FCHSD2 regulates cell death and cell adhesion by Stephanie Louise Sue A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Medical Biophysics University of Toronto © Copyright by Stephanie Louise Sue 2010 FCHSD2 regulates cell death and cell adhesion Stephanie Louise Sue Master of Science Department of Medical Biophysics University of Toronto 2010 Abstract FCH/CIP4 homology-Bin-Amphiphysin-Rvs (F-BAR) domain proteins are a subfamily of the Bin-Amphiphysin-Rvs (BAR) superfamily of proteins. They contain unique domains that bind and reshape the phospholipid bilayers of endosomal compartments during endocytosis. Using a functional assay for cell survival, we identified an F-BAR protein, FCH/CIP4 homology and double Src homology 3 domains 2 (FCHSD2), that confers drug resistance. Stable expression of shRNA against FCHSD2 in multiple cell types showed that loss of FCHSD2 sensitized cells to apoptosis by doxorubicin. Silencing of FCHSD2 also enhanced the ability of fibroblasts to grow colonies in culture. Mass spectrometry analysis of FCHSD2 protein complexes identified multiple interacting proteins that are involved in adhesion and endosome trafficking. We identified and confirmed a novel interaction between FCHSD2 and sorting nexin 18 (SNX18), a BAR domain protein that binds to endosomes. Our results suggest that FCHSD2 is involved in regulating cellular adhesion and cell death. ii Acknowledgements I owe many thanks to my colleagues who helped in my work and development as a scientist. They include: Enrico Arpaia, Thorsten Berger, Heiko Blaser, Dirk Brenner, Anne Brüstle, Gordon Duncan, Ying Ju Jang, Christiane Knobbe, Jennifer Liepa Silvester, David McIlwain, Elize Shirdel, Yuwen Su, Michael Tusche, and Drew Wakeham. I would also like to thank the Proteomics Facility at the Campbell Family Cancer Research Institute, especially to Theo Goh, for its expertise, which was instrumental to this project. Also, thanks to the Princess Margaret Hospital Flow Cytometry facility for their help and support, and to Irene Ng and Marissa Luchico. I would like to thank my supervisory committee, Dr. Vuk Stambolic and Dr. Mitsu Ikura, for their support, guidance, and encouragement. To my ʻhonoraryʼ committee member, Dr. Mark Minden, thank you for your support and collaboration on the project. Lastly, to my supervisor Dr. Tak Wah Mak, for bringing me into his science family and allowing me the freedom to find my place in it. I would not have been able to pursue this project without the unwavering love and encouragement of my friends and family. Your support means everything. Thank you. iii Table of Contents Chapter 1: Introduction#1 1. Programmed cell death#.....................................................................................1 1.1. Apoptosis#.................................................................................................1 1.2. Autophagy#................................................................................................3 1.3. Necrosis#...................................................................................................3 1.4. Mitotic catastrophe#...................................................................................4 2. Cancer#..............................................................................................................4 2.1. Leukemia#..................................................................................................5 2.1.1. Chronic lymphoblastic leukemia#.......................................................5 2.1.2. Acute lymphoblastic leukemia#..........................................................6 2.1.3. Chronic myeloid leukemia#................................................................6 2.1.4. Acute myeloid leukemia#....................................................................7 3. Endocytosis#......................................................................................................7 3.1. Endocytosis and endosomal recycling#.....................................................8 3.2. Biological functions of endosomal recycling#..........................................10 3.2.1. Growth factor signaling#...................................................................10 3.2.2. Cell adhesion and motility#..............................................................11 4. Studies of BAR superfamily proteins: insights into F-BAR protein structure and function#....................................................................................................12 4.1. Structure of BAR domains#.....................................................................12 4.2. F-BAR domain protein functions#............................................................14 5. FER/CIP4 homology and double Src homology domains 2 (FCHSD2)#..........16 5.1. Human FCHSD2#....................................................................................16 5.2. Drosophila FCHSD2/Nervous Wreck (Nwk)#..........................................17 6. Cyclical Packaging Rescue (CPR)#.................................................................18 7. Rationale of the project#...................................................................................19 8. Hypothesis#......................................................................................................20 Chapter 2: Materials and methods#21 1. Materials#.........................................................................................................21 iv 1.1. Antibodies#..............................................................................................21 1.1.1. Unconjugated#.................................................................................21 1.1.2. Conjugated#.....................................................................................21 1.2. Chemical reagents#.................................................................................21 1.3. Media and buffers#..................................................................................25 1.4. Cell lines#................................................................................................27 1.5. Plasmids#................................................................................................27 1.6. Primers#...................................................................................................28 1.7. shRNA sequences against FCHSD2#.....................................................29 1.7.1. shRNA used in NIH 3T3 cells#.........................................................29 1.7.2. shRNA used in U937 cells#..............................................................29 1.8. Kits#.........................................................................................................29 2. Methods#..........................................................................................................30 2.1. Cloning#...................................................................................................30 2.1.1. FLAG-FCHSD2#..............................................................................30 2.1.2. FCHSD2-Myc#..................................................................................31 2.1.3. HIS-SNX18#.....................................................................................31 2.1.4. shRNA vectors used in NIH 3T3 cells#............................................31 2.2. Bacterial transformation#.........................................................................32 2.3. Cell culture#.............................................................................................32 2.3.1. Cell lines#.........................................................................................32 2.3.2. Drug response assay#.....................................................................32 2.3.3. Colony formation assay#..................................................................33 2.3.4. siRNA transfection#..........................................................................33 2.3.5. U937 differentiation#.........................................................................34 2.3.6. Electroporation#...............................................................................34 2.3.7. Retroviral infection of NIH 3T3 cells#...............................................34 2.3.7.1. Retroviral supernatant production#...........................................34 2.3.7.2. Infection of NIH 3T3 cells#........................................................35 2.3.8. Lentiviral infection of U937 cells#.....................................................35 2.3.8.1. Lentiviral supernatant production#............................................35 v 2.3.8.2. Infection of U937 cells#.............................................................36 2.4. Protein expression analysis#...................................................................36 2.4.1. Cell lysis#.........................................................................................36 2.4.2. Immunoblotting#...............................................................................37 2.5. Protein biochemistry#..............................................................................37 2.5.1. Immunoprecipitation for mass spectrometry analysis#....................37 2.5.2. FCHSD2 and SNX18 immunoprecipitation#.....................................38 2.6. qRT-PCR analysis of FCHSD2 expression#............................................39 2.7. Immunocytochemistry#............................................................................39