Mechanisms Mediating Repair of Stereocilia F-actin in Sensory Hair Cells

感觉毛细胞中立体纤毛 F-肌动蛋白修复的介导机制

• 批准号: 10366129
• 负责人: Elizabeth Lee Wagner
• 金额: $ 0.87万
• 依托单位: UNIVERSITY OF VIRGINIA
• 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 2021-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10366129
• 关键词: Acoustics; Actins; Address; Affect; Amino Acids; Appearance; Area; Auditory system; Binding; Burn injury; Cell Death; Centers for Disease Control and Prevention (U.S.); Chimeric Proteins; Code; Corwin; Excision; F-Actin; Filament; Genetic Models; Hair; Hair Cells; Hearing; Human; Image; Immunohistochemistry; Inner Hair Cells; Knock-in; Knock-out; Knockout Mice; Label; Labyrinth; Lead; Length; Life; Link; Maintenance; Mechanics; Mediating; Microfilaments; Minor; Monitor; Mus; Mutation; Natural regeneration; Noise; Noise-Induced Hearing Loss; Organ; Pattern; Play; Presbycusis; Process; Proteins; Recovery; Role; Sensory; Sensory Hair; Site; Stains; Structure; System; Temporary Threshold Shift; Testing; Visualization; cell injury; cofilin; crosslink; experimental study; gamma Actin; hair cell regeneration; hearing impairment; hearing preservation; loss of function; mouse model; novel; polymerization; preservation; progressive hearing loss; reconstitution; recruit; repaired; response

Project Summary/Abstract Insult to the auditory system often results in irreparable hearing loss due to the inability of mature mammalian hair cells to regenerate (Burns, J.C. and Corwin, J.T. 2013; Groves, A.K. 2010). However, evidence suggests that minor damage to mechanotransductive hair bundles can be repaired in some cases. For example, gaps in phalloidin labeling of the F-actin in stereocilia are found after mechanical damage and appear to indicate filament breaks. Factors important for the nucleation, elongation, and crosslinking of F-actin assembly, including γ-actin, cofilin, and espin, are enriched in these sites (Belyantseva, I.A. et al. 2009). My proposal aims to investigate the mechanisms mediating this F-actin repair process. In Specific Aim 1, I will test the hypothesis that damage of stereociliar F-actin leads to the incorporation of newly synthesized F-actin in stereocilia. In order to accomplish this, I will develop mouse models for the visualization of the response of newly synthesized actin to mechanical damage, using the FLEx-Cre switch system. Repair of the F-actin core is expected to depend on proteins that mediate the nucleation, polymerization, stabilization and/or crosslinking of actin filaments. My preliminary studies suggest that XIRP2 (Xin Actin Binding Repeat Containing 2) may be involved in the stereocilia F-actin repair process, as well. We previously described XIRP2 as a novel hair cell protein enriched in the hair bundle, where it colocalizes with F-actin. Knockout of Xirp2 causes progressive hearing loss in mice (Francis, S.P. et al. 2015); moreover, heterozygous mutations in XIRP2 were identified in humans with age related hearing loss. While less severe than the hearing loss in the total Xirp2 knockout mice, heterozygous Xirp2 mice also develop progressive hearing loss. Additionally, immunostaining shows the XIRP2 is enriched in gaps in phalloidin staining, similar to those described above. In Specific Aim 2, I will test the hypothesis that XIRP2 is recruited to these gaps in as a direct response of mechanical damage. At these sites, XIRP2 may facilitate the maintenance or repair of the F-actin stereocilia core and I expect that the dysfunctional repair process in Xirp2-null mice leads to the observed progressive decline in hearing function. In order to visualize the response of XIRP2 to hair bundle damage, I will knock in a fluorescent tag at the endogenous Xirp2 locus to enable live imaging. I will determine whether XIRP2 is recruited to damaged sites in F-actin stereocilia cores using both a mechanical and genetic model of damage.

项目总结/摘要 对听觉系统的损伤通常会导致无法修复的听力损失，这是由于成熟的哺乳动物 毛细胞再生（Burns，J.C.和Corwin，J.T. 2013;格罗夫斯，A.K. 2010年）。然而，有证据表明， 在某些情况下，对机械传导毛束的轻微损伤可以修复。例如， 在机械损伤后发现静纤毛中F-肌动蛋白的鬼笔环肽标记，似乎表明 灯丝断裂。F-肌动蛋白组装成核、延伸和交联的重要因素， 包括γ-肌动蛋白、cofilin和espin在这些位点富集（Belyantseva，I. A.等人，2009年）。我的提议 目的是研究介导这种F-肌动蛋白修复过程的机制。在具体目标1中，我将测试 假设立纤毛F-肌动蛋白的损伤导致新合成的F-肌动蛋白的掺入， 静纤毛为了实现这一点，我将开发小鼠模型，用于可视化 新合成的肌动蛋白的机械损伤，使用FLEx-Cre开关系统。 预期F-肌动蛋白核心的修复依赖于介导成核、聚合 肌动蛋白丝的稳定和/或交联。初步研究表明XIRP 2（Xin Actin 含有结合重复序列2）也可能参与静纤毛F-肌动蛋白修复过程。我们之前 描述了XIRP 2作为一种新的毛细胞蛋白，富集在发束中，在那里它与F-肌动蛋白共定位。 敲除Xirp 2导致小鼠进行性听力损失（弗朗西斯，S. P.等人，2015）;此外，杂合 XIRP 2的突变在患有年龄相关性听力损失的人中被鉴定。虽然没有听证会那么严重 在总Xirp 2敲除小鼠中，杂合Xirp 2小鼠也发生进行性听力损失。 此外，免疫染色显示XIRP 2在鬼笔环肽染色的缺口中富集，类似于那些 上面描述在具体目标2中，我将测试XIRP 2被招募到这些缺口中作为一个直接的假设。 机械损伤的反应。在这些位点，XIRP 2可能促进F-肌动蛋白的维持或修复。 静纤毛核心，我预计Xirp 2缺失小鼠的功能障碍性修复过程导致观察到的 听力功能逐渐下降。为了可视化XIRP 2对毛束损伤的反应，我 将在内源性Xirp 2基因座敲入荧光标签，以实现实时成像。我将决定 XIRP 2被招募到F-肌动蛋白静纤毛核心的损伤位点，使用机械和遗传模型， 损害