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Identification of Plant SUN Domain-Interacting Tail Proteins and Analysis of Their Function in Nuclear Positioning

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Identification of Plant SUN Domain-Interacting Tail Proteins and Analysis of Their Function in Nuclear Positioning Identification of Plant SUN Domain-Interacting Tail Proteins and Analysis of Their Function in Nuclear Positioning DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Xiao Zhou, M. S. Graduate Program in Plant Cellular and Molecular Biology The Ohio State University 2013 Dissertation Committee: Professor Iris Meier, Advisor Professor Biao Ding Professor Stephen Osmani Professor R. Keith Slotkin Copyright by Xiao Zhou 2013 Abstract The nuclear envelope (NE) is a double membrane system consisting of an inner nuclear membrane (INM) and an outer nuclear membrane (ONM). Studies in opisthokonts revealed that the two membranes are bridged by protein complexes formed by the INM Sad1/UNC-84 (SUN) proteins and the ONM Klarsicht/ANC-1/Syne-1 homology (KASH) proteins. These SUN-KASH NE bridges are usually linkers of the nucleoskeleton to the cytoskeleton (LINC) conserved across eukaryotes. LINC complexes are key players in multiple cellular processes, such as nuclear and chromosomal positioning and nuclear shape determination, which in turn influence the gametogenesis and several aspects of development. Although these cellular processes have long been also known in plants, no KASH proteins are encoded in the plant genomes. I identified WPP domain interacting proteins (WIPs) as the first plant KASH protein analogs. WIPs are plant-specific ONM proteins that redundantly anchor Ran GTPase activating protein (RanGAP) to the NE. Arabidopsis thaliana WIPs (AtWIPs) interact with Arabidopsis thaliana SUN proteins (AtSUNs), which is required for both AtWIP1 and AtRanGAP1 NE localization. In addition, AtWIPs and AtSUNs are necessary for maintaining the elongated nuclear shape of Arabidopsis epidermal cells. These data ii provide a novel function of the SUN-dependent NE bridges, suggesting that a functionally diverged SUN-dependent NE bridge is conserved beyond the opisthokonts. Further analysis of AtWIPs and its binding partner Arabidopsis thaliana WPP domain- interacting tail anchored proteins (AtWITs) revealed that they are localized to the vegetative NE in pollen and play a role in the nuclear movement of the vegetative nucleus (VN) during pollen tube growth. Loss of AtWIPs or AtWITs resulted in impaired VN movement and inefficient sperm-cell-to-ovule delivery. AtWIPs and AtWITs are the first genes assigned to nuclear movement during pollen tube growth. WIPs have no similarity to known opisthokont KASH proteins, except for a C-terminal transmembrane domain, followed by a short SUN-domain interacting tail (SIT) domain terminating in a conserved four-amino-acid motif critical for the SUN-WIP interaction. This conserved pattern was used to computationally search for candidate SIT proteins. As a second criterion, I asked for conservation of the rule in most homologs of a protein family. I identified 10 new potential SIT protein families, four of which were verified for their SUN-interaction-dependent NE localization. One SIT protein, Arabidopsis thaliana AN NEMO 1, was shown to be associated with F-actin and involved in guard cell nuclear central anchorage. This study dramatically expands the number of SIT proteins and implies an independent evolution of SUN-dependent NE-bridges after the opisthokont- plant separation. iii Dedication This document is dedicated to the nerds who still have a dream in science. iv Acknowledgments I was extremely fortunate to get the opportunity to work under the instruction of my advisor, Professor Iris Meier who offered me a free-thinking research environment and guided me to an interesting research field. Without her continuous guidance, support and motivation, this dissertation would not have been possible. I am grateful to all my committee members, Professor Biao Ding who always challenged my critical thinking, Professor Steve Osmani who was very strict on my logic and data interpretation, and Professor Keith Slotkin who provided helpful advice and support on the project described in CHAPTER 3. I would also like to thank all my lab-mates through the years: Dr. Jelena Brkljacic, Dr. Sowmya Venkatakrishnan, Dr. Sivaramakrishnan Muthuswamy, Dr. Thushani Rodrigo- Dr. Peiris, Dr. Joanna Boruc, Dr. Mintu Desai, Dr. Chris DeFraia, Alex Tough, Anna Newman, Norman Groves, and other undergraduate assistants. I am grateful to their warmth, encouragement, and helpful discussion. I also want to thank Emily Yoders-Horn and Joan Leonard who took good care of my plants and set up the delightful greenhouse pond. v I am sincerely thankful to Dr. Katja Graumann and Professor David Evans for the delightful collaboration on the SUN-WIP project (CHAPTER 2). I want to thank Dr. Xianfeng Xu who left detailed information on his materials which saved me considerable time on this project. I want to thank my parents who always pointed out the negative side of my life as a researcher. This let me understand how to communicate with people outside of my research field. In the end, I want to thank Dr. Yiyi Zhang, without her encouragement, I would not have come to study in the Ohio State University. vi Vita 2004 B.S. Bioengineering, East China University of Science and Technology 2007 M.S. Biochemistry and Molecular Biology, Fudan University 2007 to present Graduate Researching Associate, Department of Molecular Genetics, The Ohio State University Publications Zhou, X., J. Boruc, and I. Meier. 2013. The Plant Nuclear Pore Complex — The Nucleocytoplasmic Barrier and Beyond. In Annual Plant Reviews. John Wiley & Sons Ltd. 57-91. Zhou, X., and I. Meier. 2013. How plants LINC the SUN to KASH. Nucleus. 4:206-215. Zhou, X., K. Graumann, D.E. Evans, and I. Meier. 2012. Novel plant SUN-KASH bridges are involved in RanGAP anchoring and nuclear shape determination. Journal of Cell Biology. 196:203-211. Boruc, J., X. Zhou, and I. Meier. 2012. Dynamics of the Plant Nuclear Envelope and Nuclear Pore. Plant Physiology. 158:78-86. Meier, I., X. Zhou, J. Brkljacic, A. Rose, Q. Zhao, and X.M. Xu. 2010. Targeting proteins to the plant nuclear envelope. Biochemical Society Transactions. 38:733-740. Fields of Study Major Field: Plant Cellular and Molecular Biology vii Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments............................................................................................................... v Vita .................................................................................................................................... vii Publications ....................................................................................................................... vii Fields of Study .................................................................................................................. vii List of Tables .................................................................................................................... xv List of Figures .................................................................................................................. xvi CHAPTER 1 Essential function of SUN-KASH complexes in opisthokont nuclear positioning and the importance of plant nuclear positioning .............................................. 1 1.1 Abstract ..................................................................................................................... 2 1.2 Introduction to SUN and KASH proteins ................................................................. 3 1.3 Function of opisthokont SUN and KASH proteins ................................................... 4 viii 1.3.1 Nuclear positioning mediated by opisthokont LINC complexes........................ 4 1.3.2 Chromosome movement mediated by opisthokont LINC complexes ................ 7 1.3.3 Nuclear pore complex assembly regulated by SUN1 ......................................... 7 1.4 Plant SUN proteins and their functions ..................................................................... 8 1.4.1 Plant SUN proteins ............................................................................................. 8 1.4.2 Possible function of plant SUN proteins in mitosis and meiosis ........................ 9 1.5 Nuclear positioning in plants................................................................................... 10 1.5.1 Nuclear movement of pollen tubes ................................................................... 11 1.5.2 Nuclear movement of root and leaf hair ........................................................... 13 1.5.3 Nuclear positioning during asymmetric cell divisions ..................................... 14 1.5.4 Root nuclear movement responding to microbes ............................................. 15 1.5.5 Nuclear positioning responding to physical stimuli ......................................... 16 1.6 Plant KASH analogs involved nuclear positioning ................................................. 17 CHAPTER 2 Novel plant nuclear envelope bridges are involved in RanGAP anchoring and nuclear shape determination ....................................................................................... 28 2.1 Abstract ................................................................................................................... 29 2.2 Introduction ............................................................................................................
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