Molecular Basis Of Transduction In Auditory Sensory Orga

听觉感觉器官转导的分子基础

• 批准号: 6965276
• 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/6965276
• 关键词: actin binding protein; actins; biological signal transduction; cell component structure /function; cytochalasins; ear hair cell; electron microscopy; electrostimulus; gene expression; genetically modified animals; green fluorescent proteins; intracellular transport; laboratory mouse; mechanical stress; microfilaments; myosins; neural information processing; organelles; polymerization; protein localization; protein transport; transfection

Stereocilia, the key cellular organelles responsible for auditory and vestibular sensory function, are organized into bundles of precisely specified rows of increasing heights forming characteristic staircase patterns. Although stereocilia are exquisitely sensitive to mechanical vibration, orderly structured, and easily damaged by over stimulation, they are maintained in proper working order for an entire lifetime. Each stereocilium is supported by a rigid paracrystalline array of several hundred parallel, uniformly polarized and regularly cross-linked actin filaments. We have previously shown that the seemingly static actin paracrystal at the core of sensory stereocilia of hair cells undergoes continuous renewal by reproducing itself at the stereocilia tips, treadmilling rearwards, and dismantling itself at the base (Schneider et al. Nature, 418: 837, 2002). We have now used the same approach of transfecting hair cells with actin-green fluorescent protein (GFP), espin-GFP, and myosin XVa-GFP to characterize the turnover process. Actin and espin are incorporated at the paracrystal tip and flow rearwards at the same rate. The flux rates (~0.002?0.04 actin subunits per second) were proportional to the stereocilia length so that the entire staircase stereocilia bundle was turned over synchronously. Actin polymerization inhibition by cytochalasin D caused stereocilia to shorten at rates matching paracrystal turnover. Myosins Ic and VIIa were localized alongside the actin paracrystal where they could drive retrograde actin flow. Myosin XVa was observed at the stereocilia tips, coinciding with a structure called the tip density, at levels proportional to stereocilia lengths. Electron microscopy analysis of the abnormally short stereocilia in the shaker 2 (myosin XVa mutant) mice did not show the characteristic tip density structure. We also show that stereocilia length is modulated by the expression levels of espin and myosin XVa as well as by local physical parameters, such as tension on the stereocilia links and on the encapsulating membrane. We argue that regulation of actin polymerization and treadmilling dynamically shapes the functional architecture of stereocilia and plays a central role in recovery from over-stimulation.

立体纤毛是负责听觉和前庭感觉功能的关键细胞器，它们被组织成一束束精确指定的高度递增的行，形成独特的楼梯图案。虽然立体纤毛对机械振动非常敏感，结构整齐，容易受到过度刺激的破坏，但它们在一生中都能保持适当的工作状态。每个立体纤毛由数百个平行的、均匀极化的、规则地交叉连接的肌动蛋白细丝组成的刚性准晶体阵列支撑。我们先前已经证明，毛细胞感觉立体纤毛核心的看似静态的肌动蛋白副晶体经历了持续的更新，通过在立体纤毛尖端复制自己，向后踏步，并在底部拆卸自己(Schneider等人)。《自然》，418：837,2002)。我们现在使用相同的方法，用肌动蛋白绿色荧光蛋白(GFP)、ESPIN-GFP和肌球蛋白XVA-GFP转染毛细胞来表征周转过程。肌动蛋白和ESpin以相同的速度结合在准晶体尖端并以相同的速度向后流动。流速(~0.002？0.04肌动蛋白亚基每秒)与纤毛长度成正比，从而使整个阶梯纤毛束同步翻转。细胞松弛素D抑制肌动蛋白聚合，导致立体纤毛以与晶体旁周转相匹配的速率缩短。肌球蛋白Ic和VIIa定位于肌动蛋白旁晶体，可驱动肌动蛋白逆行流动。肌球蛋白XVA出现在纤毛末端，与纤毛末端密度的结构一致，与纤毛长度成正比。对Shaker 2(肌球蛋白XVA突变体)小鼠异常短的立体纤毛的电子显微镜分析没有显示出典型的尖端密度结构。我们还发现，立体纤毛的长度受ESpin和肌球蛋白XVA的表达水平以及局部物理参数的调节，如立体纤毛连接和囊膜上的张力。我们认为，肌动蛋白聚合和跑步机的调节动态地塑造了立体纤毛的功能结构，并在从过度刺激中恢复起着核心作用。