCAGTG ACATTAGTGG TAAGGTCAAG CAGACTCTGG GTCTAAAAGG TCTCTTCCTC 2941 CGAGGCACAA AGCCAGGCTC GCTGGACAGT CATGCTGCTG GACAGCCCCT CCCCCGGCCC 3001 TCCGTTAGCC AGAGGCTCCT GCGGCGCACG GCCAGTGCCC CGACCAAAAG CCAGAAGCCA 3061 AGTCGCAAGG GCTTCCCAGA GCTGGCCCTG GGCACACAGG ACGCAGGCTC CGAGGGGGCA 3121 GCTGATGACG TGGCACCCTC TAGCCCCAAC CCAGCTCTGG AGGCCCCTAC TCAAGAGAGG 3181 TCAGGCAGCA GCAGCCCCCG AGGTAAGGCA CCAGGAGGAG AAGCAACAGA AGAGAGGACA 3241 CTGGCACAGG TGCGGTCCCC AAATGCCCCC GAAGGCCCTG GACCTGCAGG GATGGCCGCC 3301 ACATGCATGA AGTGTGTGGT GGGCTCCTGC GCCGGTATGG ACGTTGAGGG CCTTCAAAGG 3361 GAGCAACAAC CCAGCCCTGG GCCTGCAGGC AGCCACATGG CCATCAGCCA TCAGCCCAGG 3421 GCCCGGGTAG ATTCACTGGG GGGCCCTTGC TGCAGCCCAA GTCCTCGAGC CACCCCAGGG 3481 AGAAGCAAAG AGGCCCCCAA GGGTCCTAGG GCTCGGCGCC AGGGTCCAGG CGGCGGCTCT 3541 GTGTCCTCGG ACTCCAGCAG CCCAGACAGC CCAGGCAGCC CCAAGGTGGC CCCCTGCCAG 3601 CCGGAGGGTG CTCACAGGCA GCAGGGGGCG CTGCAGGGAG AGATGAACGC CTTGTTCGTT 3661 CAAAAGCTGG AGGAGATCAG GAGTCATTCC CCCATGTTCT CCACCGTTAG GGATTGA

The amino acid sequence encoded by the pcDNA3.1 PLCη2 plasmid (75-1238 resudes of NP_780765.2). The sequence corresponds to the 21b/22/23 splice variant.

75 MPGPQP SAASQTTGAV ACLAEVLLWV GGSVVVSPRW QLSLVVERCM 121 SAMQEGTQMV KLRGSSKGLV RFYYLDEHRS CLRWRPSRKN EKAKISIDSI QEVSEGRQSE 181 IFQRYPDSSF DPNCCFSIYH GSHRESLDLV SPSSEEARTW VTGLRYLMAG ISDEDSLARR 241 QRTRDQWLKQ TFDEADKNGD GSLSISEVLQ LLHKLNVNLP RQRVKQMFRE ADTDDHQGTL 301 GFEEFCAFYK MMSTRRDLYL LMLTYSNHKD HLDASDLQRF LEVEQKMNGV TLESCQNIIE 361 QFEPCLENKS KGMLGIDGFT NYTRSPAGDI FNPEHNRVHQ DMTQPLSHYF ITSSHNTYLV 421 GDQLMSQSRV DMYAWVLQAG CRCVEVDCWD GPDGEPIVHH GYTLTSKILF KDVIETINKY 481 AFIKNEYPVI LSIENHCSVV QQKKMAQYLT DILGDKLDLS SVSSEDATML PSPQMLKGKI 541 LVKGKKLPAN ISEDAEEGEV SDEDSADEME DDCKLLNGDA STNRKRVENI AKKKLDSLIK 601 ESKIRDCEDP NDFSVSTLSP SGKLGRKAEA KKGQSKVEED VEAGEDSGVS RQNSRLFMSS 661 FSKRKKKGSK IKKVASVEEG DETLDSPGSQ SRGTARQKKT MKLSRALSDL VKYTKSVGTH 721 DVEIEVVSSW QVSSFSETKA HQILQQKPTQ YLRFNQHQLS RIYPSSYRVD SSNYNPQPFW 781 NAGCQMVALN YQSEGRMLQL NRAKFSANGD CGYVLKPQCM CQGVFNPNSE DPLPGQLKKQ 841 LALRIISGQQ LPKPRDSVLG DRGEIIDPFV EVEVIGLPVD CSKEQTRVVD DNGFNPMWEE 901 TLVFTVHMPE IALVRFLVWD HDPIGRDFIG QRTLAFSSIM PGYRHVYLEG MEEASIFVHV 961 AVSDISGKVK QTLGLKGLFL RGTKPGSLDS HAAGQPLPRP SVSQRLLRRT ASAPTKSQKP 1021 SRKGFPELAL GTQDAGSEGA ADDVAPSSPN PALEAPTQER SGSSSPRGKA PGGEATEERT 1081 LAQVRSPNAP EGPGPAGMAA TCMKCVVGSC AGMDVEGLQR EQQPSPGPAG SHMAISHQPR 1141 ARVDSLGGPC CSPSPRATPG RSKEAPKGPR ARRQGPGGGS VSSDSSSPDS PGSPKVAPCQ 1201 PEGAHRQQGA LQGEMNALFV QKLEEIRSHS PMFSTVRD

226

CHAPTER 7. APPENDICES

Amino acid sequences of the C-terminus in the five splice variants of PLCη2 (based on Zhou et al. 2005).

21a/23 VKQTLGLKGLFLRGTKPGSLDSHAAGQPLPRPSVSQRLLRRTASAPTKSQKPSRKGFPELALGTQDAG SEGAADDVAPSSPNPALEAPTQERSGSSSPRDTRLFPLQRPISPLCSLEPIAEEPALGPGLPLQAAAP TGPSQEGSQCPVGLGAKVTSSQQTSLGAFGTLQLRIGGGRENEEPPLRPHNGGISSGPREGTSGRQTD SKSRSRVPGHLPVVRRAKSEGQVLSELSPTPAVYSDATGTDRLWQRLEPGSHRDSVSSSSSMSSNDTV IDLSLPSLGLCRSRESIPGVSLGRLTSRPCLASAARPDLPPVTKSKSNPNLRVAGGLPTAPDELQPRP LAPRLTGHHPRPPWHHLTLVGLRDCPVSAKSKSLGDLTADDFAPSFQGSTSSLSCGLGSLGVAHQVLE PGIRRDALTEQLRWLTGFQQAGDITSPTSLGPAGDGSVGGPSFLRRSSSRSQSRVRAIASRARQAQER QQRLRGQDSRGPPEEERGTPEGACSVGHEGCVDVPMPAKGAPEQVCGAADGQLLLR

21a/22/23 VKQTLGLKGLFLRGTKPGSLDSHAAGQPLPRPSVSQRLLRRTASAPTKSQKPSRKGFPELALGTQDAG SEGAADDVAPSSPNPALEAPTQERSGSSSPRVRD

21b/23 VKQTLGLKGLFLRGTKPGSLDSHAAGQPLPRPSVSQRLLRRTASAPTKSQKPSRKGFPELALGTQDAG SEGAADDVAPSSPNPALEAPTQERSGSSSPRGKAPGGEATEERTLAQVRSPNAPEGPGPAGMAATCMK CVVGSCAGMDVEGLQREQQPSPGPAGSHMAISHQPRARVDSLGGPCCSPSPRATPGRSKEAPKGPRAR RQGPGGGSVSSDSSSPDSPGSPKVAPCQPEGAHRQQGALQGEMNALFVQKLEEIRSHSPMFSTDTRLF PLQRPISPLCSLEPIAEEPALGPGLPLQAAAPTGPSQEGSQCPVGLGAKVTSSQQTSLGAFGTLQLRI GGGRENEEPPLRPHNGGISSGPREGTSGRQTDSKSRSRVPGHLPVVRRAKSEGQVLSELSPTPAVYSD ATGTDRLWQRLEPGSHRDSVSSSSSMSSNDTVIDLSLPSLGLCRSRESIPGVSLGRLTSRPCLASAAR PDLPPVTKSKSNPNLRVAGGLPTAPDELQPRPLAPRLTGHHPRPPWHHLTLVGLRDCPVSAKSKSLGD LTADDFAPSFQGSTSSLSCGLGSLGVAHQVLEPGIRRDALTEQLRWLTGFQQAGDITSPTSLGPAGDG SVGGPSFLRRSSSRSQSRVRAIASRARQAQERQQRLRGQDSRGPPEEERGTPEGACSVGHEGCVDVPM PAKGAPEQVCGAADGQLLLRL

21b/22/23 VKQTLGLKGLFLRGTKPGSLDSHAAGQPLPRPSVSQRLLRRTASAPTKSQKPSRKGFPELALGTQDAG SEGAADDVAPSSPNPALEAPTQERSGSSSPRGKAPGGEATEERTLAQVRSPNAPEGPGPAGMAATCMK CVVGSCAGMDVEGLQREQQPSPGPAGSHMAISHQPRARVDSLGGPCCSPSPRATPGRSKEAPKGPRAR RQGPGGGSVSSDSSSPDSPGSPKVAPCQPEGAHRQQGALQGEMNALFVQKLEEIRSHSPMFSTVRD

21c/22/23 VKQTLGLKGLFLRGTKPGSLDSHAAGQPLPRPSVSQRLLRRTASAPTKSQKPSRKGFPELALGTQDAG SEGAADDVAPSSPNPALEAPTQERSGSSSPRGKAPGGEATEERTLAQVRSPNAPEGPGPAGMAATCMK CVVGSCAGMDVEGLQREQQPSPGPAGSHMAISHQPRARVDSLGGPCCSPSPRATPGRSKEAPKGPRAR RQGPGGGSVSSDSSSPDSPGSPKVAPCQPEGAHRQQGALQGEMNALFVQKLEEIRSHSPMFSTGKACR SAASHALYTWHAVRD

227

CHAPTER 7. APPENDICES

Appendix B – Plasmid maps

Plasmid map for pcDNA3.1D/V5-His-TOPO used for the cloning of ΔPH-PLCη2 (insert contained a STOP codon after the coding sequence):

Figure B.1.: Plasmid map of pcDNA3.1D/V5-His-TOPO. Figure was downloaded from: http://www.invitrogen.com/1/1/10253-pcdna3-1-directional-topo-expression- kit.html.

Plasmid map for pcDNA3.1/NT-GFP-TOPO used for the cloning of PLCη2 and its PH-lacking mutant (insert contained a STOP codon after the coding sequence):

228

CHAPTER 7. APPENDICES

Figure B.2.: Plasmid map of pcDNA3.1/NT-GFP-TOPO. Figure was downloaded from: https://products.invitrogen.com/ivgn/product/K481001.

The map of the psi-H1 plasmid containing the the control shRNA:

Figure B.3.: Plasmid map of psi-H1 encoding the control shRNA. Sequences of PLCη2 shRNAs are listed in Chapter 2. Figure was taken from the product insert (Genecopeia).

229

CHAPTER 7. APPENDICES

Appendix C – Abbreviations

Abbreviations of amino acids throughout the thesis were:

Amino Acid three letter code one letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic Acid Glu E Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Table C.1 Amino acid codes.

Appendix D - Publications

230