THE ROLE OF THE ACTIN CYTOSKELETON DURING MUSCLE DEVELOPMENT IN DROSOPHILA AND MOUSE by Shannon Faye Yu A Dissertation Presented to the Faculty of the Louis V. Gerstner, Jr. Graduate School of the Biomedical Sciences in Partial Fulfillment of the Requirements of the Degree of Doctor of Philosophy New York, NY Oct, 2013 Mary K. Baylies, PhD! Date Dissertation Mentor Copyright by Shannon F. Yu 2013 ABSTRACT The actin cytoskeleton is essential for many processes within a developing organism. Unsurprisingly, actin and its regulators underpin many of the critical steps in the formation and function of muscle tissue. These include cell division during the specification of muscle progenitors, myoblast fusion, muscle elongation and attachment, and muscle maturation, including sarcomere assembly. Analysis in Drosophila has focused on regulators of actin polymerization particularly during myoblast fusion, and the conservation of many of the actin regulators required for muscle development has not yet been tested. In addition, dynamic actin processes also require the depolymerization of existing actin fibers to replenish the pool of actin monomers available for polymerization. Despite this, the role of actin depolymerization has not been described in depth in Drosophila or mammalian muscle development. ! Here, we first examine the role of the actin depolymerization factor Twinstar (Tsr) in muscle development in Drosophila. We show that Twinstar, the sole Drosophila member of the ADF/cofilin family of actin depolymerization proteins, is expressed in muscle where it is essential for development. tsr mutant embryos displayed a number of muscle defects, including muscle loss and muscle misattachment. Further, regulators of Tsr, including a Tsr-inactivating kinase, Center divider, a Tsr-activating phosphatase, Slingshot and a synergistic partner in depolymerization, Flare, are also required for embryonic muscle development. Muscle-specific depletion of tsr resulted in progressive loss of iii sarcomeric organization, overall growth defects and locomotor deficiencies. These data are the first to demonstrate a role for the ADF/cofilin family of actin depolymerizing proteins in muscle development and function in Drosophila. ! To elucidate the conservation of myoblast fusion proteins in mammalian cells, we analyzed the role of the actin regulators Dock1 and IQSec1 in C2C12 myoblasts. We demonstrate that the actin regulators, Dock1 and IQSec1, homologs of the respective fusion proteins Myoblast city and Loner, play a conserved role in mammalian myoblast fusion. Depletion of Dock1 or IQSec1 in C2C12 myoblasts, an in vitro mouse myoblast cell line, resulted in a severe fusion block. Further, we show that Dock1 is not required for myoblast differentiation, migration and alignment or adhesion and likely plays a role at the site of myoblast fusion, similar to Drosophila Myoblast city. Finally, we identify the site of fusion in mammals using an indirect reporter for the actin cytoskeleton, suggesting that actin regulation at the site of myoblast-myoblast contact is essential for the fusion process. ! Together, this work significantly extends the growing body of research indicating that myoblast fusion employs a conserved set of proteins and processes from Drosophila to mammals and underscores the importance of the actin cytoskeleton to both muscle development and maintenance. Importantly, the recent identification of mutations in Cofilin 2, the mammalian homolog of Twinstar, in patients affected with nemaline myopathy suggests that actin depolymerization is also an essential mechanism to maintain proper muscle function in humans. iv BIOGRAPHICAL SKETCH Shannon Faye Yu was born on July 5, 1985 in Augusta, Georgia, home of the Masters Tournament and twice the Georgia capital. She is the favorite daughter of Chin U and Robin Jan Yu and the older sister of Brantley Chin Yu. While growing up in Augusta, Shannon learned how to be a polite and proper Southern lady but also how to drive a manual transmission. In high school, she made good grades and made the cheerleading squad. Most importantly, however, she discovered that biology was her “thing.” ! She matriculated at the University of Georgia (UGA) in 2003 as a “pre- med” major and traded her hoop skirts for the red and black. At UGA, she developed a passion for Georgia football (Go Dawgs!) and found a group of friends (henceforth referred to as 2North) that love the Sound of Music, dance parties and 5 Star Day (R.I.P.) as much as she does. Shannon did her undergraduate thesis research with Dr. Nancy Manley studying parathyroid development in the mouse. Her experience working in the lab coupled with the fact that blood made her queasy, Shannon decided to apply to graduate programs and become a scientist. Despite her love of the South, Shannon knew in her heart that there were other, better places to live. Thus, she applied to graduate programs exclusively in New York City (NYC) and matriculated at the Louis V. Gerstner, Jr. Graduate School of the Biomedical Sciences (GSK) in July 2007. v ! In graduate school, Shannon joined the lab of Dr. Mary K. Baylies, where she studied many aspects of muscle development in mouse and Drosophila that she will tell you about shortly. She has never once regretted her choice of program or laboratory (though she may have regretted coming to graduate school at all at some points #haventweall). ! While in graduate school, Shannon met a real-life New Yorker. He taught her how to “properly” cross the street and how to do a Western blot. She taught him how to use a plate and utensils and how to make ramen from a bag. Together, they play tennis badly, enjoy visiting ballparks during their travels and turn their noses up at non-NYC bagels and pizza. In her spare time, Shannon climbs on “curtains,” reads cookbooks and Murakami novels and surfs the Internet. In addition, Shannon enjoys watching HBO every Sunday night, eating candy and reading the 2North listserv. After graduation, she is looking forward to joining the circus. vi ACKNOWLEDGEMENTS Without the love, friendship and support of a large number of individuals, I would not have started, stayed or completed graduate school. Foremost, I am grateful to my family for their unwavering belief in me and for raising me to have the same. I am also fortunate to have a second family nearby — John’s family — which has warmly welcomed me into their homes and lives for the last six years. 2North (my third family) has sustained, entertained and inspired me since we met so many years ago. To them: Thank you for being a friend. Traveled down the road and back again. Your heart is true. You’re a pal and a confidant. ! ! I would never have gotten into science without Mr. Campbell, my high school biology teacher, or into graduate school without Dr. Nancy Manley, my undergraduate thesis mentor. I am grateful that Drs. Kathryn Anderson and Lorenz Studer agreed to be a part of my Special Committee. Their comments were crucial to developing this work. I would also like to recognize the generosity of the fly community in sharing stocks, reagents and, in the case of Dr. Eric Lai, his confocal. ! Every member of my lab has provided essential comments, advice and technical assistance for many aspects of this work. For that I am incredibly appreciative. In particular, Dr. Krista Dobi taught me everything I know about basic fly husbandry during my rotation and then taught me again when I transitioned to a fly project three years into my thesis research. Her screen provided the initial impetus for studying Coronin and resulted in the work vii presented in Chapter 3 of this thesis. She has also read almost every piece of my writing (including this thesis), and her insights have improved each one tremendously. Dr. Ingo Bothe generated a library of useful stocks, which made my fly work incredibly efficient. In addition, his analysis of the requirement for PIP2 during Drosophila myoblast fusion was invaluable to my own work in C2C12 myoblasts, and his focus on myoblast fusion in the embryo was indispensable when I discovered my own fusion phenotype. Victoria Schulman has kept me company during the early morning and weekend hours for the last few years, and her strong work ethic has been an example to us all. Throughout the years, my bay mate, Kate Rochlin, has provided valuable advice on scientific (and often, not so scientific matters) and has played one of the largest roles in shaping my graduate school experience. In the future, when I reminisce on my time here, she will undoubtably be featured in many of the happy memories. Lastly, I will be forever grateful to Dr. Mary Baylies for allowing me to join her lab and for giving me the freedom (and the confidence) to define my own space within this field and to go beyond the embryo to pursue interesting phenotypes. ! But no one compares to John. John has been my closest friend, greatest supporter and easily my most important discovery in graduate school. viii TABLE OF CONTENTS LIST OF FIGURES!...............................................................................................xv LIST OF TABLES!................................................................................................xxi LIST OF ABBREVIATIONS!................................................................................xxii CHAPTER ONE: INTRODUCTION!.......................................................................1 Chapter Overview!.............................................................................................2 Muscle development