University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2015 Mechanisms of Membrane Remodeling by Peripheral Proteins and Divalent Cations Zheng Shi University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biophysics Commons, and the Physical Chemistry Commons Recommended Citation Shi, Zheng, "Mechanisms of Membrane Remodeling by Peripheral Proteins and Divalent Cations" (2015). Publicly Accessible Penn Dissertations. 2011. https://repository.upenn.edu/edissertations/2011 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2011 For more information, please contact [email protected]. Mechanisms of Membrane Remodeling by Peripheral Proteins and Divalent Cations Abstract Biological membranes undergo constant shape remodeling involving the formation of highly curved structures. As one of the most extensively studied membrane remodeling events, endocytosis is a ubiquitous eukaryotic membrane budding, vesiculation, and internalization process fulfilling numerous roles including compensation of membrane area increase after bursts of exocytosis. There are multiple independent endocytic pathways which differ by their speed as well as the proteins that are involved in. Bin/Amphiphysin/Rvs (BAR) domain proteins, such as endophilin, are responsible for sensing or generating membrane curvature in multiple endocytic pathways. In this dissertation, I elucidate the mechanisms of membrane remodeling through in vitro experimental studies with synthetic lipid bilayers. Firstly, I investigated the binding and assembly of endophilin on planar membranes. Endophilin was found to be attracted to the membrane through electrostatic forces and to subsequently oligomerize on the membrane with the help of the protein’s N-terminal helices. Next, I studied the mechanisms that govern membrane shape transitions induced by BAR domain proteins. The initiation of membrane curvature occurs at well-defined membrane tensions and protein surface densities. Importantly, the membrane budding and tubulation initiated by membrane tension reduction provides a mechanistic explanation for high speed endocytic pathways. The experimentally determined membrane shape stability diagram shows remarkable consistency with a three-parameter curvature instability model. Comparing different BAR domain proteins, the ability of three BAR domain proteins to generate high membrane curvature increases significantly from endophilin to amphiphysin, and to SNX9. The protein-protein attraction strength was identified as one of the most important factors that leads to the dramatic difference among these structurally similar proteins. Furthermore, I examined membrane interactions of α-synuclein, an intrinsically disordered protein whose aggregation is a hallmark of Parkinson’s disease. The binding of α-synuclein monomers was found to linearly expand the membrane area before the protein is able to induce membrane curvature. The area expansion is achieved by thinning of the bilayer. As I experimentally demonstrate, these features make α- synuclein a reporter of membrane tension as well as a promoter for endocytosis. Finally, I found that Ca2+ ions can induce membrane invaginations through the clustering of charged lipids, albeit less efficiently than BAR domain oteins.pr As I will discuss, this suggests an intriguing role of Ca2+ ions in the evolution of life. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Chemistry First Advisor Tobias Baumgart Keywords alpha Synuclein, BAR domain proteins, Divalent cations, Membrane curvature, Membrane dynamics, Protein membrane interactions Subject Categories Biophysics | Chemistry | Physical Chemistry This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/2011 MECHANISMS OF MEMBRANE REMODELING BY PERIPHERAL PROTEINS AND DIVALENT CATIONS Zheng Shi A DISSERTATION in Chemistry Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2015 Supervisor of Dissertation _____________________ Dr. Tobias Baumgart, Associate Professor of Chemistry Graduate Group Chairperson ______________________ Dr. Gary A. Molander, Hirschmann-Makineni Professor of Chemistry Dissertation Committee: Dr. Jeffery G. Saven, Professor of Chemistry Dr. Feng Gai, Edmund J. and Louise W. Kahn Endowed Term Professor of Chemistry Dr. E. Michael Ostap, Professor of Physiology MECHANISMS OF MEMBRANE REMODELING BY PERIPHERAL PROTEINS AND DIVALENT CATIONS COPYRIGHT 2015 Zheng Shi This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit http://creativecommons.org/licenses/by-ny-sa/2.0/ Dedicated to my parents and my sister For their unconditional love and support iii ACKNOWLEDGMENT First of all, I want to express my sincere gratitude to Professor Tobias Baumgart, who has helped me in every aspect possible through the last five years. He is the reason why I fell in love with biophysics and want to become a good scientist in the future. I simply couldn’t have asked for a better advisor. I want to thank my committee members, Professor Jeffery Saven, Professor Feng Gai, and Professor Michael Ostap, for their priceless time, comments, and encouragements. They motivated me to think deeper about my research and were there for me whenever I needed help. I want to thank everyone I have overlapped within the Baumgart Lab, for being amazing labmates and supportive friends. Particularly, I want to thank Dr. Tingting Wu, Zhiming Chen, Dr. Benjamin Capraro, and Dr. Katarzyna Jankowska. We collaborated on multiple fruitful projects and had the best time doing experiments together. I’m also grateful to Dr. Michael Heinrich, who trained me to use optical tweezers when I had little idea about how to carry out an experiment. I want to thank Dr. Chih-Jung Hsu and Dr. Wan-Ting Hsieh for being the nicest people I know and helping me in every way they could when I was new to the lab. I want to thank Ningwei Li, Jaclyn Robustelli, Dr. Zachary Graber, and Vinicius Ferreira for spending valuable time reading and commenting on my manuscripts and thesis chapters. I also want to thank Dr. Chen Zhu, Chun Liu, and Dr. Sanghamitra Deb for sharing and discussing ideas with me. I want to thank Professor Elizabeth Rhoades for her tremendous help with my α- synuclein projects. I also want to thank Professor Kathleen Stebe and Dr. Marcello Cavallaro for teaching me about liquid crystals, Professor Min Wu and Maohan Su for introducing me to FBP17. I also benefited from enlightening discussions with Professor Adam Cohen, Professor Ravi Radhakrishnan, Dr. Ramakrishnan Natesan, Ryan Bradley, Professor Jonathan Sachs, Mike Lacy, Professor James Petersson, Dr. Rebecca Wissner, John Ferrie, Professor Paul Janmey, Dr. David Slochower, Professor Tom Lubensky, Professor Wei Guo, Professor So-Jung Park, and Professor Zahra Fakhraai. I want to thank my friends at Penn Chemistry, particularly, Dr. Yu-Hsiu Wang, Dr. Zhengzheng Liao, Dr. Shi Liu, Chun-Wei Lin, Fan Zheng, Yanxin Wang, Fang Liu, Qingjie Luo, Xinle Liu, and Diomedes Saldana-Greco. They made gradschool much more fun than I thought it would to be. I will never forget the days when we went on road trips, got drunk, or had group studies together. I’m extremely grateful for my parents and my sister who have been spoiling me for more than 25 years. I know they will always be there for me and will always be proud of whatever small things I achieve. I feel bad for missing and sometimes even forgetting their birthdays and other important occasions in the past five years. I wish I will be able to visit them more frequently in the future. iv When writing his best seller ‘A Brief History of Time’, Stephen Hawking was famously warned that for every equation in the book, the readership would be halved. Therefore, at the end, I want to thank my future readers, for being a member of the special group of people who are willing to be tortured by some 90 equations in this thesis, it means a lot to me… v ABSTRACT MECHANISMS OF MEMBRANE REMODELING BY PERIPHERAL PROTEINS AND DIVALENT CATIONS Zheng Shi Tobias Baumgart Biological membranes undergo constant shape remodeling involving the formation of highly curved structures. As one of the most extensively studied membrane remodeling events, endocytosis is a ubiquitous eukaryotic membrane budding, vesiculation, and internalization process fulfilling numerous roles including compensation of membrane area increase after bursts of exocytosis. There are multiple independent endocytic pathways which differ by their speed as well as the proteins that are involved in. Bin/Amphiphysin/Rvs (BAR) domain proteins, such as endophilin, are responsible for sensing or generating membrane curvature in multiple endocytic pathways. In this dissertation, I elucidate the mechanisms of membrane remodeling through in vitro experimental studies with synthetic lipid bilayers. Firstly, I investigated the binding and assembly of endophilin on planar membranes. Endophilin was found to be attracted to the membrane through electrostatic forces and to subsequently oligomerize on the membrane with the help of the protein’s N-terminal helices. vi Next, I studied the mechanisms that govern membrane shape transitions induced by BAR domain proteins. The initiation of membrane curvature occurs at well-defined