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IDENTIFICATION and CHARACTERIZATION of ACTIN-REGULATORY PROTEINS in the HAIR CELL's CUTICULAR PLATE by LANA MARY POLLOCK Subm

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IDENTIFICATION and CHARACTERIZATION of ACTIN-REGULATORY PROTEINS in the HAIR CELL's CUTICULAR PLATE by LANA MARY POLLOCK Subm IDENTIFICATION AND CHARACTERIZATION OF ACTIN-REGULATORY PROTEINS IN THE HAIR CELL’S CUTICULAR PLATE by LANA MARY POLLOCK Submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy Dissertation advisor: Brian M. McDermott Jr., Ph.D. Department of Genetics and Genome Sciences CASE WESTERN RESERVE UNIVERSITY January 2016 Case Western Reserve University School of Graduate Studies We, the thesis committee, hereby approve the thesis/dissertation of Lana Pollock, candidate for the degree of Doctor of Philosophy (PhD).* (signed)_________Zhenghe Wang, Ph.D._________________ (chair of committee) ___________Brian McDermott, Ph.D._______________ ___________ Hua Lou, Ph.D._____________________ ___________Stephen Maricich, Ph.D., M.D.___________ ___________Anthony Wynshaw-Boris, Ph.D., M.D._____ Date of defense_____September 8th, 2015_______________ *we also certify that written approval has been obtained for release of any proprietary material contained therein 2 This thesis is dedicated to Daniel Margevicius. Thank you for your unwavering love and support. Ačiū!! 3 Table of contents List of Tables ........................................................................................................ 7 List of Figures ....................................................................................................... 8 List of abbreviations ............................................................................................ 13 Abstract .............................................................................................................. 16 Chapter 1: Background and Significance ........................................................... 18 1.1 Introduction to hearing ............................................................................... 19 1.2 The cuticular plate ..................................................................................... 24 1.2.1 Arrangement of F-actin in the cuticular plate ....................................... 24 1.2.2 Models of cuticular plate development ................................................ 30 1.2.3 Organization of stereocilia rootlets in the cuticular plate ..................... 33 1.2.4 Microtubule structures near the apical end of the hair cell .................. 34 1.2.5 Striated organelle ................................................................................ 36 1.2.6 Cuticular plate structure in cochlear hair cells ..................................... 37 1.2.7 Protein constituents of the cuticular plate ............................................ 39 1.2.8 Transport within the cuticular plate...................................................... 48 1.3 Organization of the reticular lamina of the mammalian cochlea ................ 49 1.4 The establishment of planar cell polarity in the hair cell ............................ 54 1.5 The zebrafish ear ...................................................................................... 55 1.6 New cuticular plate proteins identified in this work .................................... 59 1.6.1 ACF7 ................................................................................................... 59 1.6.2 Supervillin ........................................................................................... 61 Chapter 2: Identification of candidate proteins from the hair cell transcriptome . 65 2.1 Introduction ............................................................................................... 67 2.2 Materials and Methods .............................................................................. 68 2.2.1 Animals ............................................................................................... 68 2.2.2 Analysis of the zebrafish hair cell transcriptome ................................. 69 2.2.3 RNA-seq of the chicken hair cell transcriptome .................................. 72 2.2.4 RT-PCR .............................................................................................. 73 2.2.5 Immunolabeling ................................................................................... 75 2.2.6 Generation of pMT/SV/Pv3b/mEGFP vector ....................................... 75 4 2.2.7 Zebrafish transgenesis ........................................................................ 76 2.3 Results and Discussion ............................................................................. 77 Chapter 3: ACF7 is a hair-bundle antecedent, positioned to integrate cuticular plate actin and somatic tubulin ........................................................................... 90 3.1 Introduction: .............................................................................................. 92 3.2 Materials and Methods: ............................................................................. 93 3.2.1 Animals ............................................................................................... 93 3.2.2 Hair-cell isolation and RNA-seq .......................................................... 93 3.2.3 RT-PCR .............................................................................................. 94 3.2.4 Plasmid construction ........................................................................... 95 3.2.5 RNA in situ hybridization ..................................................................... 96 3.2.6 Fish labeling and imaging ................................................................... 96 3.2.7 Zebrafish transgenesis ........................................................................ 96 3.2.8 Labeling of mouse tissue .................................................................... 96 3.2.9 Electron microscopy and tomography ................................................. 97 3.3. Results ..................................................................................................... 98 3.3.1 Microtubule and actin crosslinking factor 1 genes are expressed in hair cells of fish, birds, and mammals ................................................................. 98 3.3.2 Acf7a encircles the fonticulus and circumscribes, underlies, and is interwoven into the CP of zebrafish ........................................................... 102 3.3.3 Localization of ACF7 in mammalian hair cells ................................... 103 3.3.4 Acf7a is a hair bundle and CP antecedent ........................................ 106 3.3.5 Electron tomography reveals filamentous linkers that couple microtubules to the actin meshwork of the CP ........................................... 110 3.4 Discussion ............................................................................................... 113 3.4.1 ACF7 links CP actin to the microtubule network ............................... 113 3.4.2 The significance of ACF7 in hair-cell polarity .................................... 114 Chapter 4: Knockout of ACF7 in the mouse ear ............................................... 116 4.1 Introduction ............................................................................................. 117 4.2 Materials and methods ............................................................................ 118 4.2.1 Animals ............................................................................................. 118 4.2.2 Genotyping ........................................................................................ 118 5 4.2.3 Whole-mount immunofluorescence ................................................... 122 4.3 Results and discussion ............................................................................ 122 Chapter 5: Actin-binding protein supervillin is a component of the hair cell’s cuticular plate and other elements of the reticula lamina .................................. 132 5.1 Introduction ............................................................................................. 134 5.2 Materials and Methods ............................................................................ 136 5.2.1 Animals ............................................................................................. 136 5.2.2 Chicken hair-cell isolation and RNA-seq ........................................... 136 5.2.3 RT-PCR ............................................................................................ 136 5.2.4 Whole mount mRNA in situ hybridization .......................................... 137 5.2.5 Whole-mount immunofluorescence of mouse tissues ....................... 138 5.2.6 Generation and preparation of zebrafish Svila antibody.................... 139 5.2.7 Zebrafish whole-mount immunofluorescence .................................... 139 5.3 Results and discussion ............................................................................ 140 5.3.1 Supervillin is expressed in vertebrate hair cells ................................ 140 5.3.2 Supervillin localizes to the CP in mouse vestibular and cochlear hair cells and to regions of supporting cells that are encompassed in the reticular lamina of the organ of Corti ........................................................................ 146 5.3.3 Supervillin localizes to the zebrafish hair cell CP .............................. 150 Chapter 6: Summary of conclusions and future directions .............................. 155 6.1 Summary of conclusions ......................................................................... 156 6.2 Future directions...................................................................................... 159 6.2.1 Determine
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