ACTIVITY-DRIVEN PLASTICITY OF THE HAIR CELL CYTOSKELETON

项目摘要/摘要 内耳毛细胞的机械敏感性取决于被称为 立体纤毛，成排排列，高度增加，具有机电转换(MET)通道 位于较短的排立体纤毛的顶端。立体纤毛的核心由高度交联的 肌动蛋白细丝的准晶阵列。当交联剂蛋白质不断更新时，交联物的更新 肌动蛋白仅限于立体纤毛尖端。我们之前报道过立体纤毛肌动蛋白核心表现出依赖于活性的可塑性(Velez-Ortega等人，eLife 2017)。我们证明了MET频道的堵塞 或者门控这些通道的尖端连接的断裂导致转换的选择性缩短 立体纤毛(即含有MET通道的立体纤毛)，而非换能器最高的排立体纤毛 保持不受影响。一旦MET阻塞被移除或尖端连接再生，立体纤毛就会重新生长。 我们的初步数据还表明，这种MET依赖的静息纤毛重塑可以影响静息状态 仪表机械内的张力以秒为单位。因此，该过程可以动态地调节灵敏度 毛细胞对声音诱发的振动的敏感度，因此，我们听力的敏感度。然而，确切的 MET依赖的静毛纤毛重塑机制仍不清楚。目前甚至还不清楚 纤毛细胞骨架的活性依赖性可塑性仅限于肌动蛋白更新活跃的区域。 或者可以扩展到这个区域之外，进入立体纤毛轴的“稳定”部分。在这里，我们假设 MET活性调节静纤毛细胞骨架经历主动肌动蛋白重塑的程度。 为了测试这一点，目标1将评估MET依赖的立体纤毛内肌动蛋白动力学的变化 角质板，一种富含肌动蛋白的结构，支撑着立体纤毛束。目标2将评估MET驱动的 立体纤毛肌动蛋白超微结构的透射电子显微镜观察 体层摄影术。由于到目前为止，对Met依赖的纤毛重建的研究只在年轻的出生后进行。 毛细胞，目标3将评估这种现象是否也存在于成熟的成人听觉毛发中。 细胞。在目标4中，我们开始探索与Met驱动的立体纤毛有关的分子玩家。 重塑，通过评估所谓的“立体纤毛识别蛋白”在突变小鼠中的表达 该模型不仅在转导立体纤毛中表现出MET依赖的肌动蛋白重塑，而且 出乎意料的是，在非换能式立体纤毛中。这项研究意义重大，因为它可能会澄清 毛细胞对立体纤毛束的结构进行微调，从而维持 我们一生中听力的敏感度。此外，立体纤毛的缩短--也许还有他们的 最终消失-可能在噪声暴露后发生(当仪表电流因尖端而降低时 链路断裂)或在某些情况下先天性耳聋(由于MET电流受损)。因此，这 研究将扩大我们对各种类型听力损失的分子机制的了解。

PROJECT SUMMARY/ABSTRACT The mechanosensitivity of the inner ear hair cells depends on cellular projections known as stereocilia, organized in rows of increasing height, with mechano-electrical transduction (MET) channels located at the tips of shorter row stereocilia. The core of stereocilia consists of a highly crosslinked paracrystalline array of actin filaments. While crosslinker proteins are constantly renewed, the renewal of actin is limited to the stereocilia tips. We previously reported that the stereocilia actin core exhibits activity-dependent plasticity (Velez-Ortega, et al., eLife 2017). We showed that the blockage of MET channels or the breakage of the tip links that gate these channels lead to the selective shortening of transducing stereocilia (i.e. the stereocilia that harbor MET channels), while the non-transducing tallest row stereocilia remain unaffected. Once the MET blockage is removed or the tip links regenerate, the stereocilia regrow. Our preliminary data also show that this MET-dependent stereocilia remodeling can affect the resting tension within the MET machinery in seconds. Thus, this process may dynamically regulate the sensitivity of hair cells to sound-induced vibrations and, hence, the sensitivity of our hearing. Yet, the exact mechanisms of MET-dependent stereocilia remodeling are still obscure. It is unknown even whether the activity-dependent plasticity of the stereocilia cytoskeleton is limited to the regions of active actin renewal or can expand beyond this region into the “stable” part of the stereocilia shaft. Here, we hypothesize that the MET activity regulates the extent of the stereocilia cytoskeleton undergoing active actin remodeling. To test this, Aim 1 will evaluate MET-dependent changes in actin dynamics within the stereocilia and the cuticular plate, an actin-rich structure supporting the stereocilia bundle. Aim 2 will evaluate MET-driven changes in the ultrastructural organization of stereocilia actin with transmission electron microscopy tomography. Since the MET-dependent stereocilia remodeling was studied so far only in young postnatal hair cells, Aim 3 will assess whether this phenomenon is present also in the mature adult auditory hair cells. In Aim 4, we begin to explore the molecular players involved in the MET-driven stereocilia remodeling, by evaluating the expression of so-called “stereocilia row identity proteins” in a mutant mouse model that exhibits MET-dependent actin remodeling not only in transducing stereocilia but also, unexpectedly, in non-transducing stereocilia. The study is significant, because it may clarify how exactly a hair cell performs fine adjustments of the architecture of the stereocilia bundle, thereby maintaining the sensitivity of our hearing throughout a lifetime. In addition, stereocilia shortening—and perhaps their eventual disappearance—could occur after noise exposure (when the MET current is reduced due to tip link breakage) or in certain cases of congenital deafness (due to impaired MET current). Therefore, this study will expand our knowledge of the molecular mechanisms of various types of hearing loss.