Mechanosensor Proteins in Hair Cell Repair

摘要 内耳的感觉毛细胞经历持续的机械和新陈代谢压力。维护和维护 毛细胞进一步受到各种其他耳毒性因素的损害，包括噪音、衰老、 基因缺陷和耳毒性药物。因为哺乳动物的听觉毛细胞不能再生，所以毛发的修复 细胞损伤对持续的听觉功能很重要。我们的研究项目尤其对 分子过程参与维系立体纤毛(F)-肌动蛋白核心。最新研究 已经得出结论，立体纤毛肌动蛋白核心在几个月内是稳定的，这意味着任何结构性损伤都必须 积极抢修。立体纤毛F-肌动蛋白核心可以承受损伤，最明显的是噪声暴露，这 在立体纤毛中，F-肌动蛋白的阴沟素标记出现了“缺口”。在初步研究中，我们发现 这些缺口在几天内就可以修复。因此，我们建议研究其分子机制。 F-肌动蛋白损伤被感知和修复。这项拟议的研究的灵感来自于 机械生物学，根据该理论，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细丝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-肌动蛋白的结构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，机械张力揭示了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。这些过程牵涉到肌动蛋白修复底物的招募和在 F-肌动蛋白纤维断裂的预防。我们推测，毛细胞可能会采用类似的策略来修复其 F-肌动蛋白为基础的立体纤毛。在寻找参与这一过程的分子时，我们把重点放在了 富含在毛细胞束中，包含潜在的机械感受器结构域，并导致进行性听力损失 在功能丧失的人或小鼠身上。我们鉴定了两种蛋白质，XIRP2(Xin肌动蛋白结合重复序列，包含 2)和CRIP3(富含半胱氨酸的蛋白3)符合这些标准。我们假设XIRP2和CRIP3是 能够感知F-肌动蛋白损伤并招募额外修复因子的机械传感器蛋白质， 从而在毛细胞立体纤毛的修复和维护中起着至关重要的作用。为了测试这一点，在SA1中，我们建议 测试XIRP2在成纤维细胞中的假想机械传感功能。在初步研究中，我们发现 XIRP2 C末端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 确定机械传感区域的实验，并探讨XIRP2‘S的机制 机械传感器的功能是受调节的。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 测试荧光标记的XIRP2是否被招募到立体纤毛病变的实验。在SA3中，为了努力 调查是否涉及额外的维修因素候选人，我们将测试机械传感器并进行维修 发束富含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.