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Molecular Basis for SH3 Domain Regulation of F-BAR-Mediated Membrane Deformation

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Molecular Basis for SH3 Domain Regulation of F-BAR-Mediated Membrane Deformation Molecular basis for SH3 domain regulation of F-BAR-mediated membrane deformation Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin By Yijian Rao From Fujian, China November, 2010 Die vorliegende Arbeit wurde in der Zeit von März 2007 bis November 2010 unter Anleitung von Prof. Dr. Volker Haucke am Institut für Chemie-Biochemie der Freien Universität Berlin im Fachbereich Biologie, Chemie, Pharmazie durchgeführt. 1st Reviewer: Prof. Dr. Volker Haucke 2nd Reviewer: Prof. Dr. Wolfram Saenger Date of defence: 13-01-2011 Acknowledgements I would like to thank all people who have helped and inspired me during my doctoral study. First and foremost, I would like to express my deep and sincere gratitude to my supervisor Prof. Dr. Volker Haucke. Thank you for giving me the opportunity to enter the world of biological science and perform my research in his lab. His contagious enthusiasm for science has motivated all people in his lab, including me. In addition, I am thankful for his understanding, encouraging and personal guidance. I would like to thank all former and current members of AG Haucke’s lab for my thesis support and for creating a homey place to work. Special thanks to Dr. Arndt Pechstein and Dr. Dmytro Puchkov for their contribution to my project. Special thanks also to Dr. Tanja Maritzen and Dr. Arndt Pechstein for their critical reading of this dissertation manuscript. In particular, I am deeply grateful to Julia Mössinger, “my neighbor in the lab” and a very nice lady. You are always accessible and willing to help me. Thank you, Julia. I gratefully thank Prof. Dr. Wolfram Saenger, Dr. Qingjun Ma and Dr. Ardeschir Vahedi-Faridi for your wonderful collaboration and contribution to my project. Without your contribution, we cannot publish the Syndapin story so fast. I also thank Prof. Dr. Wolfram Saenger for his assistance and support as my thesis committee member. I would like to thank Prof. Dr. Oleg Shupliakov and Dr. Anna Sundborger at Department of Neuroscience, Linné Center in Developmental Biology and Regenerative Medicine, Karolinska Institutet. Thanks for your collaboration and contribution to my project. I would like to thank all my friends in Berlin for spending so much happy time with me! My deepest gratitude goes to my parents and my family for their unflagging love and support throughout my life. This dissertation is simply impossible without them. Last but not least, thanks to my wife Lifeng Yang for her patience, love and support. Directory I Directory Directory ............................................................................................................ I Summary......................................................................................................... IV Zusammenfassung............................................................................................V 1 Introduction.................................................................................................1 1.1 Mechanisms of membrane curvature gerenation and membrane deformation......................................................................................................1 1.1.1 Changes in lipid composition..............................................................2 1.1.2 Influence of integral membrane proteins ............................................2 1.1.3 Cytoskeletal proteins and microtubule motor activity........................3 1.1.4 Scaffolding by peripheral membrane proteins....................................6 1.1.5 Helix insertion into membranes........................................................11 1.2 BAR domains.......................................................................................11 1.2.1 BAR/N-BAR.....................................................................................16 1.2.2 F-BAR (EFC) domain.......................................................................17 1.2.3 I-BAR domain...................................................................................21 1.3 Membrane deformation in the endocytic pathway...............................24 1.3.1 Vesicle budding.................................................................................26 1.3.2 Vesicle formation..............................................................................27 1.3.3 Vesicle fission...................................................................................28 1.4 Syndapins/Pacsins ................................................................................29 1.4.1 Interactions of the syndapin protein family ......................................30 1.4.2 Syndapins play a role in endocytic pathway.....................................31 1.4.3 Syndapin 1 shapes the plasma membrane.........................................33 2 Aims of this study......................................................................................34 3 Materials and Methods.............................................................................35 3.1 Materials...............................................................................................35 3.1.1 Chemicals and consumables .............................................................35 3.1.2 Enzymes and kits..............................................................................35 3.1.3 Markers and loading dyes .................................................................35 3.1.4 Synthetic oligonucleotides................................................................36 3.1.5 Synthetic peptides.............................................................................36 3.1.6 Bacterial strains.................................................................................36 3.1.7 Plasmids ............................................................................................36 3.1.8 Constructs..........................................................................................37 3.1.9 Mammalian cell lines........................................................................38 3.1.10 Buffers, medium and solutions......................................................38 3.1.11 Devices and equipment..................................................................39 Directory II 3.1.12 Software and internet resources.....................................................39 3.2 Molecular biology................................................................................40 3.2.1 Polymerase chain reaction (PCR) .....................................................40 3.2.2 Overlap extension PCR.....................................................................41 3.2.3 Agarose gel electrophoresis and gel extraction ................................41 3.2.4 DNA restriction digest and dephosphorylation of vector DNA........41 3.2.5 Ligation of DNA inserts into linearized vectors ...............................42 3.2.6 Preparation of chemically competent E. coli cells............................42 3.2.7 Transformation of chemically competent E. coli cells .....................42 3.2.8 Colony PCR......................................................................................43 3.2.9 Plasmid DNA mini and midi preparation .........................................43 3.2.10 DNA sequencing............................................................................43 3.2.11 Glycerol stocks..............................................................................44 3.3 Biochemistry ........................................................................................44 3.3.1 Overexpression of recombinant proteins in E. coli...........................44 3.3.2 Affinity-purification of recombinant GST........................................45 3.3.3 Affinity-purification of recombinant Hisx6-fusion proteins..............45 3.3.4 Protein quantification - Bradford assay ............................................46 3.3.5 In vitro binding Assays.....................................................................46 3.3.6 SDS polyacrylamide gel electrophoresis (SDS-PAGE)....................47 3.4 Cell Biology.........................................................................................47 3.4.1 Mammalian cell culture ....................................................................47 3.4.2 Transfection of mammalian cells with plasmid DNA ......................48 3.4.3 Live cell confocal imaging experiment.............................................48 3.4.4 Electron microscopy analysis of Cos7 cells .....................................49 3.4.5 Transferrin uptake assay ...................................................................49 3.5 Protein crystallography........................................................................49 3.5.1 Crystallizaiton ...................................................................................49 3.5.2 X-ray data collection and processing................................................51 3.5.3 Structure determination.....................................................................52 3.5.4 Model building and refinement.........................................................54 3.6 Tubulation assay in vitro ......................................................................54 3.6.1 Liposome preparation.......................................................................54 3.6.2 Electron microscopy analysis of protein-lipid tubes.........................55 3.6.3 Liposome sedimentation assay
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