The Pennsylvania State University The Graduate School Intercollege Graduate Program in Physiology MECHANISM OF CLASS III MYOSIN MEDIATED REGULATION OF ACTIN BUNDLE BASED PROTRUSIONS A Dissertation in Physiology by Manmeet H. Raval 2016 Manmeet H. Raval Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2016 The dissertation of Manmeet H. Raval was reviewed and approved* by the following: Christopher M. Yengo Associate Professor of Cellular and Molecular Physiology Dissertation Adviser Chair of Committee Lisa Shantz Associate Professor of Cellular and Molecular Physiology Scot R. Kimball Professor of Cellular and Molecular Physiology Hui-Ling Chiang Professor of Biochemistry and Molecular Biology Collin J. Barnstable Professor of Neural and Behavioral Sciences Professor of Psychiatry Research Director of Penn State Hershey Eye Center Donna Korzick Professor of Physiology and Kinesiology Chair of the Intercollege Graduate Degree Program in Physiology *Signatures are on file in the Graduate School. iii ABSTRACT Class III myosins (MYO3A and MYO3B) are actin based motors which are proposed to function as transporters in parallel actin-bundle based protrusions such as stereocilia of inner ear and calycal processes of photoreceptors. The first member of the MYO3 family was identified in Drosophila photoreceptors and called NINAC (Neither inactivation nor afterpotential C) based on its role in phototransduction. It was observed that NINAC null mutant flies undergo light and age dependent retinal degeneration. The first report describing deleterious recessive mutations in MYO3A associated with inherited human progressive hearing loss (DFNB30) was published in 2002. It is believed that MYO3 dependent transport of Espin (isoforms) from the base to the tips of the stereocilia plays a crucial role in stereocilia elongation. Previous reports have hypothesized that MYO3A and MYO3B may have overlapping functions but MYO3B can only partially compensate for MYO3A. Thus, it is critically important to reveal functional differences between isoforms to determine how the loss of MYO3A leads to deafness. Using a wide-range of cell biological and biochemical approaches, this study investigated novel aspects of class III myosins and its binding partners associated with vertebrate hearing and vision. This study reports discovery of two novel MYO3 binding proteins- MORN4 and EspinL, and provide insights into the functional mechanism of each protein in association with MYO3. It was found that the tail of MYO3 has a conserved Espin1 and EspinL binding site, whereas the iv MORN4 binding site is distinct and present only in the MYO3A tail region. Based on structural data, a novel mechanism of motor (MYO3) mediated cargo (Espin1) activation is proposed. This work demonstrates that MYO3A is uniquely engineered to regulate formation and elongation of parallel actin bundle based protrusions which does not require rapid actin remodeling. The study also revealed a correlation between MYO3 motor activity and its ability to regulate actin protrusion formation and elongation. Interestingly, the intactness of the MYO3A extended tail region was found to be crucial for stabilizing actin protrusions. Finally, the study reports the characterization of two novel deafness causing MYO3A dominant mutations- G488E and L697W. The G488E mutation leads to an interesting phenotype of a 2-fold decrease in the maximum actin-activated ATPase rate and 2-fold increase in the actin sliding velocity. Whereas, the L697W mutation leads to a ~2-fold decreases in both the maximum actin-activated ATPase rate and actin sliding velocity. These results highlight the importance of MYO3 in vertebrate sensory structures and provides novel insights into its role in length and ultrastructure maintenance of parallel actin based protrusions. v TABLE OF CONTENTS List of Figures ................................................................................................. viii List of Tables .................................................................................................. x List of Abbreviations………………………………………………………………..xi Acknowledgements ........................................................................................ xii Chapter 1 Introduction ................................................................................... 1 Types of parallel actin bundle based protrusions and associated actin regulatory proteins. ............................................................................ 4 Filopodia ............................................................................................ 5 Microvilli ............................................................................................. 6 Stereocilia .......................................................................................... 8 Discovery of Class III Myosins ................................................................. 10 Class III myosins and implications in human hereditary hearing loss ....... 12 Vertebrate inner ear physiology ......................................................... 12 Domain structure of vertebrate class III myosins ............................... 13 Role of class III myosins in vertebrate stereocilia .............................. 15 References ............................................................................................... 18 Chapter 2 Vertebrate class III myosin interact with MORN-repeat containing adaptor proteins ....................................................................................... 26 Introduction .............................................................................................. 26 Materials and Methods ............................................................................. 28 Results ..................................................................................................... 31 Discussion ................................................................................................ 35 Tables and Figures .................................................................................. 39 References ............................................................................................... 47 vi Chapter 3 Impact of the motor and tail domains of class III myosins on regulating the formation and elongation of actin protrusions .................... 50 Introduction .............................................................................................. 50 Materials and Methods ............................................................................. 54 Results ..................................................................................................... 61 Discussion ................................................................................................ 66 Tables and Figures .................................................................................. 71 References ............................................................................................... 84 Chapter 4 Characterization of a novel MYO3A missense mutation associated with a dominant form of late onset hearing loss ..................... 89 Introduction .............................................................................................. 89 Materials and Methods ............................................................................. 91 Results ..................................................................................................... 94 Discussion ................................................................................................ 98 Tables and Figures .................................................................................. 102 References ............................................................................................... 111 Chapter 5 Summary, conclusions and future directions ................................ 114 Summary and conclusions ....................................................................... 114 Discovery of novel MYO3 binding partners ........................................ 115 Characterization of differences in the motor and tail domains of MYO3A and MYO3B ................................................................... 118 Cell biological and biochemical characterization of novel MYO3A deafness causing mutations ........................................................ 120 Future Directions ...................................................................................... 121 MYO3 role in vertebrate photoreceptors ............................................ 121 Mechanism of MYO3A mediated actin protrusion regulation ............. 123 Characterization of MYO3A deafness causing mutations .................. 124 vii References ............................................................................................... 126 Appendix A Supplementary Figures-1 ..................................................... 128 Appendix B Supplementary Figures-2 ..................................................... 129 Appendix C Supplementary Figures-3 .................................................... 133 Appendix D Letters of Permission ........................................................... 135 Proof of permission for Chapter 2 ............................................................ 136 Proof of permission for Chapter 3 ............................................................ 137 Proof of permission for Chapter 5 ...........................................................