Role of myosin 1c in adaptation in the inner ear

描述（由申请人提供）：本研究的广泛、长期目标是确定听力和平衡的分子机制。内耳感觉毛细胞上充满肌动蛋白的突起或立体纤毛将声音和机械振动产生的力转化为神经冲动。机械转导的主流模型被认为对耳蜗和前庭毛细胞都是保守的，在这种模型中，相邻的立体纤毛通过称为尖端链接的细胞外细丝连接，在响应刺激时偏转，从而导致转导通道的打开或关闭。这些通道连接到一个适应马达复合体上，该复合体控制尖端连接的张力。在兴奋性刺激期间，张力最初很高，打开通道；然后，运动复合物沿着肌动蛋白细胞骨架向下滑动，降低尖端连接的张力，使通道关闭。相反，在负刺激时，张力最初很低，通道关闭；运动复合体随后上升到细胞骨架，恢复静息张力并重新打开通道。分子马达的定位，肌球蛋白1c （Myo1c）在立纤毛的战略位置；使用表达Myo1c突变体的小鼠进行的研究表明，Myo1c参与了这一称为适应的过程（Holt et al., 2002; Stauffer et al., 2005）。适应使毛细胞在长时间的刺激下对新的刺激保持敏感。本提案的目标是通过测量表达具有特定分子特性的Myo1c突变体的小鼠的适应性来确定Myo1c如何支持适应的特定方面。Myo1c突变体将包括(i)影响Myo1c适应机械负荷能力的突变体，这是本实验室先前研究预测的Myo1c的特性（Batters等，2004a; 2004b）；（ii）对Ca2+异常敏感的人，Ca2+调节Myo1c和适应。具体目的是：利用atp酶分析、运动性分析、动力学分析和单分子力学研究，在体外表达Myo1c突变体，并表征其生化和力学特性。接下来，将产生表达这些突变Myo1c分子的敲入小鼠，并在敲入小鼠的毛细胞中测量适应性。体外和动物联合研究有望为Myo1c的分子机制及其在适应中的作用提供重要的新见解。这些知识最终可能导致设计合理的诊断和治疗方法来治疗听力和/或平衡疾病。这项研究的重点是分子运动蛋白Myo1c，它与内耳毛细胞中的适应-运动复合物有关。确定毛细胞中Myo1c等转导装置关键组分的生化和生物物理特性及其在适应中的作用，对于揭示听力和平衡的分子机制具有重要意义，从而开发新的、合适的诊断和治疗方法来预防或治疗听力损失和眩晕。

DESCRIPTION (provided by applicant): The broad, long-range goal of this study is to determine the molecular mechanism of hearing and balance. Actin-filled projections, or stereocilia, on the sensory hair cells of the inner ear convert force produced by sounds and mechanical vibrations into nerve impulses. The prevailing model for mechanotransduction, thought to be well-conserved for both cochlear and vestibular hair cells, is one in which neighboring stereocilia connected by extracellular filaments called tip links, deflect in response to stimuli, thereby causing the opening or closing of transduction channels. The channels are attached to an adaptation-motor complex, which controls tip-link tension. During an excitatory stimulus, tension is initially high, opening channels; the motor complex then slips down the actin cytoskeleton, reducing tip-link tension and allowing the channels to close. By contrast, during a negative stimulus, tension is initially low and channels close; the motor complex then ascends the cytoskeleton, restoring the resting tension and reopening the channels. Localization of the molecular motor, myosin 1c (Myo1c) at strategic places in the stereocilia; and studies using mice expressing a mutant Myo1c that can be selectively inhibited have shown Myo1c's involvement in this process known as adaptation (Holt et al., 2002; Stauffer et al., 2005). Adaptation allows hair cells under prolonged stimuli to remain sensitive to new stimuli. The goal of this proposal is to determine how Myo1c supports specific aspects of adaptation by measuring adaptation in mice expressing Myo1c mutants with defined molecular properties. Myo1c mutants will include (i) those that affect the ability of Myo1c to adapt to mechanical load, a property predicted for Myo1c from previous studies in this laboratory (Batters et al., 2004a; 2004b); and (ii) those with aberrant sensitivity to Ca2+, which regulates Myo1c and adaptation. The specific aims are: to express in vitro and characterize the biochemical and mechanical properties of Myo1c mutants using ATPase assays, motility assays, kinetic analyses and single-molecule mechanical studies. Next, knock-in mice expressing these mutant Myo1c molecules will be generated and adaptation will be measured in hair cells from the knock-in mice. The combined in vitro and animal studies are expected to provide critical, new insight into the molecular mechanism of Myo1c and its role in adaptation. This knowledge could ultimately lead to the design of rational diagnostics and therapies to treat diseases of hearing and/or balance. The proposed study focuses on the molecular motor protein, Myo1c, which is implicated as the adaptation-motor complex in the hair cells of the inner ear. Determining the biochemical and biophysical properties of key components of the transduction apparatus in hair cells, like Myo1c, and its role in adaptation are of fundamental importance to revealing the molecular mechanisms of hearing and balance, so that new and appropriate diagnostics and therapies to prevent or treat hearing loss and vertigo can be developed.