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Contribution of Dysferlin-Containing Membranes to Membrane Repair in Skeletal Muscle

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Contribution of Dysferlin-Containing Membranes to Membrane Repair in Skeletal Muscle CONTRIBUTION OF DYSFERLIN-CONTAINING MEMBRANES TO MEMBRANE REPAIR IN SKELETAL MUSCLE by Joel Ryan McDade A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular and Integrative Physiology) in the University of Michigan 2014 Doctoral Committee: Associate Professor Daniel E. Michele, Chair Associate Professor Anatoli Lopatin Associate Professor Susan Brooks Herzog Associate Professor Kristen J. Verhey "Success is not a random act. It arises out of a predictable and powerful set of circumstances and opportunities." -Malcolm Gladwell This thesis is dedicated to everyone who provided me with the circumstances and opportunities necessary to succeed. ii ACKNOWLEDGEMENTS First, I would like to thank my mentor Dan, whose tremendous patience and outstanding mentoring made the work within this thesis possible. I am not only speaking for myself when I say that your guidance and support was vital for my success as a graduate student and my vision for the future. I would also like to thank the members of my committee, who have provided me with critical feedback, reagents, and support over the last several years. There are several people with whom I interacted with that were critical for my professional and personal development. When I joined the Physiology Department I was assigned an informal mentor to guide me through the ups and downs of the graduate school experience. My meetings with Ormond ended up teaching me a lot about how I should approach science, and for that I am grateful. The members of the Microscopy and Image Analysis Laboratory have guided me on all matters related to microscopy from the initial experiments in this thesis all the way through to the end. I would especially like to thank Chris Edwards, who, in addition to his knowledge of microscopy was also always good for a laugh. I would like to thank the Molecular and Integrative Physiology administrative staff for all of their assistance, especially Michele Boggs, who is always receptive to frantic emails that require immediate attention. A special thanks to all the past Michele lab members, particularly Matt Campbell and Jessica Gumerson, who came before me and showed me the ropes. Thanks to the current Michele lab members, including Joanne Garbincius, whose optimism and passion for science is contagious. Most importantly, thank you to my wonderful wife Laura, because what goes on at home is just as important as what goes on at school. iii TABLE OF CONTENTS DEDICATION....................................................................................................................ii ACKNOWLEDGEMENTS................................................................................................iii LIST OF FIGURES………................................................................................................vi CHAPTER 1 – Plasma membrane maintenance in muscular dystrophy and the role of dysferlin-mediated membrane repair in skeletal muscle..……………………….......……..1 Muscular dystrophy and plasma membrane integrity............................................1 Dysferlinopathy and a novel pathway to muscular dystrophy................................3 What is the role of dysferlin in muscle cells?.........................................................7 Membrane repair in non-muscle model-systems...................................................9 Membrane repair in muscle and the role of dysferlin...........................................13 Rationale and approach.......................................................................................17 CHAPTER 2 – Membrane damage induced vesicle-vesicle fusion of dysferlin- containing vesicles in muscle cells requires microtubules and kinesin...........................25 Abstract................................................................................................................25 Introduction..........................................................................................................26 Methods...............................................................................................................29 Results.................................................................................................................34 Discussion............................................................................................................41 Acknowledgements..............................................................................................47 CHAPTER 3 – Rapid actin cytoskeleton dependent recruitment of plasma membrane- derived dysferlin at wounds is critical for muscle membrane repair...............................57 Abstract................................................................................................................57 Introduction..........................................................................................................58 Methods...............................................................................................................60 Results.................................................................................................................64 iv Discussion............................................................................................................71 Acknowledgements..............................................................................................77 CHAPTER 4 – Conclusions and Future Directions.........................................................88 Summary of Thesis Work.....................................................................................88 Implications and future directions........................................................................97 Conclusions.......................................................................................................117 REFERENCES.............................................................................................................123 v LIST OF FIGURES 1-1) Loss of dysferlin results in muscular dystrophy independently of any effects on the DGC or membrane stability ...........................................................................................21 1-2) Phylogenic analysis and structure of ferlin proteins................................................22 1-3) Impaired membrane resealing in dysferlin-deficient muscular dystrophy................23 1-4) Hypothesized model for dysferlin-mediated membrane repair................................24 2-1) Dysferlin-eGFP localizes to a population of distinct vesicles in differentiated L6 myotubes........................................................................................................................48 2-2) Dysferlin-containing vesicles move along microtubules via KIF5B motors in differentiated L6 myotubes.............................................................................................49 2-3) Membrane damage induces fusion of dysferlin-containing vesicles leading to the formation of extremely large vesicles in L6 myotubes....................................................50 2-4) Disruption of microtubules reduces the formation of large dysferlin containing vesicles following membrane wounding in L6 myotubes................................................51 2-5) Expression of dnKIF5 inhibits formation of large dysferlin-containing vesicles following membrane wounding in L6 myotubes..............................................................52 2-6) Dysferlin-containing vesicles interact with lysosomal membranes following mechanical wounding in differentiated L6 myotubes......................................................53 2-7) Dysferlin-containing vesicles do not label with KIF5C in differentiated L6 myotubes........................................................................................................................54 2-8) Pharmacological inhibition of actomyosin interaction does not affect formation of large dysferlin-containing vesicles following wounding in differentiated L6 myotubes...55 2-9) Effect of dominant negative KIF5 on the organization of the actin-cytoskeleton, Golgi-complex, and lysosomes in differentiated L6 myotubes........................................56 3-1) Dysferlin-pHGFP transgenic reporter mouse..........................................................78 vi 3-2) Dysf-pHGFP is localized at the lateral sarcolemma and striated internal membranes in adult skeletal muscle fibers from dysf-pHGFP TG mice similar to endogenous dysferlin in wild-type mice..........................................................................79 3-3) Dysferlin is highly enriched in the plasma membrane and transverse tubules in adult skeletal muscle fibers............................................................................................80 3-4) Sarcolemma-derived dysferlin is recruited to membrane lesions following wounding in adult skeletal muscle fibers........................................................................................81 3-5) Membrane damage induces formation of acidic dysferlin-containing vesicles in adult skeletal muscle fibers............................................................................................82 3-6) Disruption of actin filaments impairs recruitment of sarcolemma-derived dysferlin without affecting damage-induced endocytosis of dysferlin...........................................83 3-7) Disruption of the actin cytoskeleton increases FM1-43 uptake following membrane wounding in adult wild-type skeletal muscle fibers.........................................................84
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