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Characterization of the Inherent Electrophysiology of Zebrafish Hair Cells and the Effect of Mutations in Met Channel Candidate Genes

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Characterization of the Inherent Electrophysiology of Zebrafish Hair Cells and the Effect of Mutations in Met Channel Candidate Genes CHARACTERIZATION OF THE INHERENT ELECTROPHYSIOLOGY OF ZEBRAFISH HAIR CELLS AND THE EFFECT OF MUTATIONS IN MET CHANNEL CANDIDATE GENES By KAYLA JEANNE KINDIG Submitted in partial fulfillment of the requirements for the degree of Master of Science Thesis Advisor: Dr. Brian McDermott Jr. Department of Biology CASE WESTERN RESERVE UNIVERSITY May 2019 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Kayla Kindig candidate for the degree of Master of Science*. Committee Chair Nicole Crown Committee Member Brian McDermott Committee Member Ruben Stepanyan Committee Member Hillel Chiel Committee Member Susan Burden-Gulley Date of Defense March 22nd, 2019 *We also certify that written approval has been obtained for any proprietary material contained therein. 1 Table of Contents List of Tables.......................................................................................................................3 List of Figures.....................................................................................................................4 Acknowledgments..............................................................................................................5 Abstract...............................................................................................................................6 Introduction........................................................................................................................7 The Human Ear...............................................................................................................7 Sensory Hair Cells.........................................................................................................13 The Mechanotransduction Channel............................................................................16 Properties of the MET Channel...........................................................................16 Candidate Channel Proteins TMC1 and TMC2................................................17 Other Potential MET Channel Proteins.............................................................21 Important Components of the MET Complex...................................................23 Zebrafish and the Lateral Line System........................................................................26 Materials and Methods....................................................................................................32 Zebrafish Breeding........................................................................................................32 Lateral Line Microphonic Potential Recordings........................................................32 Inner Ear Microphonic Potential Recordings.............................................................33 Statistics and Software..................................................................................................34 Results...............................................................................................................................35 Hair Cell Directionality of Wildtype PrimI Neuromasts..........................................35 Tmcs in the Inner Ear and Lateral Line.....................................................................37 Tmc1......................................................................................................................37 Tmc2a and Tmc2b................................................................................................38 Tmc1, Tmc2a, and Tmc2b...................................................................................42 Discussion.........................................................................................................................48 Channel Composition May Vary Between Hair Cells of the Lateral Line............48 TMCs May Combine to Form the MET Complex...................................................52 Appendix...........................................................................................................................57 References.........................................................................................................................60 2 List of Tables Table 1...............................................................................................................................43 3 List of Figures Figure 1................................................................................................................................8 Figure 2..............................................................................................................................11 Figure 3..............................................................................................................................11 Figure 4..............................................................................................................................12 Figure 5..............................................................................................................................18 Figure 6..............................................................................................................................24 Figure 7..............................................................................................................................29 Figure 8..............................................................................................................................30 Figure 9..............................................................................................................................31 Figure 10............................................................................................................................36 Figure 11............................................................................................................................40 Figure 12............................................................................................................................41 Figure 13............................................................................................................................43 Figure 14............................................................................................................................44 Figure 15............................................................................................................................45 Figure 16............................................................................................................................46 Figure 17............................................................................................................................47 Figure 18............................................................................................................................57 Figure 19............................................................................................................................58 Figure 20............................................................................................................................59 Figure 21............................................................................................................................60 Figure 22............................................................................................................................61 4 Acknowledgements I would foremost like to thank Robin Woods-Davis for her patience in training me and for answering my many questions. Acknowledgment and sincere appreciation is necessary for every member of the McDermott lab, especially those who generated the mutant fish I used in my research. Of course, I would like to thank Dr. Brian McDermott for allowing me to work in his lab and Dr. Ruben Stepanyan for allowing me to use his equipment, as well as his guidance with electrophysiology. I also need to thank my mom and my sister Felica for all their efforts in maintaining my sanity. An extra big thanks to my dear friend Alex Grossman, who proofread this document and gave me great feedback. 5 Characterization of the Inherent Electrophysiology of Zebrafish Hair Cells and the Effect of Mutations in MET Channel Candidate Genes KAYLA JEANNE KINDIG Abstract Mechanotransduction is vital for the senses of hearing and balance. Mechanotransduction occurs when a physical stimulus causes mechanically-gated channels of a sensory cell to open, allowing ions to enter the cell, thus converting a mechanical signal into an electrical one. We know that the mechanoelectrical transduction (MET) channel of sensory hair cells of the inner ear is located at the tips of actin-based stereocilia, but the identity of the pore-forming protein of the channel is unknown. It is also uncertain whether the proteins composing the channel are constant, or if they vary between hair cells based on differences in physiological requirements. In this thesis, I measure stimulus-evoked microphonic potentials of zebrafish hair cells to first determine the electrophysiological response amplitude of wildtype lateral line neuromasts, and then I use this method to examine how the mutation of certain hair cell genes affects mechanotransduction of the lateral line and inner ear. I find evidence to suggest that the MET channel components vary between hair cells, and that the proteins at the pore of the channel differ between mammals and zebrafish. This information may allow us to better understand hair cell tuning at the level of the MET complex and how the proteins necessary for mechanotransduction vary among vertebrates.
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