KERATIN REMODELLING IN STRESS TAN TONG SAN NATIONAL UNIVERSITY OF SINGAPORE 2012 i KERATIN REMODELLING IN STRESS TAN TONG SAN (B. Sc. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS Graduate School for Integrative Sciences and Engineering NATIONAL UNIVERSITY OF SINGAPORE 2012 ii iii ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my supervisor, Prof. Birgit Lane, for her guidance and mentorship, and for giving me the opportunity, independence and resources to conduct my research. Her depth of knowledge and passion for scientific discovery have been a great source of inspiration and motivation for me throughout these four years of PhD endeavour, and will continue to be in the future. I would also like to thank my thesis advisory committee, Prof. Michael Raghunath and A/Prof. Edward Manser, for their critical feedback along the way. My sincere thanks and appreciation also go to John Common, for his insightful comments and suggestions throughout the course of this project. I would also like to acknowledge Ildiko for initiating the keratin phosphorylation project. Special thanks also go to Cedric, Chida, Darren, Gopi, Nama, John Lim, Delina, Chye Ling, Huijia and Declan, for their help in research techniques. I am most fortunate to be part of the EBL lab, a stimulating and lively place to work in. Thanks go to Kenneth, Zhou Fan, Paula, Chai Ling, Christine, Yi Zhen, Rosita, Vivien and Giorgiana, fellow students who join me in the pursuit of a doctorate. Appreciation is also extended to Iskandar, Jun Xian, Carol, Anita, Yi Ling, Ting Ting and other lab members for their support and help. In addition, I would like to thank A*STAR for awarding me this A*STAR Graduate Scholarship, without which I would not be able to come this far. Finally, I would like to express my heartfelt appreciation to my family, both immediate and extended, for being so understanding and encouraging in this whole process. Their constant support and faith in me have made the difference in my life – and it is to them I dedicate this thesis. i TABLE OF CONTENTS ACKNOWLEDGEMENTS………………………………………………………i TABLE OF CONTENTS…………………………………………………………ii SUMMARY.............................................................................................................. x LIST OF TABLES………………………………………………………………... xii LIST OF FIGURES................................................................................................. xiii LIST OF APPENDICES…………………………………………………………. xv LIST OF ABBREVIATIONS……………………………………………………. xvi CHAPTER 1: INTRODUCTION...........................................................................1 1.1 The importance of skin…………………………………………………….. 2 1.1.1 Barrier functions of the epidermis…………………………………. 2 1.1.2 Homeostasis of the epidermis……………………………………… 3 1.1.3 Mechanical resilience of the skin…………………………………...3 1.2 Skin composition and functions……………………………………………. 4 1.2.1 The hypodermis……………………………………………………. 4 1.2.2 The dermis…………………………………………………………. 4 1.2.3 The basement membrane…………………………………………... 5 1.2.4 The epidermis……………………………………………………….7 1.2.4.1 Cell types within epidermis……………………………... 8 1.2.4.2 The stratum basale………………………………………. 9 1.2.4.3 The stratum spinosum…………………………………… 9 1.2.4.4 The stratum granulosum………………………………… 10 1.2.4.5 The stratum corneum……………………………………. 10 1.3 Anchoring junctions mediating cell-cell adhesions………………………... 14 1.3.1 Desmosomes……………………………………………………….. 14 1.3.1.1 Desmosomal cadherins………………………………….. 15 1.3.1.2 Desmosomal armadillo proteins………………………… 16 1.3.1.3 Desmosomal plakin proteins…………………………….. 18 1.3.1.4 Calcium pumps regulating desmosomal adhesions……... 20 ii 1.3.2 Adherens junctions………………………………………………….24 1.3.2.1 Cadherins………………………………………………... 24 1.3.3 Gap junctions………………………………………………………. 26 1.3.4 Tight junctions……………………………………………………... 28 1.4 Anchoring junctions mediating cell-matrix adhesions…………………….. 30 1.4.1 Hemidesmosomes………………………………………………….. 30 1.4.1.1 α6β4 integrins…………………………………………… 31 1.4.1.2 Laminin-332……………………………………………... 32 1.4.1.3 BP180…………………………………………………… 33 1.4.1.4 BP230…………………………………………………… 34 1.4.1.5 Plectin…………………………………………………… 35 1.4.2 Hemidesmosomal assembly………………………………………... 36 1.4.3 Focal contacts……………………………………………………… 38 1.4.3.1 α3β1 integrins…………………………………………… 38 1.4.3.2 α-actinin…………………………………………………. 38 1.4.3.3 Vinculin………………………………………………….. 39 1.4.3.4 Paxillin…………………………………………………... 39 1.4.3.5 Talin……………………………………………………... 39 1.4.3.6 Kindlin…………………………………………………... 40 1.5 Keratin intermediate filaments……………………………………………...44 1.5.1 Keratin expression in epidermis…………………………………….44 1.5.1.1 Stratification process…………………………………….. 45 1.5.1.2 Wound healing (re-epithelialization)……………………. 46 1.5.2 Keratin structure and assembly…………………………………….. 47 1.5.2.1 Keratin intermediate filament family……………………. 48 1.5.2.2 Primary structure of keratin……………………………... 49 1.5.2.3 Secondary structure of keratin…………………………... 50 1.5.2.4 Keratin intermediate filament formation………………... 52 iii 1.6 Post-translational modifications of keratin intermediate filament ………… 56 1.6.1 Phosphorylation……………………………………………………. 56 1.6.1.1 Kinases regulating keratin organization…………………. 56 1.6.1.2 Phosphatases regulating keratin organization…………… 59 1.6.2 Glycosylation………………………………………………………. 60 1.6.3 Ubiquitylation……………………………………………………… 60 1.6.4 Sumoylation………………………………………………………... 61 1.7 Keratinopathies (mutations in keratins)……………………………………. 62 1.7.1 Spectrum of disease phenotypes of keratin mutations……………... 62 1.7.1.1 Phenotypic variations between different mutations in the same gene………………………………………………... 63 1.7.1.2 Phenotypic variations between different mutations of the same amino acid residue in the same gene………………..64 1.7.1.3 Phenotypic variations due to genetic modifiers………….. 64 1.7.1.4 Phenotypic variations due to functional redundancy for keratins…………………………………………………… 65 1.8 Classification of epidermolysis bullosa……………………………………. 65 1.9 Classification of epidermolysis bullosa simplex……………………………70 1.9.1 Characterization of EBS phenotypes………………………………. 70 1.9.1.1 Genotype-phenotype correlation of EBS………………… 72 1.10 Other functions of keratin intermediate filament………………………….. 74 1.10.1 A role for keratins in regulating cell size and protein synthesis… 74 1.10.2 A role for keratins in regulating stress kinase activity………….. 74 1.11 Aims and outline of thesis…………………………………………………. 75 CHAPTER 2: MATERIALS AND METHODS…………………………..……. 77 2.1 Materials…………………………………………………………………… 78 2.1.1 Chemicals and reagents used in this thesis………………………… 78 2.1.2 Antibodies used in this thesis…...…………………………………..79 2.1.3 Plasmids……………………………………………………………. 80 2.1.4 Cell lines and cell culture…………………………………………...80 iv 2.1.5 Cryopreservation of cell lines……………………………………… 81 2.1.6 Thawing of cell lines………………………………………………..82 2.2 Drug/EGF treatment methodology………………………………………… 82 2.3 Molecular biology methodology…………………………………………… 83 2.3.1 Site-directed mutagenesis………………………………………….. 83 2.3.2 Cloning…………………………………………………………….. 85 2.3.2.1 Restriction digestion of DNA…………………………… 85 2.3.2.2 DNA agarose gel electrophoresis………………………... 88 2.3.2.3 DNA gel extraction……………………………………… 88 2.3.2.4 Dephosphorylation of plasmid DNA……………………. 88 2.3.2.5 Ligation of DNA………………………………………… 89 2.3.3 Transformation of chemically competent cells…………………….. 89 2.3.4 PCR analysis of transformants……………………………………... 89 2.3.5 Amplification and isolation of plasmid DNA from bacteria (Mini-prep)………………………………………………………….90 2.3.6 DNA sequencing…………………………………………………… 90 2.3.7 Bacterial DNA maxi-prep using Endofree® Plasmid Maxi Kit……. 91 2.3.8 Preparation of bacteria stocks……………………………………… 91 2.3.9 Transient transfection……………………………………………… 92 2.3.10 Viral packaging and collection…………………………………….. 92 2.3.11 Viral titration of N/TERT-1 cells and quantification……………… 93 2.3.12 Viral infection of N/TERT-1 cells to generate stable cell lines……. 93 2.3.13 Transient infection of N/TERT-1 cells…………………………….. 94 2.4 Gene expression analysis…………………………………………………... 94 2.4.1 RNA extraction…………………………………………………….. 94 2.4.2 cDNA synthesis……………………………………………………. 95 2.4.3 Quantitative real-time PCR (qPCR)………………………………...95 2.4.4 Over-expression studies……………………………………………. 96 2.4.5 RNA interference (siRNA)………………………………………… 96 v 2.5 Protein expression analysis………………………………………………… 98 2.5.1 Cell lysis and protein quantification……………………………….. 98 2.5.2 Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE)………………………………………………………. 99 2.5.3 Protein detection and chemiluminesence…………………………... 99 2.5.4 Immunocytochemistry……………………………………………... 99 2.6 Time-lapse microscopy imaging and image processing…………………… 100 2.6.1 Differential interference contrast (DIC) live-cell imaging………… 100 2.6.2 Fluorescence live-cell imaging…………………………………….. 101 2.6.3 Phase contrast live-cell imaging of scratch wounds using IncuCyteTM………………………………………………………… 102 2.6.4 Confocal live-cell imaging using MEK1/2 inhibitor treatment……. 103 2.7 Functional studies………………………………………………………….. 104 2.7.1 Osmotic stress assay……………………………………………….. 104 2.7.2 Confluence assay…………………………………………………... 104 2.7.3 Multiple scratch wound assay……………………………………… 104 2.8 Statistical analysis………………………………………………………….. 105 2.8.1 Differential interference contrast (DIC) live-cell imaging………… 105 2.8.2 Confluence assay…………………………………………………... 105 2.8.3 Drug/EGF treatment……………………………………………….. 105 2.8.4 Phase contrast live-cell imaging of scratch wounds using IncuCyteTM………………………………………………………… 105 2.8.5 Co-localization study………………………………………………. 106 2.8.6 MEK1/2 inhibitor treatment………………………………………...106