The overall goal of this collaborative project is to determine whether myosin 1b is the adaptation motor, to ascertain the role of myosin 1b in other hair cell functions, and to dissect the hair cell roles of myosins VI, VIIa, and XV. In their approach, the investigators developed a mutant/inhibitor strategy that allows them to test the role of a myosin isozyme in a given cellular function. By mutating myosin in a manner that maintains normal ATP hydrolytic and chemomechanical activity, yet greatly enhances binding of an inhibitor, they sensitize myosin to the inhibitor. They then show that the inhibitor has an effect on the hair cell function of interest only when the sensitized myosin transgene is expressed in hair cells. During the initial funding period, they designed such a mutation in myosin 1b, Y61G, which sensitizes the mutant protein to N6-modified ADP analogs. In this renewal application, they propose to complete their examination of the role of myosin 1b in slow adaptation, examining the effects of the ADP analogs in hair cells that express Y61G myosin 1b using transgenic or knock-in methods. They will also determine whether myosin 1b sets hair cell resting tension and examine whether it participates in restoration of functional transduction after the hair cell's tip links are broken. The latter two goals will require development of new myosin inhibitors that can freely pass across cell membranes. Finally, they propose to use the knowledge of effective mutant/inhibitor combinations developed for myosin 1b to examine roles for three myosin isozymes that are essential for hair cell function, myosins VI, VIIa, and XV. After developing myosin mutants and selective inhibitors, they will use assays for transduction and adaptation, bundle development, apical endocytosis, and aminoglycoside uptake to determine where and when these myosin isozymes are required in hair cells.
| Karcher, Ryan L; Provance, D William; Gillespie, Peter G et al. (2007) Chemical-genetic inhibition of sensitized mutant unconventional myosins. Methods Mol Biol 392:231-40 |
| Senften, Mathias; Schwander, Martin; Kazmierczak, Piotr et al. (2006) Physical and functional interaction between protocadherin 15 and myosin VIIa in mechanosensory hair cells. J Neurosci 26:2060-71 |
| LeMasurier, Meredith; Gillespie, Peter G (2005) Hair-cell mechanotransduction and cochlear amplification. Neuron 48:403-15 |
| Stauffer, Eric A; Scarborough, John D; Hirono, Moritoshi et al. (2005) Fast adaptation in vestibular hair cells requires myosin-1c activity. Neuron 47:541-53 |
| Gillespie, Peter G; Dumont, Rachel A; Kachar, Bechara (2005) Have we found the tip link, transduction channel, and gating spring of the hair cell? Curr Opin Neurobiol 15:389-96 |
| Gillespie, Peter G (2004) Myosin I and adaptation of mechanical transduction by the inner ear. Philos Trans R Soc Lond B Biol Sci 359:1945-51 |
| Provance Jr, D William; Gourley, Christopher R; Silan, Colleen M et al. (2004) Chemical-genetic inhibition of a sensitized mutant myosin Vb demonstrates a role in peripheral-pericentriolar membrane traffic. Proc Natl Acad Sci U S A 101:1868-73 |
| Colclasure, J Chris; Holt, Jeffrey R (2003) Transduction and adaptation in sensory hair cells of the mammalian vestibular system. Gravit Space Biol Bull 16:61-70 |
| Strassmaier, Meredith; Gillespie, Peter G (2002) The hair cell's transduction channel. Curr Opin Neurobiol 12:380-6 |
| Dumont, Rachel A; Zhao, Yi-Dong; Holt, Jeffrey R et al. (2002) Myosin-I isozymes in neonatal rodent auditory and vestibular epithelia. J Assoc Res Otolaryngol 3:375-89 |
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