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Formin3 Regulates Dendritic Architecture and Is Required for Somatosensory Nociceptive Behavior

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Formin3 Regulates Dendritic Architecture and Is Required for Somatosensory Nociceptive Behavior Georgia State University ScholarWorks @ Georgia State University Neuroscience Institute Dissertations Neuroscience Institute 12-15-2016 Formin3 Regulates Dendritic Architecture and is Required for Somatosensory Nociceptive Behavior Ravi Das Follow this and additional works at: https://scholarworks.gsu.edu/neurosci_diss Recommended Citation Das, Ravi, "Formin3 Regulates Dendritic Architecture and is Required for Somatosensory Nociceptive Behavior." Dissertation, Georgia State University, 2016. https://scholarworks.gsu.edu/neurosci_diss/27 This Dissertation is brought to you for free and open access by the Neuroscience Institute at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Neuroscience Institute Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. FORMIN3 REGULATES DENDRITIC ARCHITECTURE AND IS REQUIRED FOR SOMATOSENSORY NOCICEPTIVE BEHAVIOR by RAVI DAS Under the Direction of Daniel N. Cox, PhD ABSTRACT Cell-type specific dendritic morphologies emerge via complex growth mechanisms modulated by intrinsic and extrinsic signaling coupled with activity-dependent regulation. Combined, these processes converge on cytoskeletal effectors to direct dendritic arbor development, stabilize mature architecture, and facilitate structural plasticity. Transcription factors (TFs) function as essential cell intrinsic regulators of dendritogenesis involving both combinatorial and cell-type specific effects, however the molecular mechanisms via which these TFs govern arbor development and dynamics remain poorly understood. Studies in Drosophila dendritic arborization (da) sensory neurons have revealed combinatorial roles of the TFs Cut and Knot in modulating dendritic morphology, however putative convergent nodal points of Cut/Knot cytoskeletal regulation remain elusive. Here we use a combined neurogenomic, bioinformatic, and genetic approach to identify and molecularly characterize downstream effectors of these TFs. From these analyses, we identified Formin3 (Form3) as a convergent transcriptional target of both Cut and Knot. We demonstrate that Form3 functions cell-autonomously in class IV (CIV) da neurons to stabilize distal higher order branching along the proximal-distal axis of dendritic arbors. Furthermore, live confocal imaging of multi-fluor cytoskeletal reporters and IHC analyses reveal that form3 mutants exhibit a specific collapse of the dendritic microtubule (MT) cytoskeleton, the functional consequences of which include defective dendritic trafficking of mitochondria and satellite Golgi. Biochemical analyses reveal Form3 directly interacts with MTs via the FH1/FH2 domains. Form3 is predicted to interact with two alpha-tubulin N-acetyltransferases (ATAT1) suggesting it may promote MT stabilization via acetylation. Analyses of acetylated dendritic MTs supports this hypothesis as defects in form3 lead to reductions, whereas overexpression promotes increases in MT acetylation. Neurologically, mutations in Inverted Formin 2 (INF2; the human ortholog of form3) have been causally linked to dominant intermediate Charcot-Marie-Tooth (CMT) disease E. CMT sensory neuropathies lead to distal sensory loss resulting in a reduced ability to sense heat, cold, and pain. Intriguingly, disruption of form3 function in CIV nociceptive neurons results in a severe impairment in nocifensive behavior in response to noxious heat, which can be rescued by expression of INF2 revealing shared primordial functions in regulating nociception and providing novel mechanistic insights into the potential etiological bases of CMT sensory neuropathies. INDEX WORDS: Formin3, CMT, Cytoskeleton, Transcription factors, Dendrite development, Microtubule stabilization. FORMIN3 REGULATES DENDRITIC ARCHITECTURE AND IS REQUIRED FOR SOMATOSENSORY NOCICEPTIVE BEHAVIOR by RAVI DAS A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the College of Arts and Sciences Georgia State University 2016 Copyright by Ravi Das 2016 FORMIN3 REGULATES DENDRITIC ARCHITECTURE AND IS REQUIRED FOR SOMATOSENSORY NOCICEPTIVE BEHAVIOR by RAVI DAS Committee Chair: Daniel Cox Committee: Nancy Forger Walter Walthall Giorgio Ascoli Electronic Version Approved: Office of Graduate Studies College of Arts and Sciences Georgia State University December 2016 iv DEDICATION I would like to dedicate this to my family. I would like to thank my mom, Ajit Kaur, who gave me the most unscientific support of all - believing that I could do anything I set out to do without needing any facts to buttress this belief. I would like to thank my brother, sister, brother-in-law, sister-in-law and my dad for their moral support and encouragement. One person who has journeyed with me throughout my PhD from the day one is my wife to be Aurora Sánchez Palacio. I would like her to know that the most difficult chapter of my PhD was to pursue it being far from her, but, her unwavering support and encouragement to pursue my passion for science made this day possible. Thank You Aury, this is for you. Finally, but definitely not least, I would like to thank my dear friends, Aaditya Prakash and Brijesh Mallik, who from my childhood onwards have always been there for me, despite the distance. v ACKNOWLEDGEMENTS The successful completion of this study required a lot of guidance, collaboration and assistance from many people. Whatever I have been able to do today is only due to all the brilliant people I had the chance to work with - each and every Professor, colleague and student around me these years has been an inspiration and made me think deeper about how to improve my project. First and foremost, I would like to express my deepest appreciation and gratitude to my mentor and friend, Dr. Daniel Cox, for believing in me and motivating me to strive to produce the best work possible. His constant supervision while in turn giving me leeway to propose new ideas helped me complete this work. He has helped me develop the ability to ask the right questions and to set out the path to answer them, and more importantly, he has taught me to deepen my fascination for scientific inquiry. He has always been available for my questions, and the positivity and generosity of his time, together with his vast knowledge kept me motivated to work harder every day. He always knew where to direct me for the answers to the obstacles I had to face. Joining his lab has been one of the best decisions I have made for my professional life, and seeing how me and my colleagues are becoming more and more accomplished scientists every day convinces me that his hard work and devotion to his students makes him the best mentor that anybody can ever ask for. Secondly, I would like to thank my two rockstar undergraduates, Jenna Harris and Hansley Bobo for all their hard work and time commitment during the course of my PhD. Without their help in hours and hours of neural reconstructions, fly genetics and painstaking fly behavior analyses, the completion of this work would have been immeasurably more difficult. Thirdly, I would like to thank Shatabdi Bhattacharjee and Atit Patel for their help with the screen; Surajit Bhattacharya for his help with the bioinformatics; Sarah Trunnell for the Xmas flies vi (GMA-Jupiter); Uy Nguyen for his help in neural reconstructions; Darshan Patel for developing Flyboys and all the Cox lab members for their moral support and feedback. I would like to extend my appreciation to my committee members, Drs. Nancy Forger, Bill Walthall, and Giorgio A. Ascoli for their consistent support, illuminating suggestions, encouragement, constructive criticism and direction during the course of this dissertation. I owe a big and special thank you to Dr. Akinao Nose for having kept the Formin3 reagents for over a decade and generously gifting them to us which served invaluable for my project. I would also like to thank Sumit Nanda and Dr. Ascoli for their help and collaboration with the computational aspects of this dissertation, and Drs. Istvan Foldi and József Mihály for their help and collaboration work for the Formin project. Finally, I would like to thank Georgia State University, which has been my home for the past three years, the Brains and Behavior program and the Neuroscience Institute for providing me this great opportunity to complete my study here. vii TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................. v LIST OF TABLES .......................................................................................................... xii LIST OF FIGURES ....................................................................................................... xiii LIST OF ABBREVIATION.......................................................................................... xvi 1 INTRODUCTION ........................................................................................................ 1 1.1 Overview – Dendrites and the Cytoskeleton ..................................................... 1 1.2 Biomedical Relevance .......................................................................................... 5 1.3 Developmental Neurogenetics ............................................................................ 6 1.4 Neurogenetic and Neurogenomic Techniques .................................................. 8 1.5 Advancements in microscopy ..........................................................................
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