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The Role of Kinesin-2 in Navigating Microtubule Obstacles: Implications for the Regulation of Axonal Transport Gregory Hoeprich University of Vermont

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The Role of Kinesin-2 in Navigating Microtubule Obstacles: Implications for the Regulation of Axonal Transport Gregory Hoeprich University of Vermont University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2016 The Role Of Kinesin-2 In Navigating Microtubule Obstacles: Implications For The Regulation Of Axonal Transport Gregory Hoeprich University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Biophysics Commons Recommended Citation Hoeprich, Gregory, "The Role Of Kinesin-2 In Navigating Microtubule Obstacles: Implications For The Regulation Of Axonal Transport" (2016). Graduate College Dissertations and Theses. 558. https://scholarworks.uvm.edu/graddis/558 This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. THE ROLE OF KINESIN-2 IN NAVIGATING MICROTUBULE OBSTACLES: IMPLICATIONS FOR THE REGULATION OF AXONAL TRANSPORT A Dissertation Presented by Gregory Joseph Hoeprich to The Faculty of the Graduate College of The University of Vermont In Partial Fulfilment of the Requirements For the Degree of Doctor of Philosophy Specializing in Molecular Physiology and Biophysics May, 2016 Defense Date: March 8, 2016 Dissertation Examination Committee: Christopher Berger, Ph.D., Advisor Victor May, Ph.D., Chairperson David Warshaw, Ph.D. Teresa Ruiz, Ph.D. Jason Stumpff, Ph.D. Cynthia J. Forehand, Ph.D., Dean of the Graduate College ABSTRACT Neurons are specialized cells that transmit information through electrical and chemical signals using structural processes known as dendrites and axons. Dendrites receive information for the cell to interpret while the exceedingly long axon transmits the processed information to its target destination. To ensure the neuron properly carries out its extracellular functions, the orchestration of intracellular cargo (e.g. mitochondria) is critical. This is especially true in the axon, which can be up to a meter in length. There are many challenges involved in the spatial and temporal regulation of cargo over such vast cellular distances. In order to accomplish cargo transport between the cell body and axon terminus the neuron has developed an efficient process to overcome this challenge called axonal transport. Axonal transport utilizes a system of molecular motors coupled to cargo, creating a multi-motor complex, which walks along a set of tracks to position the cargo at the right time and place. One class of molecular motors, called kinesin, are used to traffic cargo away from the cell body and walk along microtubule tracks. These motors work in teams to navigate a complex microtubule landscape that is rich in microtubule-associated proteins (MAPs). One MAP abundantly found within the axon is called Tau and is implicated in a variety of neurodegenerative disorders (e.g. Alzheimer’s disease). Much attention has been focused on the kinesin-1 motor while investigating the axonal transport process. However, kinesin-2 plays an equally important role and is not as well characterized as kinesin-1. Previously, it has been demonstrated, in vitro, that Tau disrupts kinesin-1 transport, even below physiological concentrations, however, in vivo evidence suggests the contrary. Given this discrepancy, there are likely other cellular systems in place to provide the necessary navigation of Tau obstacles. One solution may involve multi-motor complexes using two kinesin family members attached to cargo, as both kinesin-1 and kinesin-2 have been observed coupled to cargo. In order to peel away the complex layers of kinesin-1 and kinesin-2 coupled cargo inside the axon, single-molecule imaging techniques were employed to observe the individual behavior of both kinesin-1 and kinesin-2, in vitro. Further, using a combination of genetic engineering, single-molecule analysis and mathematical modeling has helped elucidate differences between these two motors. Kinesin-2 was found to be insensitive to Tau obstacles, unlike kinesin-1, which is in part due to a longer region of the motor called the neck-linker. This region connects the motor domain, which interfaces with the microtubule track, to the coiled-coil stock, which interfaces with the cargo. When the neck- linker lengths were swapped between the motors their behavior in the presence of Tau also switched, and kinesin-2 became sensitive to Tau. To understand kinesin-2’s mechanism of navigating Tau obstacles, we looked at the lateral stepping characteristics of both motors. We observed kinesin-2’s lateral stepping frequency to be higher than kinesin-1 and independent of the microtubule obstacle concentration. Thus, kinein-2’s longer neck- linker allows a more agile walk along the microtubule surface to navigate obstacles more efficiently than kinesin-1. In a multi-motor complex containing both motors, kinesin-2 is more efficient at maneuvering around MAPs while kinesin-1, which has previously been demonstrated to sustain a higher stall force, is more efficient at towing cargo. This work demonstrates how teams of directionally similar motors may work together to position cargo during axonal transport. CITATION Material from this dissertation has been published in the following form: Hoeprich, G. J., Thompson, A.R., McVicker, D.P., Hancock, W.O., Berger, C.L.. (2014). Kinesin's Neck-Linker Determines its Ability to Navigate Obstacles on the Microtubule Surface. Biophysical Journal, 106(8), 1691-1700. ii DEDICATION To my entire family, especially my parents (Michael and Marie) and my brothers (Stephen, Mark and David), I will always be indebted to you for all your help, understanding, and support throughout my schooling. Above all, I would like to thank my fiancée Audrey DiMauro who has always provided me with love and support. Audrey, you have helped me stay levelheaded throughout this whole process and I could not have done this without you! This dissertation is for all of you! iii ACKNOWLEDGMENTS I am grateful to my advisor and mentor, Dr. Christopher Berger, for his guidance and wisdom during my time at UVM. He has been instrumental in teaching me how to become an independent scientist and has helped shape the person and scientist I am today. I thank my committee members Dr. Victor May, Dr. David Warshaw, Dr. Teresa Ruiz, Dr. Jason Stumpff and Dr. Christopher Berger for their support and insight. I thank all of the members of the Berger Lab past and present for their support: Dr. Justin Decarreau, Dr. Andrew Thompson, Dr. Derrick McVicker, Lynn Chrin, Jamie Stern, Rehan Ali, Miranda Redmond and Dominique Lessard. I also thank our collaborators and others who have been invaluable throughout this process: Dr. Gerardo Morfini, Dr. William Hancock, all members from the Warshaw and Stumpff Labs and MPBP graduate students. I thank the Department of Molecular Physiology and Biophysics (especially Julie Lovelette, Margaret Corson and Guy Kennedy) and the Cellular, Molecular and Biomedical Sciences Program for all their support. iv TABLE OF CONTENTS Page CITATION ........................................................................................................................ ii DEDICATION.................................................................................................................. iii ACKNOWLEGEMENTS ............................................................................................... iv LIST OF TABLES ......................................................................................................... viii LIST OF FIGURES ......................................................................................................... ix LIST OF ABBREVIATIONS ......................................................................................... xi CHAPTER 1: INTRODUCTION .....................................................................................1 AXONAL TRANSPORT ................................................................................................1 MICROTUBULES ..........................................................................................................4 Microtubule-Associated Proteins (MAPs).............................................................. 10 Tau Protein ..............................................................................................................13 KINESIN MOTORS .....................................................................................................16 Kinesin-1 .................................................................................................................17 Kinesin-2 .................................................................................................................20 MULTI-MOTOR COMPLEXES ..................................................................................24 Adaptor Proteins ....................................................................................................24 Motor Regulation During Axonal Transport ..........................................................25 SCOPE AND PURPOSE .............................................................................................29 CHAPTER 2: KINESIN’S NECK-LINKER DETERMINES ITS ABILITY TO NAVIGATE OBSTACLES ON THE MICROTUBULE SURFACE .................32 ABSTRACT .................................................................................................................33 INTRODUCTION ........................................................................................................34
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