Mechanosensor Proteins in Hair Cell Repair

摘要 内耳的感觉毛细胞承受持续的机械和代谢压力。的维护 毛细胞进一步受到各种其他耳毒性因素损害的挑战，包括噪音、衰老、 遗传缺陷和耳毒性药物。由于哺乳动物的听毛细胞不会再生，毛发的修复 细胞损伤对于持续的听觉功能很重要。我们的研究计划特别感兴趣 参与维持静纤毛丝状 (F)-肌动蛋白核心的分子过程。最近的研究 得出的结论是，静纤毛肌动蛋白核心在数月内保持稳定，这意味着任何结构损坏都必须 予以积极修复。静纤毛 F-肌动蛋白核心可能会受到损害，最明显的是噪音暴露，这 被证明会导致静纤毛中 F-肌动蛋白的鬼笔环肽标记出现“缺口”。在初步研究中，我们发现 这些差距在几天之内就可以修复。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。这项研究的灵感来自于一个新兴概念 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丝与效应蛋白的相互作用。用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通过力来调节。然而，LIM 结构域蛋白的一个子集 其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和 预防F-肌动蛋白纤维断裂。我们推断毛细胞可能采用类似的策略来修复其 基于 F-肌动蛋白的静纤毛。在我们寻找参与这个过程的分子时，我们关注的是蛋白质 富含毛细胞束，包含潜在的机械传感器域，并导致进行性听力损失 在功能丧失的人类或小鼠中。我们鉴定了两种蛋白质，XIRP2（Xin Actin Binding Repeat Containing 2) 和 CRIP3（富含半胱氨酸的蛋白 3）满足这些标准。我们假设 XIRP2 和 CRIP3 是 机械传感器蛋白能够感知 F-肌动蛋白损伤并招募额外的修复因子， 从而在毛细胞静纤毛的修复和维护中发挥重要作用。为了测试这一点，在 SA1 中，我们建议 测试 XIRP2 在成纤维细胞中假设的机械传感器功能。在初步研究中，我们发现 XIRP2 C 末端的新型机械传感器结构域。我们将使用活细胞激光消融和细胞拉伸 定义机械传感器区域的实验，并研究 XIRP2 的机制 机械传感器功能受到调节。在SA2中，我们建议测试机械传感器和修复功能 XIRP2 体内实验，使用缺乏机械传感器域的小鼠模型。我们还将进行离体 实验测试荧光标记的 XIRP2 是否被募集到静纤毛损伤处。在 SA3 中，努力 调查其他修复因子候选者的参与情况，我们将测试机械传感器和修复 发束富集 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.