University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2018 Cargo Specific Regulation Of Cytoplasmic Dynein By Effector Proteins Mara Olenick University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry Commons, Biophysics Commons, and the Cell Biology Commons Recommended Citation Olenick, Mara, "Cargo Specific Regulation Of Cytoplasmic Dynein By Effector Proteins" (2018). Publicly Accessible Penn Dissertations. 3167. https://repository.upenn.edu/edissertations/3167 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/3167 For more information, please contact [email protected]. Cargo Specific Regulation Of Cytoplasmic Dynein By Effector Proteins Abstract Axonal transport is vital for the development and survival of neurons. The transport of cargo and organelles from the axon to the cell body is driven almost completely by the molecular motor, cytoplasmic dynein. Yet, it remains unclear how dynein is spatially and temporally regulated given the variety of cargo that must be properly localized to maintain cellular function. Previous work has suggested that adaptor proteins provide a mechanism for cargo-specific egulationr of motors. During my thesis work, I have investigated the role of mammalian Hook proteins, Hook1 and Hook3, as potential motor adaptors. Using optogenetic and single molecule assays, I found that Hook proteins interact with both dynein and dynactin, to effectively activate dynein motility, inducing longer run lengths and higher velocities than the previously characterized dynein activator, BICD2. In addition, I found that complex formation requires the N-terminal domain of Hook proteins, which resembles the calponin-homology domain of EB proteins yet cannot bind directly to microtubules. In collaborative studies, we found the Hook domain directly interacts with a helix of the dynein light intermediate chain and this interaction is important for Hook-induced processive motility of dynein. In my final project, I found that Hook1 mediates the transport of TrkB-BDNF signaling endosomes in primary hippocampal neurons. Using live cell microscopy and microfluidic devices, Hook1 depletion resulted in a significant decrease in the flux and processivity of BDNF-Qdots along the mid-axon, an effect specific for Hook1 but not Hook3. ogetherT , my work suggests that dynein effectors like Hook proteins can differentially regulate dynein to allow for organelle-specific tuning of the motor for precise intracellular trafficking. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Biochemistry & Molecular Biophysics First Advisor Erika L. Holzbaur Subject Categories Biochemistry | Biophysics | Cell Biology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/3167 CARGO SPECIFIC REGULATION OF CYTOPLASMIC DYNEIN BY EFFECTOR PROTEINS Mara Alizabeth Olenick A DISSERTATION in Biochemistry and Molecular Biophysics Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2018 Supervisor of Dissertation __________________________ Erika L. F. Holzbaur, PhD, Professor of Physiology Graduate Group Chairperson __________________________ Kim A. Sharp, PhD, Associate Professor of Biochemistry and Biophysics Dissertation Committee E. Michael Ostap, PhD, Professor of Physiology Roberto Dominguez, PhD, Professor of Physiology Michael A. Lampson, PhD, Associate Professor of Biology Erfei Bi, PhD, Professor of Cell and Developmental Biology CARGO SPECIFIC REGULATION OF CYTOPLASMIC DYNEIN BY EFFECTOR PROTEINS COPYRIGHT 2018 Mara Alizabeth Olenick ACKNOWLEDGMENT Getting a PhD can be a very long journey, full of ups and downs but having a good support system can make it an amazing ride. First, I would like to thank my mentor, Erika Holzbaur for always being positive and encouraging throughout my thesis. Without her encouragement and support along the way, I wouldn’t have the confidence in myself as a scientist that I have today. I would also like to thank my committee members who always pushed me to think more analytically and be more rigorous with my science. Special thanks to Roberto Dominquez for being a great collaborator throughout my thesis. His scientific advice and structural expertise was critical to my thesis work. I want to thank the Biochemistry and Molecular Biophysics Graduate Group facility and staff for being supportive and helpful, especially during the early years when you are just trying to get use to the graduate school life style. A big thank you to the Pennsylvania Muscle Institute, a community with such collaborative spirit and excitement for science. The many years of PMI journal club have taught me so much about critical thinking and the art of science, which no class could ever achieve. I would not be writing this thesis without the tremendous support of the Holzbaur lab members of past and present. First, the biggest thank you to Mariko Tokito, our lab manager for being the cloning master and keeping our lab running with the upmost precision. I would also like to thank Karen Wallace Jahn for being the best lab veterinarian and providing the wit and humor that keeps all of us grounded in the real world. These two women provide such experience and expertise that is invaluable to the Holzbaur lab. Over the years, I have had the pleasure of working with the most talented post-docs and graduate students, who provided a variety of expertise and experience to learn from. Special thank you to Meredith Wilson, Swathi Ayloo, Sandra Maday and Amy Ghiretti for being my original lab lunch crew and helping me through the first half of my graduate school career. Their support and friendship were vital during those early years. Another big thank you to Pallavi Gopal, Chantell Evans, and Andrea Stavoe for being my second lab lunch crew and helping me make it to the graduate school finishing line. Our afternoon iii crossword breaks kept me sane when the science was driving me insane. I would like to thank everyone in the Holzbaur lab, past and present, for making lab a great place to grow as a scientist. Of course, I could not have survived the many years of graduate school without the support from my family and friends. I would like to thank all the wonderful friends that I have gained in graduate school over the years. A special thanks to my fellow 2013 BMB incoming class members, also known as BAMB. Not only did we make it through those first two years of class together, but we gained friendship that will last a lifetime. I would also like to thank my family members who got me away from work every now and then to keep me grounded. A special thank you to my parents, Jodie and William Olenick for always believing in me and telling me to shoot for the stars. Finally, I would like to thank my partner in crime and light of my life, Kevin Walker for always being supportive and loving even when I’m stressed and cranky. Thank you to everyone who encouraged me to keep going! iv ABSTRACT CARGO SPECIFIC REGULATION OF CYTOPLASMIC DYNEIN BY EFFECTOR PROTEINS Mara A. Olenick Dr. Erika L.F. Holzbaur Axonal transport is vital for the development and survival of neurons. The transport of cargo and organelles from the axon to the cell body is driven almost completely by the molecular motor, cytoplasmic dynein. Yet, it remains unclear how dynein is spatially and temporally regulated given the variety of cargo that must be properly localized to maintain cellular function. Previous work has suggested that adaptor proteins provide a mechanism for cargo-specific regulation of motors. During my thesis work, I have investigated the role of mammalian Hook proteins, Hook1 and Hook3, as potential motor adaptors. Using optogenetic and single molecule assays, I found that Hook proteins interact with both dynein and dynactin, to effectively activate dynein motility, inducing longer run lengths and higher velocities than the previously characterized dynein activator, BICD2. In addition, I found that complex formation requires the N-terminal domain of Hook proteins, which resembles the calponin- homology domain of EB proteins yet cannot bind directly to microtubules. In collaborative studies, we found the Hook domain directly interacts with a helix of the dynein light intermediate chain and this interaction is important for Hook-induced processive motility of dynein. In my final project, I found that Hook1 mediates the transport of TrkB-BDNF signaling endosomes in primary hippocampal neurons. Using live cell microscopy and microfluidic devices, Hook1 depletion resulted in a significant decrease in the flux and processivity of BDNF-Qdots along the mid-axon, an effect v specific for Hook1 but not Hook3. Together, my work suggests that dynein effectors like Hook proteins can differentially regulate dynein to allow for organelle-specific tuning of the motor for precise intracellular trafficking. vi TABLE OF CONTENTS ACKNOWLEDGMENT ................................................................................................................... III ABSTRACT ..................................................................................................................................... V LIST OF FIGURES ......................................................................................................................... IX CHAPTER 1: INTRODUCTION