A fundamental challenge of auditory neuroscience is to understand how hair cells, sensory cells of the inner ear, transduce mechanical displacements with subnanometer resolution and at kilohertz rates. For this purpose, the hair cell deploys its transduction apparatus, an integrated collection of components that includes the transduction channel, tip link, and adaptation motor. To understand mechanical transduction, we must identify molecules that contribute to the transduction apparatus and determine how they interact. One group of related proteins that is essential for hair cells in the myosin family. Three myosin isozymes appear to play specialized roles in hair cells, including: myosin VIIa, mutations in which underlies the human deafness Usher syndrome IB and are responsible for deafness in the shaker-1 mouse; myosin VI, mutations of which are responsible for deafness in the Snell's waltzer mouse; and myosin 1B, which is thought to play a pivotal role in transduction, mediating adaptation to sustained mechanical stimuli. We intend to identify bundle molecules that associate with hair-bundle myosin isozymes and to establish which of these are essential for transduction or adaptation. At least three types of important molecules might bind to the myosin isozyme that mediates adaptation: (i) linker proteins, which could couple myosin molecules to the transduction apparatus; (ii) the extent spring, which restrains the transduction apparatus during large displacements; or (iii) the transduction channel itself. We plan to identify myosin-binding proteins by screening expression libraries; we will then demonstrate the relevance of cloned proteins by delivering to hair cells inhibitory protein fragments, derived from bundle myosin-binding proteins, expecting these fragments will act to block transduction or adaptation. Another set of experiments will test the role of myosins 1B, VI, and VIIa in tip-link regeneration following calcium-chelator treatment. Assembly of the transduction apparatus--which appears to take place during tip-link regeneration--may rely on myosin molecules, and we will test that assertion using biochemical morphological, and electrophysiological experiments. Finally, we will examine myosin motility in hair cells using fluorescence recovery after photobleaching (FRAP). We expect that these dynamics experiments with FRAP will tie together properties of myosin isozymes and their binding proteins, both at steady-state and during tip-link regeneration.
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