University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2016 Molecular and Cellular Approaches Toward Understanding Dynein-Driven Motility Swathi Ayloo University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biophysics Commons, Cell Biology Commons, and the Molecular Biology Commons Recommended Citation Ayloo, Swathi, "Molecular and Cellular Approaches Toward Understanding Dynein-Driven Motility" (2016). Publicly Accessible Penn Dissertations. 1595. https://repository.upenn.edu/edissertations/1595 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1595 For more information, please contact [email protected]. Molecular and Cellular Approaches Toward Understanding Dynein-Driven Motility Abstract Active transport is integral to organelle localization and their distribution within the cell. Kinesins, myosins and dynein are the molecular motors that drive this long range transport on the actin and microtubule cytoskeleton. Although several families of kinesins and myosins have evolved, there is only one form of cytoplasmic dynein driving active retrograde transport in cells. While dynactin is an essential co-factor for most cellular functions of dynein, the mechanistic basis for this evolutionarily well conserved interaction remains unclear. Here, I use single molecule approaches with purified dynein ot reconstitute processes in vitro, and implement an optogenetic tool in neurons to further dissect regulatory mechanisms of dynein- driven transport in cells. I demonstrate for the first time, at the single molecule level, that dynactin functions as a tether to enhance the initial recruitment of dynein onto microtubules but also acts as a brake to slow the motor. I then extend this work in neurons to understand regulation of the dynein motor at the cellular level. Neurons are particulary dependent on long-range transport as organelles and macromolecules must be efficiently vmo ed over the extended length of the axon and further, have mechanisms in place for the compartment-specific egulationr of trafficking in onsax and dendrites. I use a light-inducible dimerization tool to recruit motor proteins or motor adaptors to organelles in real time to examine downstream effects of organelle motility and compartment-specific regulation of motors. I find that while dynein works efficiently in both onsax and dendrites, kinesins are differentially regulated in a compartment-specific manner. I further demonstrate that dynein-driven motility in neurons is largely governed by microtubule orientation and requires microtubule dynamics for efficient navigation in axons and dendrites. Together, this work sheds light on the molecular and cellular mechanisms of dynein function both in vitro and in vivo using a combination of approaches. My findings converge to a model wherein dynactin enhances the recruitment of dynein onto microtubule plus ends, leading to efficient minus-end directed motility of dynein. This becomes especially critical in neuronal growth cones and dendrites owing to the large number of highly dynamic microtubules in these compartments. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Biology First Advisor Erika L. Holzbaur Subject Categories Biophysics | Cell Biology | Molecular Biology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1595 MOLECULAR AND CELLULAR APPROACHES TOWARD UNDERSTANDING DYNEIN-DRIVEN MOTILITY Swathi Ayloo A DISSERTATION in Biology Presented to the Faculties of the University of Pennsylvania In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2016 Supervisor of Dissertation _______________________________ Erika L.F. Holzbaur, PhD, Professor of Physiology Graduate Group Chairperson _______________________________ Michael A. Lampson, PhD, Associate Professor of Biology Dissertation Committee Erfei Bi, PhD, Professor of Cell and Developmental Biology E. Michael Ostap, PhD, Professor of Physiology Tatyana Svitkina, PhD, Professor of Biology Michael A. Lampson, PhD, Associate Professor of Biology MOLECULAR AND CELLULAR APPROACHES TOWARD UNDERSTANDING DYNEIN-DRIVEN MOTILITY COPYRIGHT 2016 Swathi Ayloo ACKNOWLEDGMENT This work would not have been possible without the support and help from several people. I would first like to acknowledge my advisor, Erika Holzbaur for her wonderful mentorship. She has always held me to high standards and constantly challenged me to think deeply about my science. She has always encouraged me and I greatly appreciate all the opportunities she provided me with, ensuring my success. Thank you, Erika, for your faith in me and teaching me several things throughout my graduate training. I would also like to thank my thesis committee members: Erfei Bi, Mike Ostap, Tanya Svitkina and Mike Lampson for their insights and advice. Special thanks to Mike Ostap and Mike Lampson for terrific collaborations on both my projects. Both of them were instrumental in providing key reagents and scientific advice which made this thesis work possible. I would especially like to thank all the past and current members of the Holzbaur lab: Mariko Tokito for making DNA constructs and for all the endless conversations she had with me about protein purification and several other biochemical assays; Karen Wallace for her assistance with mouse work; Adam Hendricks who first introduced me to single molecule assays and was my go-to person for all things biophysics; Meredith Wilson, Betsy McIntosh, Alison Twelvetrees, Amy Ghiretti, Mara Olenick and Eva Klinman for their friendship and for putting up with several of my random bouts of craziness. Special thanks to Sandra Maday who has been a great friend and has made this journey a lot more fun. I have learned so much from Sandy about data analysis, giving talks and scientific writing. Besides the scientific stuff, I thoroughly enjoyed our late-night working sessions motivating each other, hour-long conversations about random things, sometimes even as late as 3 am and more recently our tea breaks. Thank you Sandy for being there for me through everything and I will truly miss you. iii I would also like to thank Aditya Dodda and Ed Ballister for their fantastic collaborations. Aditya spent several hours and days looking at my data to develop new ways to analyze it. What started out with Aditya fixing errors in the basic code I wrote, turned into him developing new algorithms and simulations for my data. Ed Ballister developed the light- inducible dimerization system and was the first one to think about applying it to motors. Working with him was a lot of fun and without him, my second project would never exist. I would also like to acknowledge the Pennsylvania Muscle Institute (PMI) at Penn, especially Mike Ostap, Yale Goldman, Katya Grishchuck and their lab members. Being part of the PMI gave me access to several key resources and opportunities. I always enjoyed our journal clubs and learned how to think about science through these meetings. The PMI community made me feel at home and inculcated a sense of belonging in me. Big thanks to all my friends in Boston who was my family away from home. Needless to say, I am quite excited to be surrounded by them the next few years. Their friendship through the years, since my undergrad days has been invaluable. I had a lot of fun with you guys! I am grateful to all my family members – parents, uncle and aunt, my siblings and my cousins. They were always there for me and made everything possible for me. I would not be where I am today without them. Thank you for believing in me and giving me the freedom to pursue what I wanted. Finally, I would like to thank Rama, my strength and my pillar of support. iv ABSTRACT MOLECULAR AND CELLULAR APPROACHES TOWARD UNDERSTANDING DYNEIN-DRIVEN MOTILITY Swathi Ayloo Erika L.F. Holzbaur Active transport is integral to organelle localization and their distribution within the cell. Kinesins, myosins and dynein are the molecular motors that drive this long range transport on the actin and microtubule cytoskeleton. Although several families of kinesins and myosins have evolved, there is only one form of cytoplasmic dynein driving active retrograde transport in cells. While dynactin is an essential co-factor for most cellular functions of dynein, the mechanistic basis for this evolutionarily well conserved interaction remains unclear. Here, I use single molecule approaches with purified dynein to reconstitute processes in vitro, and implement an optogenetic tool in neurons to further dissect regulatory mechanisms of dynein-driven transport in cells. I demonstrate for the first time, at the single molecule level, that dynactin functions as a tether to enhance the initial recruitment of dynein onto microtubules but also acts as a brake to slow the motor. I then extend this work in neurons to understand regulation of the dynein motor at the cellular level. Neurons are particulary dependent on long-range transport as organelles and macromolecules must be efficiently moved over the extended length of the axon and further, have mechanisms in place for the compartment-specific regulation of trafficking in axons and dendrites. I use a light-inducible dimerization tool to recruit motor proteins or motor adaptors to organelles in real time to examine downstream effects of organelle motility and compartment-specific regulation of motors. I find that