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Investigating Novel Regulators of Golgi Membrane Tubulation

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Investigating Novel Regulators of Golgi Membrane Tubulation INVESTIGATING NOVEL REGULATORS OF GOLGI MEMBRANE TUBULATION A Dissertation Presented to the Faculty of the Graduate School of Cornell University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Kevin Dinh Ha August 2012 © 2012 Kevin Dinh Ha INVESTIGATING NOVEL REGULATORS OF GOLGI MEMBRANE TUBULATION Kevin Dinh Ha, Ph.D. Cornell University 2012 The Golgi complex serves as a vital organelle from which proteins and membrane lipids are modified, sorted, and trafficked to various destinations. Mutations that cause defects in structural maintenance or membrane trafficking at the Golgi are commonly linked to neurodegeneration, metabolic disease, and reproductive disorders. Both structural maintenance and membrane trafficking rely on cooperative efforts of coated vesicles and membrane tubules. Although extensive information is available for membrane coated vesicle traffic, knowledge of membrane tubules remains comparably deficient. Understanding the regulatory mechanisms behind membrane tubules may help elucidate how Golgi tubule biogenesis can respond to varying physiological stimuli such as increased secretory loads. I utilized an siRNA library against all known and purported human kinases, or the kinome, in a high throughput, microscopy-based screen that identified proteins involved in Brefeldin A (BFA)-induced Golgi membrane tubulation. This screen successfully identified siRNAs that significantly inhibited or enhanced the effects of BFA-induced Golgi tubulation. Among the identified hits, I further characterized two inhibitory siRNA that targeted Protein- Associating with the Carboxyl-terminal domain of Ezrin (PACE1) and diacylglycerol kinase γ (DGK-γ), and determined that they play important roles in maintaining intact Golgi ribbon structures through regulating Golgi membrane tubule biogenesis. I found that these proteins also facilitate Golgi reassembly and anterograde membrane trafficking of both soluble and transmembrane proteins, further buttressing the importance of membrane tubules in multiple, cellular processes. The results of my studies will be useful not only in contributing to the understanding of existing regulatory mechanisms behind membrane tubulation but also in discovering novel mechanisms. Further studies on membrane tubulation will help shed light on physiological functions and possibly lead to translational advances in diseases that depend on membrane tubulation and/or trafficking. BIOGRAPHICAL SKETCH Kevin D. Ha was born in Sacramento, California. He attended the University of California, San Diego, where he obtained his B.S. in Molecular Biology and conducted undergraduate research studying the biochemical mechanisms underlying early onset of Alzheimer’s disease. He subsequently worked as an associate research scientist with the Discovery Team within the Department of Cell Biology at Pfizer in La Jolla, California, investigating novel therapeutic antibodies against numerous cancers. While at Pfizer, Kevin harnessed a deep interest in cellular biology and advance technologies. He subsequently joined the Graduate Field of Biochemsitry, Molecular and Cell Biology in the department of Molecular Biology and Genetics at Cornell University in 2006. He later joined the laboratory of Dr. Bill Brown in 2007, where he studied Golgi membrane tubulation. He successfully defended his Ph.D. dissertation in 2012 and accepted a postdoctoral fellowship at the University of California, San Francisco, under the mentorship of Dr. Bin Liu, where he will resume translational studies on cancers, particularly discovering novel therapeutic antibodies via a phage display screen. Kevin used to have numerous hobbies, including computers, computer software programming, web-design, photography, music production, turntablism, screenwriting, cinematography, weight lifting, fishing, following practically all sports, and cooking. The arrival of Kevin’s two children, Atticus and Melia, saw the departure of his hobbies. His hobbies were quickly replaced with diaper-changing, spoon-feeding, book readings, stroller walks, picking up toys from the ground, and collecting spittle and drool on his clothing. iii For my loving family, Samantha, Atticus, and Melia For my supportive family, Eric, Tony, Ruby, Lisa, and my mom But especially for my father and Davis The winds carry the ashes peacefully to the skies iv ACKNOWLEDGEMENTS I would like to kindly thank my principal investigator, Dr. Bill Brown, in providing me the opportunity to conduct research within his laboratory and for providing me intellectual and personal mentorship. Dr. Brown has been thoroughly supportive of me during my entire stay and I truly appreciate his style of diplomacy, his great sense of humor, and of course, his pies and cakes. I would also like to thank my committee members, Dr. Volker Vogt and Dr. Rick Cerione. I am grateful that Dr. Vogt recruited me to Cornell University, provided me invaluable advice, allowed me to rotate in his laboratory, and allowed me to be his teaching assistant in his cell biology undergraduate course, all of which I still carry fond memories of. I thank Dr. Cerione for his exceptional advice and for granting me the opportunity to rotate within his laboratory. I would like to thank Dr. Fenghua Hu for generously providing me training and usage of her excellent equipment. Without Dr. Hu, much of my work would not be feasibly possible. I would like to thank Raghuvir “Ram” Viswanatha, Felipe Santiago, and Damien Garbett from the Bretscher lab for providing reagents. I would like to thank my good friends at Cornell University, Matt Bratkowski, Lynda Goh, Justin Sibert, Zach Via, Rich McCloskey, Brady O’Hanlon, Benjamin Clarke, Jeremy Bird, and Amber Krauchunas for providing all sorts of entertainment. I would like to especially thank Karl Ruggeberg, my best friend at Cornell, for providing countless days of friendship. I would like to thank my lab colleagues, both former and current: Amy Antosh for being not only a great mentor in electron microscopy but also a good friend, Danielle Kalkofen, Marie v Bechler, John Schmidt, Elysa Goldberg, and Ben Clarke for their thoughtful research advice, and Eda Dou, Ina Chen, and Griselda Yvone for providing a dynamic and interesting environment at work. I am also truly grateful for the presence of Sricharan Murugesan in the lab, as he provided countless discussions on everything sports-related. I will surely miss the presence of a sports fanatic at work. I would like to thank my large family, Eric, Tony, Ruby, Lisa, and my mom. Although I moved away far across the country, my family still managed to maintain a close relationship. Lastly, I would like to thank my own family, Samantha Kim Truong, Atticus Eamon Ha, and Melia Katelyn Ha for their loving, moral support. vi TABLE OF CONTENTS BIOGRAPHICAL SKETCH ......................................................................................................... iii DEDICATION .............................................................................................................................. iv ACKNOWLEDGEMENTS .............................................................................................................v TABLE OF CONTENTS .............................................................................................................. vii LIST OF FIGURES ....................................................................................................................... ix LIST OF TABLES ...........................................................................................................................x CHAPTER 1. Mechanisms of Membrane Trafficking 1.1. Abstract ............................................................................................................................... 2 1.2. Introduction ......................................................................................................................... 3 1.3. Models of intra-Golgi trafficking ........................................................................................ 4 1.4. Significance of membrane tubules ...................................................................................... 6 1.5. Involvement of lipid-modifying enzymes in membrane tubulation .................................... 9 1.6. Review of lipid-modifying enzymes in Golgi membrane dynamics ................................ 10 1.7. PLA and LPAT enzymes remodel phospholipids and the Golgi complex ....................... 14 1.7.1. PAFAHIb ................................................................................................................ 18 1.7.2. cPLA2α .................................................................................................................... 24 1.7.3. PLA2G6/iPLA2-β .................................................................................................... 27 1.7.4. iPLA1γ ..................................................................................................................... 29 1.7.5.Lysophospholipid acyltransferases .......................................................................... 30 1.8. Integration of cPLA2α and AGPAT3/LPAAT3 to influence Golgi COPI vesicle and membrane tubule formation ..................................................................................................... 35 1.9. Conclusions and future directions ..................................................................................... 41 REFERENCES .............................................................................................................................
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