Mechanosensor Proteins in Hair Cell Repair

摘要 内耳的感觉毛细胞经历持续的机械和代谢应激。维持 毛细胞还受到多种其它耳毒性因素的损害的挑战，这些因素包括噪音，老化， 遗传缺陷和耳毒性药物因为哺乳动物的听毛细胞不会再生， 细胞损伤对于持续的听觉功能是重要的。我们的研究项目特别关注 参与静纤毛丝状（F）-肌动蛋白核心维持的分子过程。最近的研究 我得出的结论是，静纤毛肌动蛋白核心是稳定的几个月，这意味着任何结构损伤必须 积极修复。静纤毛F-肌动蛋白核心可以承受损伤，最明显的是噪音暴露， 在静纤毛中的F-肌动蛋白鬼笔环肽标记中显示出"间隙"。在初步研究中，我们发现， 这些缺口在几天内就能修复。因此，我们建议研究分子机制， F-肌动蛋白损伤被感知并修复。这项拟议的研究受到了一个新兴概念的启发， 机械生物学，根据其F-肌动蛋白具有内在的机械感觉特性。在这个模型中， 机械应变调节肌动蛋白丝与效应蛋白的相互作用。对于多种肌动蛋白结合 蛋白质，它们与F-肌动蛋白的组成性结合仅仅是通过力来调节的。然而，LIM结构域蛋白的一个子集 独特之处在于机械应变揭示了F-肌动蛋白上先前隐藏的结合位点，提供了一个开/关开关 用于下游工艺。这些过程涉及肌动蛋白修复底物的募集， 防止F-actin纤维断裂。我们推断毛细胞可能采用类似的策略来修复其 F-肌动蛋白基静纤毛。在我们寻找参与这一过程的分子时，我们专注于蛋白质， 在毛细胞束中富集，含有潜在的机械传感器域，并导致进行性听力损失 在功能丧失的人或小鼠中。我们鉴定了两种蛋白，XIRP2（Xin Actin Binding Repeat Containing 2)和CRIP3（富含半胱氨酸的蛋白3）。我们假设XIRP2和CRIP3是 能够感测F-肌动蛋白损伤并募集额外的修复因子的机械传感器蛋白， 从而在毛细胞静纤毛的修复和维护中发挥重要作用。为了验证这一点，在SA1中，我们建议 以测试成纤维细胞中XIRP2的假设机械传感器功能。在初步研究中，我们发现 XIRP2 C-末端的一个新的机械传感器结构域。我们将使用活细胞激光消融和细胞拉伸 实验来定义机械传感器区域，并研究XIRP2的机制。 机械传感器功能被调节。在SA2中，我们建议测试机械传感器和修复功能， XIRP2在体内，使用缺乏机械传感器结构域的小鼠模型。我们还将进行体外实验 实验以测试荧光标记的XIRP2是否被募集到静纤毛损伤。在SA3中，为了 调查参与额外的修复因子候选人，我们将测试机械传感器和修复 毛束富集的LIM结构域蛋白CRIP3的功能。

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.