Development of the stereocilia and kinocilia in the mammalian organ of Corti as directed by Rab11a Público
Knapp, Lindsey (Spring 2019)
Abstract
The organ of Corti in the mammalian cochlea is precisely designed for its function of auditory perception. Sensory hair cells have apical projections consisting of stereocilia organized into a V-shaped bundle and a kinocilium at the vertex representing the intrinsic polarity of the hair cell. Coordinated polarity of the cell is established through the planar cell polarity pathway. In order for the intrinsic and coordinated polarity of the hair cell to be established, proteins involved in these pathways must be specifically trafficked to the correct cellular compartments. Rab11a is a protein of the recycling endosome also necessary for ciliation that is positioned to play a role in cochlear hair cell development. Using Rab11a conditional knockout mice, we investigated the role that Rab11a plays in the formation and maintenance of the hair cell. In cultured cells we found that Rab11a was necessary for ciliogenesis. However, in the hair cell, Rab11a regulates kinocilia formation through a genetic interaction with IFT88. Stereocilia formation is affected in Rab11a conditional knockouts leading to stereocilia bundled with an altered intrinsic polarity no longer resembling the V-shaped bundle but instead have fragmented bundles. Additionally, Rab11a loss leads to stereocilia degeneration and hearing loss in adult mice. Loss of Rab11a did not alter apical protein trafficking in the cochlea suggesting the malformation of stereocilia was not due to a global loss of apical protein trafficking. We found that expression of dominant negative Rab11a construct in culture altered the trafficking of Usher protein Cadherin23 from the apical domain of the cell to throughout the entire cell suggesting that Rab11a may be responsible for trafficking inter-stereocilia link proteins responsible for stereocilia adhesion. Additionally, the planar cell polarity pathway is regulated downstream of core PCP proteins by Rab11a and Ankrd6, the mammalian homolog of Diego. Together, these data show that Rab11a plays vital roles in hair cell formation and maintenance through its roles in kinocilia formation, regulation of the planar cell polarity pathway, stereocilia formation, and ultimately hearing suggesting that intracellular trafficking in hair cells is a vital part of hair cell development.
Table of Contents
TABLE OF CONTENTS
Chapter 1: The Inner Ear………………………………………………….………….1
Development of the Cochlea………………………………………….…….…2
Morphological Development of the Organ of Corti…………………..2
Hair Cell Development and Patterning………………………………..4
Planar Cell Polarity……………………………………..…........4
The Kinocilia and Intrinsic Polarity……………………..……..9
Additional Intrinsic Polarity Proteins…………….…………...11
Building the Stereocilia………………………………….........13
Stereocilia Adhesion Complexes and Usher Proteins .…..14
Hearing and Auditory Perception…………………………………………….16
Summary………………………………………………………………………..18
Figures………………………………………………………………………….19
Chapter 2: Rab11a and Trafficking in Cell Biology……………………..……...24
Rab11a in Protein Trafficking………………………………………………...25
Rab11a in Ciliation…………………………………………………………….28
Rab11a in Apical Surface Projects: Microvilli……………………………….29
Trafficking and Rab11a’s Potential Roles in the Inner Ear….…………….30
Summary………………………………………………………………………..33
Figures………………………………………………………………………….35
Chapter 3: Rab11a plays a role in the development of the stereocilia and kinocilia in the mammalian organ of Corti …37
Introduction …………………………………………………………………….38
Results …………………………………………………………………………45
Rab11a is localized adjacent to the basal body in cochlear hair cells……45
Inactivation of Rab11a leads to defects in stereocilia bundles of
HCs …………………………………………………………………….46
The influence of Rab11a inactivation on hair bundle morphology
varies along the longitudinal and mediolateral axes of the cochlea
…………………………………………………………………………...47
Rab11a plays a role in ciliogenesis and interacts genetically with
IFT88 in the formation of the kinocilia in HCs ……………………...48
Ultra structure of Rab11a reveals clear stereocilia fragmentation
and kinocilia loss in the cochlea ……………………………………..50
Protein partitioning along the PCP axis or the apical- basal axis of the organ of Corti is not apparently affected by Rab11a conditional knockout ... 51
Rab11a regulates PCP downstream of core PCP proteins.………55
Rab11a is required for hearing and adults have hair cell degeneration ... 56
Rab11a is required for Cadherin23 localization to stereocilia-like extensions in cultured cells …56
Discussion ……………………………………………………………………..57
Rab11a in apical protein localization ………………………………..58
Rab11a in ciliogenesis in the cochlea ………………………………60
Rab11a in sculpting the stereocilia …………………………………61
Rab11a in planar cell polarity ………………………………………..63
Materials and Methods ……………………………………………………….65
Figures …………………………………………………………………………72
Chapter 4: Ankrd6 Regulates Planar Cell Polarity in the Mammalian Organ of Corti …97
Introduction …………………………………………………………………….98
Results …………………………………………………………………………99
Ankrd6 is asymmetrically polarized in the cochlea …………….….99
Ankrd6 interacts genetically with Vangl2 to coordinate PCP in the
Organ of Corti ………………………………………………………..100
Discussion ……………………………………………………………………101
Ankrd6 as a PCP protein in the mammalian organ of Corti……..101
Materials and Methods ……………………………………………………...102
Figures ………………………………………………………………………..104
Chapter 5: Conclusions and Future Perspectives ………….………………..109
Rab11a alone is not responsible for global apical protein trafficking in the
organ of Corti ………………………………………………………………...110
Regulation of kinocilia of the hair cell by Rab11a ………………………..112
Regulation of stereocilia formation in the hair cell by Rab11a ………….115
Further understanding of the planar cell polarity pathway form trafficking to
downstream effectors ……………………………………………………….119
Summary………………………………………………………………………121
References………………………………………………………………................ 124
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