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Masterarbeit MASTERARBEIT Titel der Masterarbeit „Studying Potential Interactions of LAP2with Chromatin Modifiers“ verfasst von Michael Peter Skoruppa BSc angestrebter akademischer Grad Master of Science (MSc) Wien, 2015 Studienkennzahl lt. Studienblatt: A 066 834 Studienrichtung lt. Studienblatt: Masterstudium Molekulare Biologie Betreut von: Ao. Univ. Prof. Dipl. Ing. Dr. techn. Roland Foisner Table of Contents STATUTORY DECLARATION __________________________________________________________ 4 ABBREVIATIONS ____________________________________________________________________ 5 1. INTRODUCTION _________________________________________________________________ 7 1.1 THE CELL AND THE NUCLEUS _____________________________________________________ 7 1.2 THE NUCLEAR ENVELOPE _______________________________________________________ 10 1.2.1 Integral Nuclear Membrane Proteins _________________________________________ 11 1.2.1.1 Lamina-Associated-Polypeptide 2 (LAP2) Family _____________________________________ 12 1.2.1.2 Lamin-associated-Polypeptide 2(LAP2)__________________________________________ 13 1.2.1.3 Functions of LAP2 ___________________________________________________________ 16 1.2.2 Nuclear Lamins _________________________________________________________ 18 1.2.2.1 Nucleoplasmic Lamins _________________________________________________________ 18 1.2.2.2 Lamin-Chromatin Interactions ____________________________________________________ 20 1.3 SEARCH FOR LAP2 INTERACTING PROTEINS THAT ARE INVOLVED IN CHROMATIN ORGANIZATION __ 23 1.3.1 BioID Experiments _______________________________________________________ 23 1.3.1.1 Prohibitin ____________________________________________________________________ 24 1.3.1.2 SMARCA4 ___________________________________________________________________ 25 1.3.1.3 CHD4 ______________________________________________________________________ 25 1.3.1.4 KI-67 _______________________________________________________________________ 26 1.3.1.5 Sin3A ______________________________________________________________________ 26 1.3.1.6 NuMA ______________________________________________________________________ 26 1.4 SCIENTIFIC AIM OF THIS PROJECT ________________________________________________ 28 2. MATERIALS AND METHODS _____________________________________________________ 29 2.1 CELL LINES AND GENERAL CELL CULTURE __________________________________________ 29 2.2 PREPARATION OF CELL LYSATES __________________________________________________ 30 2.3 BCA-ASSAY ________________________________________________________________ 31 2.4 ANTIBODIES USED IN THIS PROJECT ________________________________________________ 32 2.5 CO-IMMUNOPRECIPITATION _____________________________________________________ 34 2.6 SDS POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE) AND IMMUNOBLOT __________________ 37 2.7 IMMUNOFLUORESCENCE MICROSCOPY _____________________________________________ 39 2.8 PROXIMITY LIGATION ASSAY (PLA) ________________________________________________ 40 3. RESULTS _____________________________________________________________________ 43 3.1 IMMUNOFLUORESCENCE ________________________________________________________ 44 3.1.1 Human cell lines – HeLa shLAP2 and shLuciferase co-cultivated _________________ 45 3.1.2 Immortalized Murine Dermal Fibroblasts – imMDF LAP2 (+/+) and LAP2 (-/-) ________ 48 3.2 WESTERN BLOT ANALYSIS ______________________________________________________ 52 3.2.1 Human cell lines – HeLa shLAP2 and shLuciferase ____________________________ 52 3.2.1.1 LAP2 Immunoprecipitation _____________________________________________________ 52 3.2.1.2 Co-immunoprecipitation of potential LAP2 interaction-partners _________________________ 53 3.2.1.2.1 SMARCA4 ________________________________________________________________ 55 3.2.1.2.2 CHD4 ____________________________________________________________________ 56 3.2.1.3 Co-Immunoprecipitation of LAP2with SMARCA4 and CHD4 ___________________________ 57 3.2.1.4 Comparison of CHD4, SMARCA4 and LAP2 with controls _____________________________ 58 3.2.2 Immortalized Murine Dermal Fibroblasts – imMDF LAP2 (+/+) and LAP2 (-/-) ________ 60 3.3 PROXIMITY LIGATION ASSAY _____________________________________________________ 62 3.3.1 Human cell lines – HeLa unmodified _________________________________________ 63 3.3.2 Immortalized Murine Dermal Fibroblasts – imMDF LAP2 (+/+) and LAP2 (-/-) ________ 66 4. DISCUSSION __________________________________________________________________ 71 5. REFERENCES _________________________________________________________________ 74 6. TABLE OF FIGURES ____________________________________________________________ 82 7. ACKNOWLEDGEMENTS _________________________________________________________ 83 8. APPENDIX ____________________________________________________________________ 85 8.1 USER MANUALS FOR ASSAY KITS _________________________________________________ 85 8.1.1 PierceTM BCA Protein Assay Kit from Life-Technologies by Thermo Fisher Scientific ___ 85 8.1.2 PLA Duolink® in Situ – Fluorescence ________________________________________ 85 8.2 ABSTRACT __________________________________________________________________ 86 8.3 ZUSAMMENFASSUNG __________________________________________________________ 87 8.4 CURRICULUM VITAE ___________________________________________________________ 88 Statutory Declaration I hereby declare that this master thesis has been written only by the undersigned and without any assistance from third parties. Furthermore, I confirm that no sources have been used in the preparation of this thesis other than those indicated in the thesis itself. I also state that all copyrights have been respected and met. Nonetheless, if infringements of any kind should emerge I solicit you courteously to contact me first before taking any further (legal) actions. Date: 24.09.15 Signature: _____________________________________________ Michael Peter Skoruppa, BSc 4 | P a g e Abbreviations BAF Barrier to Autointegration Factor BioID Proximity-Dependent Biotin Identification BRD4 Bromodomain-Containing Protein 4 CBX3 Chromobox Protein Homolog 3 or HP1- CHD4 Chromodomain-helicase-DNA-binding protein 4 CHD8 Chromodomain Helicase DNA Binding Protein 8 CTR C-Terminal Region DABCO 1, 4 diazabizyclo [2.2.2] octane DamID DNA Adenine Methyltransferase Identification DMEM Dulbecco’s Modified Eagle Medium DPBS Dulbecco’s Phosphor Buffered Saline DTT Dithiothreitol ECM Extracellular Matrix EDTA Etyhlenediaminetetraacetic Acid EGTA Ethyleneglycolgetraacetic Acid ER Endoplasmic Reticulum FACE2 Farnesylated Proteins-Converting Enzyme 2 (CaaX prenyl protease) FCS Fetal Calf Serum HP1 Heterochromatin-Protein 1 ICMT Isoprenylcysteine Methyltransferase INM Inner Nuclear Membrane KASH Klarsicht, ANC1, Syne Homology, conserved C-terminal regions KD Knockdown KI-67 Marker of Proliferation KI-67 KO Knockout LAD Lamina Associated Domain LAP1 Lamin-Associated Polypeptide 1 LAP2 Lamin-Associated Polypeptide 2 LBR Lamin-B Receptor LEM LAP2-EMERIN-MAN Domain 5 | P a g e LINC Linker of Nucleoskeleton and Cytoskeleton LMNB1/2 Lamin B1/2 MAN1 LEM Domain-Containing Protein 3 (LEMD3) MAPK Mitogen Activated Protein Kinase Mb Mega Bases NLS Nuclear Localization Sequence NP – 40 Nonyl – Phenoxypolyethoxylethanol NPC Nuclear Pore Complex NTR N-Terminal Region NuMA Nuclear Mitotic Apparatus Protein 1 NuRD Nucleosome Remodeling and Deacetylase Complex ONM Outer Nuclear Membrane p53BP p53 Binding Protein PI Protease Inhibitors PLA Proximity Ligation Assay PMSF Phenylmethanesulfonylfluoride pRB Retinoblastoma Protein rRNA Ribosomal RNA RT Room Temperature Sin3a SIN3 Transcription Regulator Family Member A; Paired Amphipathic Helix Protein Sin3a SMARCA4/5 SWI/SNF Related Matrix-Associated Actin-Dependent Regulator of Chromatin Subfamily a, Member 4/5 SN Supernatant SREBP-1 Sterol Response Element Binding Protein 1 STAT1 Signal Transducer and Activator of Transcription 1 SUN Sad1p, UNC-84 domains, conserved C-terminal regions TEMED Tetramethylethylenediamine TGF- Transforming Growth Factor TMPO Thymopoeitin (LAP2) TRIS Tris-(hydroxymethyl)-aminomethane WB Western Blot WT Wildtype ZMPSTE24 Zinc Metallopeptidase STE24 6 | P a g e 1. Introduction 1.1 The Cell and the Nucleus If you look at a whole organism, the cell is defined as the smallest unit being able to organize and perform all activities necessary for life. In fact, all the complex biochemical and physiological processes as well as the coordinative and motor skills within our organism, depend on the functionality and communication between single cells (Campbell et al. 2014). For us, the daily walk to the supermarket, picking up groceries or even reading these lines seem to be a simple task we are just able to do, although this simple procedure is based on an unfathomable amount of biochemical processes and communications, happening in a split second between a vast number of different cells (Campbell et al. 2014). Unsurprisingly, all cells have major differences, but also share certain common characteristics. Every cell, for example, is enclosed by a membrane, which controls the exchange of various substances between the cell itself and its environment. However, it is possible to distinguish between two major forms of life: eukaryotic and prokaryotic cells. The names eukaryotic and prokaryotic are derived from Greek, meaning “true nucleus” and “before nucleus”, respectively, referring to their developmental stage in evolution (Campbell et al. 2014). While the eukaryotic cell harbors membrane-enclosed organelles such as the nucleus,
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