Role of myosin 1c in adaptation in the inner ear

描述(由申请人提供)：这项研究的广泛、长期的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投射，或称立体纤毛，将声音和机械振动产生的力转化为神经冲动。机械转导的流行模型被认为对耳蜗毛细胞和前庭毛细胞都很保守，在这种模型中，相邻的由称为尖端连接的细胞外细丝连接的立体纤毛对刺激做出反应而偏转，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-运动复合体，该复合体控制尖端链接的张力。在兴奋性刺激期间，最初的张力很高，打开了通道；然后，运动复合体滑下肌动蛋白细胞骨架，减少尖端连接的张力，使通道关闭。相比之下，在负性刺激期间，最初的张力很低，通道关闭；然后运动复合体上升到细胞骨架，恢复静止的张力，重新打开通道。分子马达肌球蛋白1c(Myo1c)在立体纤毛中的关键位置的定位；以及对表达可选择性抑制的突变Myo1c的小鼠的研究表明，Myo1c参与了这一称为适应的过程(Holt等人，2002年；Stauffer等人，2005年)。适应使毛细胞在长时间的刺激下对新的刺激保持敏感。这项建议的目标是通过测量表达具有特定分子特性的Myo1c突变的小鼠的适应，确定Myo1c如何支持适应的特定方面。Myo1c突变将包括(I)那些影响Myo1c适应机械负荷的能力的突变，这是本实验室以前的研究(Batters等人，2004a；2004b)预测的Myo1c的特性；以及(Ii)对调节Myo1c和适应的钙离子异常敏感的突变。其具体目的是：利用ATPase分析、运动分析、动力学分析和单分子力学研究，在体外表达和表征Myo1c突变体的生化和力学性质。下一步，将产生表达这些突变Myo1c分子的转基因小鼠，并将在转基因小鼠的毛细胞中测量适应情况。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关键的、新的见解。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。这项拟议的研究重点是分子马达蛋白Myo1c，它被认为是内耳毛细胞中的适应-马达复合体。确定毛细胞转导装置的关键成分，如Myo1c的生化和生物物理性质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，从而可以开发新的适当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.