Molecular Basis of Transduction in Auditory Sensory Orga

听觉感觉器官转导的分子基础

• 批准号: 7297791
• 负责人: BECHARA KACHAR
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7297791
• 关键词: Molecular; Basis; Transduction; Auditory; Sensory

Stereocilia, are specialized actin protrusions on the surface of the sensory cells of the inner ear where the key step in the process of mechanical to electrical transduction takes place. In each cell, stereocilia are organized in bundles of precisely adjusted graded lengths forming characteristic staircase patterns. Recent studies on actin incorporation and turnover in stereocilia, as well as the identification of novel proteins that when mutated cause deafness through stereocilia disruption, provide unique insights into the mechanisms and molecules involved in the regulation of their steady state length. Our current research focuses on the role played by several stereocilia proteins that are associated with the regulation of the different compartments of the stereocilia structure. Class III myosins share structural features with myosins XVa, VIIa, and Ic including a canonical motor domain and a short regulatory neck domain containing a variable number of IQ motifs. Myosin III proteins are unique in that they contain an N-terminal kinase domain that undergoes auto-phosphorylation, and a region in their tail domain that functions as a second actin-binding site. Myosin IIIa has been implicated in DFNB30, a form of inherited progressive hearing loss. We used immunolabeling to show that myosin IIIa is localized at stereocilia tips. Myosin IIIa progressively accumulates during stereocilia maturation in a thimble-like pattern around the stereocilia tip. This pattern is distinct from the cap-like localization of myosin XVa and the shaft localization of myosin Ic. Overexpression of deletion mutants for functional domains of GFP-myosin IIIa shows that the motor domain, but not the actin-binding tail domain, is required for stereocilia tip localization. Deletion of the kinase domain produces stereocilia elongation and bulging of the stereocilia tips. We also evaluated the distribution and effects of GFP-myosin IIIa and the various myosin IIIa mutants on filopodia in transfected COS-7 cells. The spatial and temporal patterns of myosin IIIa localization at stereocilia tips and the effects of deletion mutants of myosin IIIa on stereocilia and other actin protrusions suggest that the pathophysiology of DFNB30 is likely to involve disruption of stereocilia structure and function. The thimble-like localization and the influence myosin IIIa has on stereocilia shape reveal a previously unrecognized molecular and functional compartment at the distal end of stereocilia, the site of both actin polymerization and operation of the mechanoelectrical transduction apparatus.

静纤毛是内耳感觉细胞表面上的特化肌动蛋白突起，在机械到电转换过程中的关键步骤发生。在每个细胞中，静纤毛被组织成束的精确调整梯度长度形成特征的楼梯图案。最近的研究肌动蛋白的掺入和营业额静纤毛，以及新的蛋白质，突变时，通过静纤毛中断导致耳聋的鉴定，提供了独特的见解的机制和分子参与调节其稳态长度。我们目前的研究集中在几个静纤毛蛋白，与静纤毛结构的不同隔室的调节所发挥的作用。III类肌球蛋白与肌球蛋白XVa、VIIa和Ic共享结构特征，包括典型运动域和含有可变数目的IQ基序的短调节颈域。肌球蛋白III蛋白的独特之处在于它们含有一个经历自磷酸化的N-末端激酶结构域，以及在其尾部结构域中作为第二肌动蛋白结合位点的区域。肌球蛋白IIIa与DFNB 30有关，DFNB 30是一种遗传性进行性听力损失。我们使用免疫标记显示肌球蛋白IIIa位于静纤毛尖端。肌球蛋白IIIa在静纤毛成熟期间以顶针状模式逐渐积聚在静纤毛尖端周围。这种模式是不同的帽状定位的肌球蛋白XVa和轴定位的肌球蛋白Ic。GFP-肌球蛋白IIIa功能域的缺失突变体的过表达表明，静纤毛尖端定位所需的是马达结构域，而不是肌动蛋白结合尾结构域。激酶结构域的缺失产生静纤毛伸长和静纤毛尖端的凸出。我们还评估了GFP-肌球蛋白IIIa和各种肌球蛋白IIIa突变体在转染的COS-7细胞中对丝状伪足的分布和影响。肌球蛋白IIIa定位于静纤毛尖端的空间和时间模式以及肌球蛋白IIIa缺失突变体对静纤毛和其他肌动蛋白突起的影响表明，DFNB 30的病理生理学可能涉及静纤毛结构和功能的破坏。顶针样定位和肌球蛋白IIIa的影响，静纤毛的形状揭示了一个以前未被认识的分子和功能区室的远端静纤毛，两个肌动蛋白聚合和操作的机电转换装置的网站。