University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 10-24-2013 Localization and Dynamics of Myosin Vb in Mammalian Cells Eun-Ji Kim [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Kim, Eun-Ji, "Localization and Dynamics of Myosin Vb in Mammalian Cells" (2013). Doctoral Dissertations. 250. https://opencommons.uconn.edu/dissertations/250 Localization and Dynamics of Myosin Vb in Mammalian Cells Eun-Ji Kim, Ph.D. University of Connecticut, 2013 Abstract The myosin superfamily is a large and diverse family of proteins that are capable of binding to actin filaments and generating mechanical forces by hydrolyzing ATP. Myosins perform a diverse set of functions in cell, including endocytosis, exocytosis, movement of pigment granules or mRNA, and cell motility1. More specifically, Class-V myosins are a group of barbed-end directed motors that have been implicated in organelle transport. For example, in vitro motility assays suggest that myosin Va is a processive motor with a step size of 36 nm2-4 and moves at the speed of 0.6 µm/sec, which is consistent with a functional role in actin-based transport process. Although the properties of myosin V in vitro are relatively well-studied, much less is understood about the motility and protein-protein interaction of myosin V in vivo. Previous studies of molecular motor proteins in vivo have analyzed intracellular cargoes, which are decorated with multiple types of motor proteins5,6, making it difficult to deduce the exact dynamics and the function of a specific motor protein. In this work, we carried out experiments to analyze the localization and the dynamics of myosin Vb in live mammalian cells at both the ensemble level and the level of individual myosin Vb molecules. We found that: 1) motor molecules localize and accumulate at regions enriched in F-actin i Eun-Ji Kim-University of Connecticut, 2013 barbed-ends; 2) individual motor molecules have a high stalling rate when bound to F-actin and do not undergo continuous long-distance movement in cells and 3) over-expression of myosin Vb motor domains promotes filopodia growth. Based on these observations, we propose a novel cellular function of myosin Vb proteins: myosin Vb facilitates linear F-actin growth by moving towards, and eventually binding to, the barbed-ends of individual actin filaments, which in turn prevents capping of the F-actin. Therefore, our study provides new insights into the mechanism of myosin’s function in filopodia formation. ii Localization and Dynamics of Myosin Vb in Mammalian Cells Eun-Ji Kim, Ph.D. B.S. Chonnam National University, 2004 M.A. Central Connecticut State University, 2008 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut [2013] iii Copyright by Eun-Ji Kim [2013] iv APPROVAL PAGE Doctor of Philosophy Dissertation Localization and Dynamics of Myosin Vb in Mammalian Cells. Presented by Eun-Ji Kim, B.S.,M.A. Major Advisor __________________________________________________ Dr. Ji Yu Associate Advisor _______________________________________________ Dr. John H. Carson Associate Advisor _______________________________________________ Dr. Vladimir Rodionov Associate Advisor ________________________________________________ Dr. Srdjan D. Antic University of Connecticut [2013] v Acknowledgements Five years have passed since I joined Dr. Ji Yu’s lab. While in Dr. Ji Yu lab, I met a lot of amazing people including Dr. Sulagna Das, Qingfen Yang, Dr. Jingqiao Zhang, Dr. Veda Tatarvarty, Dr. DongMyung Oh, Erika Hoyos Ramirez, Olena Marchenko, Cliff Locke, Ahmed Elmokadem, Yei feng. They were extremely helpful and patient with me. Without their help and support, my life during PhD training would have been more difficult. I would like to thank Dr. Ji Yu, I feel very lucky to be under his mentorship. He is a brilliant scientist with a wide range of knowledge including Chemistry, Biology, Physics, Mathematics, Programming, etc. His brain blowing ideas towards solving problems in biology kept me awake overnight with curiosity. I would also like to thank my committee members, who were very tough, but very cool. I am a very lucky person to have my committee member, Dr. Vladimir Rodionov whose passion for science has directly transformed me during my PhD training. He has increased my excitement in and helped me greatly in understanding the functioning of motor proteins due to his expertise and experience. He invited amazing scientists whose works are directly related to my work, I was able to meet them and learn from them. I feel very lucky to have him as my committee member. I would like to thank Dr. John Carson, who is very supportive in the opinions of the students and provide continuous motivations while increasing our curiosity by asking a lot of tough questions. His attitude towards science and mentorship had a huge impact on me personally. I feel very lucky to have an opportunity to get close to him. I would like to thank Dr. Srdjan Antic, his one on one approach to students, giving priceless feedback and sincere vi empathy about our performance deemed very useful, and I am very lucky to have met him. I would like to thank Sofya Borinskaya, as her research talks increased my thesis progress enormously. She is a great peer and we working on similar topic helped to increased my scientific knowledge. I would like to thank Dr. Akeisha Belgrave, I start PhD training with her, we went through a lot of things together, and are finally finishing up at the same time. Cheers to both of us. I would like to thank Dr. Yi Wu and Dr. Taofei Yin. They helped me with lot of things including reagents and very valuable feedback. I would like to thank Jian-Ping Haung and Dr. Xin-Ming Ma who give me a lot of help and mentorship. Special thanks to JP! I would like to thank the other people in CCAM including Dr. Raquell Homes, Dr. Jing Yang, Dr. Cibelle Falkenberg. Dr. Ann Cowan, Dr. Leslie Loew, Dr. Boris Slepchenko, Dr. Igor Novak, Dr. Michael Blinov, Dr. Ion Moraru, Dr. Charles Wogemuth, Dutton Jeff, and CCAM students Rene Norman, Abhijit Deb Roy. Lastly, I would like to thank my parents, and my brother. My parents are my biggest support, my role models, my best friends. I really appreciate their supports and endless love. I would like to thank my husband (my unofficial committee member), who taught me a lot of things and helped me a lot to finish up. He, dedicated his time to me, helped my unstable mood, tolerated my busy life and supported my dream. vii TABLE OF CONTENTS ACKNOWLEDGEMENTS………………………………………………………………… vi LIST OF FIGURES ……………………………………………………………………….. xi ABBREVIATIONS…………………………………………………………………………. xii Chapter 1..……………………………………………………………………………… 1 1.1 Introduction to Myosin Motor Proteins………...………………………….…….. 1 1.1.1 Structure and function of Myosins………………...…………………………… 1 1.1.2 In vitro properties of Myosin Vb …………………..……………………………. 3 1.1.3 In vivo function of Myosin Vb…………………..………………………….......... 5 1.2 Myosin Vb’s role in intracellular transport………………..…………………….. 7 1.2.1 Processivity and in vivo cargo transport……………………..………………… 7 1.2.2 Regulation of Myosin Vb cargo binding………………………………..………. 8 1.3 Myosin Vb’s role in filopodia formation ………………………………….……… 9 1.3.1 Actin cytoskeleton ……………………………………………...………………... 9 1.3.2 Mechanism of filopodia formation………………………………………..……... 11 1.3.3 Myosin Vb is required for microvilli formation……………………….………… 13 1.3.4 Other myosins implicated in filopodia formation.……………………………… 14 1.4 Single molecule imaging of Single Myosin Motor in cells………………...….. 15 1.5 Thesis hypothesis and objectives……………………………………………..….. 16 Chapter 2……………………………………………………….….. 18 2.1 Abstract………………………………………………………………………………… 18 viii 2.2 Introduction…………………………………………………………………………… 18 2.3 Material and method…………………………………………………………………. 20 2.3.1 Cloning and Mutagenesis…………………………………………………….….. 20 2.3.2 Cell culture and transfection……………………………………………………… 21 2.3.3 Drug treatment…………………………………………………………………….. 21 2.3.4 Western blotting…………………………………………………………………… 21 2.3.5 Single molecule Imaging and analysis………………………………………….. 22 2.3.6 MSD analysis………………………………………………………………………. 23 2.4 Result…………………………………………………………………………………… 23 2.4.1 Myosin Vb localizes to cell edge………………………………………………… 23 2.4.2 Cell edge localization of myosin Vb depends on motor activity……………… 25 2.4.3 Single molecule quantification of myosin Vb motility in live cells…………….. 25 2.4.4 Myosin Vb processivity…………………………………………………………… 28 2.4.5 Rif expression increase dissociation rate of myosin molecules from cortical actin………………………………………………………………………………… 29 2.4.6 Myosin Vb tethering at actin barbed-end……………………………………….. 30 2.5 Discussion…………………………………………………………………………….. 32 Chapter 3……………………………………………………….….. 34 3.1 Abstract………………………………………………………………………………… 34 3.2 Introduction…………………………………………………………………………… 34 3.3 Material and methods……………………………………………………………….. 36 3.3.1 Cloning and constructs…………………………………………………………… 36 3.3.2 Cell culture and transfection……………………………………………………… 37 ix 3.3.3 Immunofluorescence and antibodies……………………………………………. 37 3.3.4 Total internal reflective fluorescent (TIRF) imaging and live cell imaging…... 38 3.3.5 Confocal imaging and fluorescent recovery after photobleaching (FRAP)…. 38 3.4 Results………………………………………………………………………………..... 39 3.4.1 Myosin Vb induce filopodia formation………………………………………....... 39 3.4.2 Myosin Vb forms tight focus at the growing filopodia