Supplement for Mechanotransduction-Dependent Remodeling of the Stereocilia Cytoskeleton

立体纤毛细胞骨架的机械传导依赖性重塑的补充

• 批准号: 10170923
• 负责人: Alejandra Catalina Velez Ortega
• 金额: $ 7.41万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10170923
• 关键词: 3-Dimensional; Actins; Address; Administrative Supplement; Affect; Antibodies; Architecture; Area; Auditory; Automobile Driving; Buffers; Calcium; Cells; Childbirth; Cilia; Cochlea; Cytoskeleton; Data Analyses; Data Set; Exhibits; Goals; Growth; Hair; Hair Cells; Hearing; Height; Human Resources; Image; Ions; Label; Labyrinth; Lead; Link; Maintenance; Manuscripts; Measurement; Molecular; Morphology; Motor; Mus; Mutant Strains Mice; Mutation; Myosin ATPase; Natural regeneration; Noise-Induced Hearing Loss; Outer Hair Cells; Plasmids; Preparation; Protein Isoforms; Proteins; Regulation; Resources; Rest; Role; Scanning Electron Microscopy; Sensory; Sensory Hair; Shapes; Testing; Time; Variant; age related; base; beta Actin; depolymerization; experimental study; falls; gamma Actin; hearing impairment; mechanotransduction; microscopic imaging; mutant; new therapeutic target; parent grant; polymerization; prevent; response; treadmill

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.

项目总结/摘要 静纤毛是内耳感觉毛细胞的感觉机制。我们发现， 这些充满肌动蛋白的投射依赖于钙通过机械-电 位于静纤毛顶端的MET通道（Velez-Ortega，et al.，eLife，2017）。 我们发现，MET通道的阻塞或其门控尖端连接的断裂导致 静纤毛缩短;但一旦堵塞被移除或尖端链接再生，静纤毛 能够再生。根据静纤毛高度对静息MET变化的反应， 目前，我假设，静纤毛肌动蛋白细胞骨架表现出活性依赖的可塑性。 母基金（R21 DC 017247）的目标2一直在研究肌球蛋白XV亚型的贡献 在MET依赖性调节静纤毛高度使用几个肌球蛋白XV突变小鼠。我们的结果 到目前为止，这两种类型的哺乳动物听觉毛细胞（内毛细胞与外毛细胞）， 不同的分子机制驱动静纤毛细胞骨架中的MET依赖性变化。在 此外，我们还观察到了有趣的MET依赖性变化，即毛束的静止倾斜， 可能由静纤毛根和/或表皮板水平的变化引起。 本行政补充文件的目的是获得资源（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） 回答了一些问题，传统的SEM成像一直无法解决，由于困难， 在一些肌球蛋白XV突变体中获得极短静纤毛的精确3D测量;以及， 使用相同的FIB-SEM数据集，（ii）进一步探索机械转导依赖性 静纤毛根和/或表皮板的重排。此外，这一补充将使 博士Velez-Ortega花更少的时间在实验台上进行实验，相反，更多的时间在 数据分析和稿件准备。Velez-Ortega博士工作重点的这种“转变”将 最大限度地减少父母补助金的进展，由于即将出生的孩子在秋季 2020年