Our senses of hearing and balance rely on the proper development, function and maintenance of the hair bundle at the apical surface of inner ear hair cells. Hair bundles are composed of distinct multi-protein complexes of defined function and architecture that work together and that are responsible for mechanoelectrical transduction, adaptation and possibly signal amplification. Failure of function of any of these machineries leads to deafness or various degrees of hearing and balance impairments. Our goal is to visualize these stereocilia molecular machines in their native cellular context at molecular resolution. Our rationale is that the determination of the molecular 3D organization of these stereocilia complexes will ultimately lead to a mechanistic understanding of development, function and maintenance of this precision organelle. Specifically, we propose specifically to study four regions in stereocilia from lower (frog, zebrafish) and higher (mouse, guinea pig) vertebrates, each of which hosts a particular type of multi-protein complex, and plays a distinct role in hair bundle formation, function and/or maintenance. The four regions are 1.) the tip complex, 2.) the actin core, 3.) the adaptation complex, as well as 4.) the tapered region and the rootlets.
Our specific aims are as follows:
Specific Aim 1 : Determine 3D organization of sterecilia tip complex, the site of mechanotransduction and actin bundle nucleation Specific Aim 2: Determine 3D organization of side plaque, the site of adaptation and possibly signal amplification.
Specific Aim 3 : Determine 3D organization of the actin core, including the actin-actin cross linkers and the actin bundle-membrane connectors.
Specific Aim 4 : Determine the 3D organization of molecular machines residing in the tapered stereocilia region, the rootlets and the cuticular plate We will employ electron tomography of either high-pressure frozen, freeze-substituted and resin-embedded hair bundle sections or of frozen-hydrated isolated individual stereocilia. The 3D architecture of the bundle multiprotein complexes will aid in a mechanistic understanding of our senses or hearing and balance and provide a baseline for future analysis of diseased tissue.

Public Health Relevance

Our study will result in a detailed model of the 3D organization of the hair bundle and its constituent machineries, and will provide a structural framework in which genetic, immunolocalization and electrophysiological studies can be interpreted and will thus have profound implications for our understanding of hair bundle physiology and therefore lead to a fundamental understanding of our senses of hearing and balance.

Agency
National Institute of Health (NIH)
Type
Research Program Projects (P01)
Project #
5P01GM051487-19
Application #
8666759
Study Section
Special Emphasis Panel (ZRG1)
Project Start
Project End
Budget Start
Budget End
Support Year
19
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Lawrence Berkeley National Laboratory
Department
Type
DUNS #
City
Berkeley
State
CA
Country
United States
Zip Code
94720
Holmes, Dawn E; Giloteaux, Ludovic; Chaurasia, Akhilesh K et al. (2015) Evidence of Geobacter-associated phage in a uranium-contaminated aquifer. ISME J 9:333-46
Tsai, Wen-Ting; Hassan, Ahmed; Sarkar, Purbasha et al. (2014) From voxels to knowledge: a practical guide to the segmentation of complex electron microscopy 3D-data. J Vis Exp :e51673
Nakamura, Muneaki; Chen, Lu; Howes, Stuart C et al. (2014) Remote control of myosin and kinesin motors using light-activated gearshifting. Nat Nanotechnol 9:693-7
Antonellis, Patrick J; Pollock, Lana M; Chou, Shih-Wei et al. (2014) ACF7 is a hair-bundle antecedent, positioned to integrate cuticular plate actin and somatic tubulin. J Neurosci 34:305-12
Beers, Keith M; Yakovlev, Sergey; Jackson, Andrew et al. (2014) Absence of Schroeder's paradox in a nanostructured block copolymer electrolyte membrane. J Phys Chem B 118:6785-91
Chen, X Chelsea; Wong, David T; Yakovlev, Sergey et al. (2014) Effect of morphology of nanoscale hydrated channels on proton conductivity in block copolymer electrolyte membranes. Nano Lett 14:4058-64
Shamir, Eliah R; Pappalardo, Elisa; Jorgens, Danielle M et al. (2014) Twist1-induced dissemination preserves epithelial identity and requires E-cadherin. J Cell Biol 204:839-56
Howes, Stuart C; Alushin, Gregory M; Shida, Toshinobu et al. (2014) Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure. Mol Biol Cell 25:257-66
Remis, Jonathan P; Wei, Dongguang; Gorur, Amita et al. (2014) Bacterial social networks: structure and composition of Myxococcus xanthus outer membrane vesicle chains. Environ Microbiol 16:598-610
Alushin, Gregory M; Lander, Gabriel C; Kellogg, Elizabeth H et al. (2014) High-resolution microtubule structures reveal the structural transitions in ??-tubulin upon GTP hydrolysis. Cell 157:1117-29

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