University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2017 Engineered Cytoskeletal Arrays Reveal Mechanisms Of Membrane Transport And Tubulation Betsy Buechler Mcintosh 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 Mcintosh, Betsy Buechler, "Engineered Cytoskeletal Arrays Reveal Mechanisms Of Membrane Transport And Tubulation" (2017). Publicly Accessible Penn Dissertations. 2463. https://repository.upenn.edu/edissertations/2463 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2463 For more information, please contact [email protected]. Engineered Cytoskeletal Arrays Reveal Mechanisms Of Membrane Transport And Tubulation Abstract Within the cell, cytoskeletal molecular motors transport and remodel membrane-bound cargos along microtubule and actin filament tracks. Typically, there are multiple actin and microtubule motors attached to the same cargo, which must coordinate to navigate a complex cytoskeletal environment and deliver their cargos to specific locations. eW used an engineering, in vitro reconstitution, approach to investigate the interplay between a processive, microtubule-based motor, kinesin-1, and a non-processive, actin filament-based motor, Myo1c, in a simplified environment with increasing physiological complexity. First, we examined the interplay between purified motors attached to a membrane-coated bead at individual actin filament/microtubule intersections on the surface of a coverslip. We found that Myo1c is capable of initiating and terminating microtubule-based, kinesin-1-driven runs at actin filament/microtubule intersections. This ability of Myo1c to affect kinesin-1 motility at actin intersections is inhibited by the presence of nonmuscle tropomyosin Tm2 at the actin intersection. This suggests that tropomyosin may regulate Myo1c tethering of kinesin-1-driven cargo within cells by preventing termination of motility until reaching the highly dynamic actin just beneath the plasma membrane, sorting cargo to distinct subcellular domains. Next, we investigated the interplay between Myo1c and kinesin-1 on deformable giant unilamellar vesicles (GUVs) at physiologically relevant micropatterned arrays of sparse microtubules crossing dense actin filaments. eW found that the lipid composition of GUVs regulates its frequency of tubulation along microtubules by kinesin-1 and actin filaments yb Myo1c. GUVs containing a PtdIns(4,5)P2-rich lipid composition (PIP2-GUVs) tend to deform at actin/microtubule intersections along the microtubule, yet, the BAR domain protein endophilin is necessary for robust tubulation. Alternatively, in the presence of a physiological lipid mixture (LM-GUVs), kinesin-1 can readily tubulate Myo1c-tethered cargo at actin/microtubule intersections, with no significant change upon addition of endophilin. Myo1c can also transport both PIP2-GUVs and LM-GUVs along actin, yet significantly more deformation and tubulation occurs with LM-GUVs. In both cases, the presence of endophilin increases the frequency of tubulation along actin filaments by Myo1c. Overall, the ability of Myo1c and kinesin-1 to transport, sort, and deform vesicles along microtubules and actin filaments depends on the type of actin track, scaffolding-type membrane deformation-factors like endophilin, and the lipid composition of the vesicle. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor E. M. Ostap Second Advisor Erika L. Holzbaur Keywords actin filament, intracellular transport, kinesin, microtubule, myosin, tubulation Subject Categories Biophysics | Cell Biology | Molecular Biology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/2463 ENGINEERED CYTOSKELETAL ARRAYS REVEAL MECHANISMS OF MEMBRANE TRANSPORT AND TUBULATION Betsy Buechler McIntosh A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2017 Supervisor of Dissertation Co-Supervisor of Dissertation ____________________________ ____________________________ E. Michael Ostap, Ph.D. Erika L. F. Holzbaur, Ph.D. Professor of Physiology William Maul Measey Professor in Physiology Graduate Group Chairperson ____________________________ Daniel S. Kessler, Ph.D., Associate Professor of Cell and Developmental Biology Dissertation Committee Erfei Bi, Ph.D., Professor of Cell and Developmental Biology Yale E. Goldman, M.D., Ph.D., Professor of Physiology Roberto Dominguez, Ph.D., Professor of Physiology Tatyana Svitkina, Ph.D., Professor of Biology ENGINEERED CYTOSKELETAL ARRAYS REVEAL MECHANISMS OF MEMBRANE TRANSPORT AND TUBULATION COPYRIGHT 2017 Betsy Buechler McIntosh This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit https://creativecommons.org/licenses/by-nc-sa/3.0/us/ I dedicate this Ph.D. dissertation to my husband, Logan McIntosh, my parents, Dennis and Lisa Buechler, and my sister, Jennifer Buechler. Thank you for unconditional support and love. I wouldn’t be here without you! iii ACKNOWLEDGMENTS By the very nature of my highly collaborative thesis work, this project would not have been possible without a great number of people. First, I would like to thank my co-advisors, Drs. Mike Ostap and Erika Holzbaur. I was very fortunate to be able to work directly with two such skilled and passionate researchers, who teamed-up to guide me through experimental challenges and successes, grants, papers, presentations, and the day-to-day nitty gritty of academic biomedical science. I have also appreciated and highly benefitted from the many opportunities for leadership roles, interactions with visiting speakers, and attendance to scientific meetings that you both provided. Mike, I appreciate your endless patience with my many interruptions of scientific questions big and small, and the fact that I have someone with whom I can share enthusiasm for snow and mountains. Erika, thank you for your endless energy and enthusiasm and for providing a strong, successful, female scientist role model. Mike and Erika, through your collegial interactions during our weekly meetings and the many journal clubs and seminars in between, I have learned endlessly about the rewards of successful collaboration, which I will to take with me in my future endeavors. This dual mentorship gave me limitless access to incredibly intelligent and skilled researchers, and more tools and reagents than even I could manage to use. Thank you to all members of the Ostap and Holzbaur laboratories, past and present; you all positively impacted my project and graduate experience. In particular, I would like to express my gratitude to Michael Woody, Mara Olenick, Drs. Adam Hendricks, Sandy Maday, Swathi Ayloo, Meredith Wilson, Jake Lazarus, Mariko Tokito, Ionas Pyrpassopoulos, Michael Greenberg, Allison Zajac, Abbey Weith, Beth Feeser, Dan Safer, Tianming Lin, and Liqiong Chen for thoughtful conversation and technical expertise throughout my thesis work. Thank you also to Swathi, Greenberg, Woody, Abbey, and Beth for invigorating conversation about politics, the world, life, and other random topics (like the weather). I appreciate Lisa Davidson, Riley Payne, and Erin Masucci for enriching my science with mentorship opportunities, and to Erin for serious experimental assistance during this last scramble to finish my dissertation work. I would like to thank Eric Johnston and Brock Peterson from the iv Singh Center for Nanotechnology for their help with the Excimer laser patterning. Additionally, I would like to acknowledge the scientific support and reagents I received from: Dr. Henry Shuman about optics and optical trapping; Dr. Yale Goldman and his lab members Drs. Lisa Lippert, Matt Caporizzo, and Jody Dantzig Brody; Dr. Katya Grischuk and her lab members Drs. Anatoly Zaytsev and Suvranta Tripathy; Dr. Roberto Dominguez and his lab members Dr. David Kast and Greg Rebowski; Dr. Tobias Baumgart and his lab members Jaclyn Robustelli and Dr. Zhiming Chen; and the many other participants of the Pennsylvania Muscle Institute. I cannot express how much I learned from each and every member during informal discussion, journal clubs, and seminars. Thank you also to my thesis committee members Drs. Erfei Bi, Tanya Svitkina, Yale Goldman, and Roberto Dominguez for always pushing me to think harder about my research. To my funding sources at the American Heart Association and National Institutes of Health, I greatly appreciate the opportunities that were afforded to me by your patronage. These organizations enable astounding training opportunities and life-saving and life-enriching scientific advances. I hope to continue to apply these experiences in the future. I would like to extend a huge thank you to Ashley Douglass of the Pennsylvania Muscle Institute for managing the many annoying and essential administrative tasks that have made doing researcher efficient, timely, pleasant, and possible! Thank you for ordering the food, ordering reagents/supplies/chasing down suppliers, organizing everything, for the many chats, and all the support. I also appreciate the Physiology Department front office staff, and Roz Schorr and Dr. Kevin