Supplement for Mechanotransduction-Dependent Remodeling of the Stereocilia Cytoskeleton

项目摘要/摘要 立体纤毛是内耳感觉毛细胞的感觉机械。我们发现，它的稳定性 这些充满肌动蛋白的投射依赖于钙通过机械-电子的持续流入。 位于立体纤毛顶端的转导(MET)通道(Velez-Ortega等人，eLife，2017)。 我们发现，MET通道的堵塞或其门控尖端连接的断裂会导致 立体纤毛缩短；但一旦阻塞被移除或尖端连接再生，立体纤毛 能够再生的。基于静息状态下纤毛高度的变化 目前，我假设立体纤毛肌动蛋白细胞骨架表现出活性依赖的可塑性。 父母资助的目标2(R21 DC017247)一直在研究肌球蛋白XV亚型的贡献 在使用几个肌球蛋白XV突变小鼠的MET依赖的立体纤毛高度调节中。我们的结果 到目前为止，表明哺乳动物的两种类型的听觉毛细胞(内毛细胞和外毛细胞)具有 不同的分子机制驱动甲硫氨酸依赖的细胞骨架的改变。在……里面 此外，我们还观察到了有趣的依赖MET的发束静止倾斜的变化 可由立体纤毛细根和/或角质板水平的变化引起。 本行政补编的目标是获得资源(用于人员和用品) 将允许我们执行聚焦离子束扫描电子显微镜(FIB-SEM)成像以(I) 回答一些传统的扫描电子显微镜成像由于困难而无法解决的问题 获得一些肌球蛋白XV突变体中极短的立体纤毛的准确3D测量；以及， 使用相同的FIB-SEM数据集，以(II)进一步探讨机械转导依赖 立体纤毛根束和/或角质板的重新排列。此外，本补编还将允许 Velez-Ortega博士将花更少的时间在长椅上进行实验，而是花更多的时间 数据分析和稿件准备。Velez-Ortega博士的努力重点的这种“转移”将 尽量减少由于秋季即将出生的孩子而导致的父母补助进度的下降 2020年。

PROJECT SUMMARY/ABSTRACT Stereocilia are the sensory machinery of the inner ear sensory hair cells. We found that the stability of these actin-filled projections depends on the constant influx of calcium through the mechano-electrical transduction (MET) channels that are located at the tips of stereocilia (Velez-Ortega, et al., eLife, 2017). We found that the blockage of MET channels or the breakage of their gating tip links leads to the shortening of stereocilia; but once the blockage is removed or the tip links regenerate, stereocilia are able to regrow. Based on the changes in stereocilia height in response to variations in the resting MET current, I hypothesized that the stereocilia actin cytoskeleton exhibits activity-dependent plasticity. Aim 2 of the parent grant (R21 DC017247) has been studying the contribution of myosin XV isoforms in the MET-dependent regulation of stereocilia height using several myosin XV mutant mice. Our results thus far indicate that the two types of mammalian auditory hair cells (inner vs. outer hair cells) have different molecular mechanisms driving the MET-dependent changes in the stereocilia cytoskeleton. In addition, we have observed interesting MET-dependent changes to the resting tilt of the hair bundle that could arise from changes at the level of the stereocilia rootlets and/or the cuticular plate. The goal of this Administrative Supplement is to obtain resources (for personnel and supplies) that would allow us to perform focused ion beam scanning electron microscopy (FIB-SEM) imaging to (i) answer some questions that traditional SEM imaging has not been able to address due to the difficulty in obtaining accurate 3D measurements of the extremely short stereocilia in some myosin XV mutants; and, using the same FIB-SEM data sets, to (ii) further explore the mechanotransduction-dependent rearrangements of the stereocilia rootlets and/or cuticular plate. In addition, this supplement would allow Dr. Velez-Ortega to spend less time on the bench performing experiments and, instead, more time on data analysis and manuscript preparation. This `shift' in the focus of Dr. Velez-Ortega's effort would minimize a decrease in the progress of the parent grant due to the upcoming birth of her child in the Fall of 2020.