The Pennsylvania State University The Graduate School Department of Physiology A SILENT SYMPHONY: UNCONVENTIONAL MYOSIN 3A AND HUMAN HEREDITARY HEARING LOSS A Thesis in Anatomy by Lina Jamis © 2015, Lina Jamis Submitted in Partial Fulfillment of the Requirements for the degree of Master of Science August 2015 The thesis of Lina Jamis was reviewed and *approved by the following: Christopher Yengo Associate Professor of Cellular and Molecular Physiology Thesis Advisor Alistair Barber Associate Professor of Ophthalmology and Cellular & Molecular Physiology Sarah Bronson Professor and Director of Research Development and Interdisciplinary Research Patricia McLaughlin Professor of Neural and Behavioral Sciences Chair of the Graduate Program in Anatomy *Signatures are on file in the Graduate School. ii A Silent Symphony: Unconventional Myosin3A and Human Hereditary Hearing Loss Lina Jamis (ABSTRACT) The perception of sound is a process that converts mechanical sound waves to nerve impulses and relies heavily on actin-based protrusions in inner ear hair cells called stereocilia. Unconventional class III myosins (MYO3A and MYO3B) have been found to play a role in the maintenance of stereocilia length, which is essential for hearing. A number of proteins have been implicated in stereocilia length maintenance and many are associated with non-syndromic hereditary hearing loss. ESPIN1 is an actin bundling protein that is transported by MYO3 to the barbed-end of the stereocilia where it plays a role in cross-linking and regulating actin filament length. Recessive loss-of-function mutations in MYO3A are the cause of the inherited hearing impairment phenotype DFNB30 (Walsh et al., 2002), possibly because of the failed MYO3A-dependent ESPIN1 transport to the stereocilia tips. A mouse model of DFNB30 results in age-dependent degeneration of the stereocilia of inner ear hair cells (Walsh, 2010). An uncharacterized missense mutation (G488E) in MYO3A was recently found to be associated with human deafness (Grati et al., unpublished). This mutation is located near the switch I loop, responsible for the steric availability of myosin’s nucleotide-binding pocket. We proposed that the mutation may alter the motor ATPase cycle and motility, which were examined directly with biochemical studies. Another goal of the study was to characterize the point mutation in a cell culture model, which examines MYO3A and ESPIN1 in filopodia, actin-based protrusions with similar properties to stereocilia. We hypothesized that the mutation may prevent normal motility to the filopodial tips and proper elongation of the filopodia. MYO3A transport of ESPIN1 was tested by examining the localization of fluorescently tagged versions of MYO3A and actin bundling in COS7 cells. Since wild- type MYO3A accumulates at filopodia tips, this model provides a well-defined spatial compartment where potential interactions can be clearly visualized. Live imaging of COS7 cells transfected with WT GFP.MYO3A.ΔK and ESPIN1 showed dynamic co- localization at the filopodia tips. In contrast, GFP.MYO3A.ΔK.G488E co-expression with ESPIN1 showed a significant reduction in filopodial tip localization of both proteins. Steady-state actin-activated ATPase activity of baculovirus-expressed MYO3A. ΔK.2IQ wildtype and mutant were performed with an NADH-coupled assay. The maximum ATPase rate of the mutant was shown to be significantly reduced as compared to the wildtype, suggesting that the mutation results in the loss of MYO3A’s force- generating function. Motor activity of purified GFP-labeled MYO3A was directly examined using an in-vitro motility assay. Wild-type MYO3A was able to generate actin sliding while the mutant myosin is still is progress. The cumulative results from these experiments suggest that the mutation located on switch I and near to the conserved nucleotide binding region, disrupts this structural element that is critical for motor function. The introduction of this mutation is sufficient to produce defects in MYO3A-based ESPIN1 translocation, filopodial length, and filopodia density. The findings in the filopodia of COS7 cells may hold true for iii stereocilia in which mutant MYO3A prevents normal motility to stereocilia tips, causing height defects and degeneration of the stereocilia in the inner ear hair cells. iv TABLE OF CONTENTS LIST OF FIGURES……………………………………………………………….viii LIST OF TABLES…………………………………………………………………ix LIST OF ABBREVIATIONS……………………………………………………...x ACKNOWLEDGEMENTS………………………………………………………..xii Chapter 1: INTRODUCTION: HEARING, STEREOCILIA, AND MYOSIN…..1 1.1 Purpose………………………………………………………………...1 1.2 Human Hereditary Deafness…………………………………………..1 1.3 The Cell Biology of Hearing…………………………………………..2 1.4 Stereocilia…………………………….………………………………..4 1.5 Cytoarchitecture and organization…………………………..................5 1.6 ESPIN1 and maintenance of actin filaments…………………………...7 1.7 Class III Myosins………………………………………………………8 1.8 The Myosin Superfamily………………………………………………9 1.9 The Myosin Structure………………………………………………….10 1.9.1 Motor Head…………………………………………………..11 1.9.2 Nucleotide-binding Pocket………...…………………………11 1.9.3 Lever Arm……………………………………………………12 1.9.4 Tail……………………………………………………………13 1.10 Class III Unconventional Myosins…………………………………….13 1.11 Myosin 3A Kinetics……………………………………………………17 1.12 Myosin 3B Subtype……………………………………………………18 v 1.13 The Myosin Nucleotide-binding Pocket………………………………19 1.14 Remaining Questions………………………………………………….21 1.15 Conclusions……………………………………………………………21 Chapter 2: RESEARCH OBJECTIVES……………………………………………22 Chapter 3: EXPERIMENTAL METHODOLOGY………………………………...23 3.1 Reagents………………………………………………………………...23 3.2 Expression Plasmids……………………………………………………23 3.2.1 Biochemistry………………………………………………….23 3.2.2 Cell biology………………………………………………...…23 3.2.3 Site-directed mutagenesis………………………………….…24 3.3 Protein Expression and Purification……………………………………24 3.4 Sequence Analysis……………………………………………………...25 3.5 Cell Culture and Transient Transfection………………………………..25 3.6 Live Cell Imaging of Cells Expressing Fluorescent Proteins…………..26 3.7 Data Analysis, Statistical Analysis, and Software Used……………….26 3.8 Steady-state ATPase Activity………….……………………………….27 3.9 In-Vitro Motility……………………….…………………………….....27 Chapter 4: RESULTS………………………………………………………………29 4.1 Sequence Analysis……………………………………………………...29 4.2 Quantification of MYO3A Tip Localization, Filopodia Length, and Density in COS7 Cells………………………………………………….29 4.3 MYO3A Steady-state ATPase Activity………………………………...31 4.4 MYO3A In-Vitro Motility………………………………………………31 Chapter 5: DISCUSSION……………………………………………………….......42 vi Chapter 6: CONCLUSION AND FUTURE DIRECTIONS……………..…………50 WORKS CITED……………………………………………………………….......54 APPENDIX………………………………………………………………………58 vii LIST OF FIGURES Figure 1.1 The Organ of Corti 3 Figure 1.2 Auditory transmission and the internal structure of the ear 5 Figure 1.3 The actin tread-milling mechanism 6 Figure 1.4 Cargo Transporter, Myosin V structure and important domains 10 Figure 1.5 Ribbon representation of the S1 structure of the myosin motor 11 Figure 1.6 Myosin 3A structure and important domains 15 Figure 1.7 The actomyosin ATPase cycle reaction scheme 18 Figure 1.8 Space-filling model of the myosin protein and ATP 20 Figure 4.1 Sequence alignments of amino acid residues from the Switch I 32 region of human MYO3A in comparison with human MYO3B, MYO5A, MYO7A, and Drosophila NINAC Figure 4.2 Locations of WT and mutant MYO3A in COS7 cells 34 Figure 4.3 Impact of the G488E mutation on MYO3A filopodia elongation 35 Figure 4.4 Impact of the G488E mutation on filopodia induction in COS7 36 cells Figure 4.5 The presence of ESPIN1 increases tip-localization of wildtype 37 MYO3A Figure 4.6 Mutant MYO3A can function as a dominant-negative 38 Figure 4.7 Actin-activated ATPase activity of MYO3A.ΔK.2IQ and 39 MYO3A.ΔK.G488E.2IQ Figure 4.8 In-vitro motility assay of WT MYO3A 40 Figure 5.1. WT MYO3A.ΔK is able to tip-localize on COS7 filopodia, while 41 mutant MYO3A.ΔK.G488E is not viii LIST OF TABLES Table 1.1 Function, targeting, and regulation of unconventional myosin, 16 organized by myosin class Table 3.1 Experimental myosin constructs 24 Table 4.1 Actin-activated ATPase activity of MYO3A.ΔK.2IQ constructs 39 Table 4.2 Sliding velocity of actin filaments on MYO3A.ΔK.2IQ constructs 40 ix LIST OF ABBREVIATIONS ADP Adenosine diphosphate AM Acto-myosin ATP Adenosine triphosphate Bac Baculovirus C° Centigrade DNA Deoxyribonucleic acid DTT Dithiothreitol EGTA Ethylene glycol tetraacetic acid Epsin1-un Unlabeled ESPIN1 GFP Green fluorescent protein IHC Inner hair cell ΔK Delta kinase F-actin Filamentous-actin Katpase Catalytic ATPase rate Kcat Catalytic rate constant KM Kinase motor MD Motor dead min Minute mL Milliliter MYO3A Myosin IIIA MYO3B Myosin IIIB MYO5 Myosin V x n Number NADH Nicotinamide adenine dinucleotide NBP Nucleotide binding pocket NINAC Neither inactivation nor afterpotential C N-terminus Amino terminus OHC Outer hair cell Pi Inorganic phosphate PBS Phosphate buffered saline pFB pFastBac S.E. Standard Error S1 Subunit 1 THDI Tail homology domain I THDII Tail homology domain II TCBR Tip to cell body ratio µg Microgram µL Microliter µm Micrometer µM Micromolar WT Wildtype xi ACKNOWLEDGEMENTS This study was made possible by funding from the National Institute of Health. Special thanks should be given to the committee responsible for mentoring and assisting in the revisions of the experimental design. This committee included Dr. Christopher Yengo, Dr. Alistair Barber, and Dr. Sarah Bronson. Their knowledge and research experience were essential