Mechanosensor Proteins in Hair Cell Repair

毛细胞修复中的机械传感器蛋白

• 批准号: 10718860
• 负责人: Jung-Bum Shin
• 金额: $ 47.86万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10718860
• 关键词: Ablation; Actin-Binding Protein; Actins; Age; Aging; Auditory; Binding; Binding Proteins; Binding Sites; C-terminal; Cardiac Myocytes; Cell Death; Cell Line; Cell Maintenance; Cells; Characteristics; Complex; Data; Equilibrium; Exhibits; Exposure to; F-Actin; Fiber; Fibroblasts; Genetic; Hair; Hair Cells; Hearing; Human; Inner Hair Cells; Intercalated disc; Knock-in Mouse; Knockout Mice; LIM Domain; LIM Domain Protein; Label; Labyrinth; Lasers; Lesion; Life; Loudness; Maintenance; Mechanical Stress; Mechanics; Mediating; Metabolic stress; Microfilaments; Modeling; Molecular; Mus; Mutation; Noise; Noise-Induced Hearing Loss; Pharmaceutical Preparations; Play; Prevention; Process; Property; Protein Isoforms; Proteins; Proteomics; RNA Splicing; Reporting; Research; Role; Sensory Hair; Site; Stress Fibers; Stretching; Testing; Thymus Gland; Time; Torsion; Vinculin; alpha catenin; beta Actin; cell injury; cofilin; cysteine rich protein; depolymerization; experience; experimental study; gamma Actin; genome wide association study; hearing impairment; in vivo; interest; live cell imaging; loss of function; mouse model; muscle LIM protein; noise exposure; novel; ototoxicity; paralogous gene; prevent; programs; progressive hearing loss; recruit; repair function; repaired; response; transcriptome sequencing; virtual

Abstract Sensory hair cells of the inner ear experience continuous mechanical and metabolic stress. The maintenance of hair cells is further challenged by damage from a variety of other ototoxic factors, including loud noise, aging, genetic defects, and ototoxic drugs. Because mammalian auditory hair cells do not regenerate, the repair of hair cell damage is important for continued auditory function. Our research program is especially interested in molecular processes involved in the maintenance of the stereocilia filamentous (F)-actin core. Recent studies have concluded that the stereocilia actin core is stable over months, implying that any structural damage must be actively repaired. The stereocilia F-actin core can sustain damage, most notably by noise exposure, which was shown to cause “gaps” in phalloidin labeling of F-actin in stereocilia. In preliminary studies, we found that these gaps are repaired in days. We therefore propose to investigate the molecular mechanisms by which the F-actin lesions are sensed and repaired. The proposed study was inspired by an emerging concept in mechanobiology, according to which F-actin possesses intrinsic mechanosensory properties. In this model, mechanical strain modulates the interaction of actin filaments with effector proteins. For a variety of actin binding proteins, their constitutive binding to F-actin is merely tuned by force. A subset of LIM domain proteins however are unique in that mechanical strain reveals previously hidden binding sites on F-actin, providing an on/off switch for downstream processes. These processes were implicated in the recruitment of actin repair substrates and in the prevention of F-actin fiber breakage. We reasoned that hair cells might employ a similar strategy to repair its F-actin-based stereocilia. In our search for molecules involved in this process, we focused on proteins that are enriched in the hair cell bundle, contain potential mechanosensor domains, and cause progressive hearing loss in human or mice with loss of function. We identified two proteins, XIRP2 (Xin Actin Binding Repeat Containing 2) and CRIP3 (cysteine rich protein 3) that fulfill these criteria. We hypothesize that XIRP2 and CRIP3 are mechanosensor proteins capable of sensing F-actin damage and recruiting additional repair factors, thus playing essential roles in hair cell stereocilia repair and maintenance. To test this, in SA1, we propose to test the hypothesized mechanosensor function of XIRP2 in fibroblasts. In preliminary studies, we discovered a novel mechanosensor domain in the C-terminus of XIRP2. We will use live cell laser ablation and cell stretch experiments to define the mechanosensor region, and investigate the mechanisms by which XIRP2’s mechanosensor function is regulated. In SA2, we propose to test the mechanosensor and repair function of XIRP2 in vivo, using a mouse model that lacks the mechanosensor domain. We will also perform ex vivo experiments to test whether fluorescently tagged XIRP2 is recruited to stereocilia lesions. In SA3, in an effort to investigate the involvement of additional repair factor candidates, we will test the mechanosensor and repair function of the hair bundle enriched LIM domain protein CRIP3.

抽象的 内耳的感觉毛细胞经历连续的机械和代谢应力。维护 毛细胞受到各种其他耳毒性因素的破坏，包括大声噪音，衰老， 遗传缺陷和耳毒性药物。因为哺乳动物的听觉毛细胞不会再生 细胞损伤对于持续听觉功能很重要。我们的研究计划特别感兴趣 参与立体丝菌（F） - 肌动蛋白核心维持的分子过程。最近的研究 已经得出结论，立体尾肌动蛋白核心在几个月内都是稳定的，这意味着任何结构性损害都必须 积极维修。立体cil肌F-肌动蛋白核可以维持损害，最著名的是噪声暴露，这是 在初步研究中，我们发现显示“差距”在立体cil的F-肌动蛋白标记中引起“差距”。 这些差距在几天内修复。因此，我们建议研究分子机制 感测并修复F-肌动蛋白病变。拟议的研究灵感来自于新兴概念 机理生物学，根据该机构，F-肌动蛋白具有内在的机械感应特性。在这个模型中， 机械应变调节肌动蛋白与效应蛋白的相互作用。用于各种肌动蛋白结合 蛋白质，它们与F-肌动蛋白的组成型结合仅通过力调节。但是，LIM结构蛋白的子集 它是独一无二的，因为机械应变揭示了以前在F-肌动蛋白上隐藏的结合位点，提供了开/关开关 用于下游过程。这些过程在肌动蛋白维修底物的募集中暗示 预防F-肌动蛋白纤维断裂。我们认为毛细胞可能会采用类似的策略来修复其 基于F-肌动蛋白的立体胶质。在我们寻找参与此过程的分子时，我们专注于 富含毛细胞捆绑包，包含潜在的机械学域，并导致渐进的听力损失 在人类或小鼠中失去功能。我们鉴定了两种蛋白质XIRP2（XIN肌动蛋白结合重复重复） 2）和CRIP3（富含半胱氨酸蛋白3）符合这些标准。我们假设XIRP2和CRIP3是 机械传感器蛋白能够感测F-肌动蛋白损伤并募集其他修复因子， 在毛细胞立体胶质修复和维护中扮演重要角色。为了测试这一点，在SA1中，我们提出了 测试成纤维细胞中XIRP2的假设的机理经验函数。在初步研究中，我们发现了 XIRP2的C末端中的一种新型机械试剂域。我们将使用活细胞激光消融和细胞拉伸 定义机理区域的实验，并研究XIRP2的机制 机理传感器功能受到调节。在SA2中，我们建议测试 XIRP2在体内，使用缺乏机械力体域的小鼠模型。我们还将进行体内 测试荧光标记的XIRP2是否募集到立体病变的实验。在SA3中，为了 调查其他维修因子候选物的参与，我们将测试机理经验器和维修 富含头发的LIM结构蛋白CRIP3的功能。