MYOSIN I BETA DISRUPTION & INNER EAR STRUCTURE/FUNCTION

描述(改编自申请人的摘要)：这项工作的总体目标 合作项目是确定肌球蛋白1b是否是适应 以确定肌球蛋白1b在其他毛细胞功能中的作用，并 剖析肌球蛋白VI、VIIa和XV在毛细胞中的作用。在他们的方法中， 研究人员开发了一种突变/抑制策略，允许他们测试 肌球蛋白同工酶在特定细胞功能中的作用。通过突变肌球蛋白 以保持正常的ATP水解性和化学机械活性的方式， 然而极大地增强了抑制剂的结合，它们使肌球蛋白对 抑制剂。然后，他们表明这种抑制物对毛细胞有影响。 仅当致敏的肌球蛋白转基因在 毛细胞。在最初的资金阶段，他们设计了这样一个突变 肌球蛋白1b，Y61G，使突变蛋白对N6修饰的ADP敏感 类比。在这份续期申请中，他们建议完成他们的 检测肌球蛋白1b在慢适应中的作用及其影响 利用转基因技术在表达Y61G肌球蛋白1b的毛细胞中的ADP类似物 或敲入法。他们还将确定肌球蛋白1b是否会设置毛细胞 静息张力，并检查它是否参与恢复 毛细胞尖端连接断裂后的功能转导。后者 两个目标将需要开发新的肌球蛋白抑制剂，这种药物可以自由 穿过细胞膜。最后，他们建议利用 为肌球蛋白1b开发的有效突变体/抑制剂组合用于检测 对毛细胞功能至关重要的三种肌球蛋白同工酶的作用， 肌球蛋白VI、VIIa和XV。在培育肌球蛋白突变体和选择性 抑制剂，他们将使用转导和适应的分析，捆绑 发育、心尖内吞作用和氨基糖苷摄取以确定 当毛细胞需要这些肌球蛋白同工酶时。

DESCRIPTION (adapted from applicant's abstract): 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.