Molecular roles in active and passive mechanics in cochlear hair bundles

描述（由申请人提供）：声音振动通过耳道进入外耳，由中耳转化为压力波。内耳的压力波通过感觉毛细胞毛束的机电转导（MET）过程转化为电信号；这种电信号驱动突触传递，从而将信息传递到大脑。在这个过程中失败会导致听力损失和耳聋。多种人类基因突变在MET过程中表现出缺陷。了解MET的基本特性将有助于更好地了解遗传性耳聋，从而导致有针对性的治疗和治疗。越来越多的关于哺乳动物耳蜗毛细胞MET特性的数据与现有的MET分子模型不相容。具体来说，适应是MET的一个关键过程，被普遍认为是由钙信号发出的，但它似乎不是由钙离子进入驱动的，因此对当前的适应模型提出了挑战。为了更好地了解耳蜗MET的潜在机制，需要以与耳蜗毛细胞中MET过程的快速速率相匹配的速率测量毛束的机械变化。在本提案中，为了克服当前的技术限制，开发了新的微机电系统（MEMS）设备来专门测量耳蜗毛束力学。利用哺乳动物耳蜗毛细胞的全细胞电压钳记录以及新的MEMS器件，将测量快速耳蜗MET过程的动力学和力学。这些数据将用于生成新的耳蜗MET模型。位于顶端连接区域的肌球蛋白运动被认为是MET的重要组成部分。新的实验将在耳蜗中进行，使用这些新颖的MEMS设备来表征毛束在改变运动活动时的力学特性。从这些实验中，分子马达以及耳蜗毛细胞上尖端连接区域在MET过程中的作用将被确定。在声损伤过程中，毛束受到过度刺激的压力，导致毛束的僵硬变化。为了表征哺乳动物毛束的力学特性，本研究旨在通过药物应用和缺乏特定结构的遗传小鼠模型，量化立体纤毛连接和立体纤毛根对被动毛束刚度的贡献。本实验将进一步加深我们对哺乳动物耳蜗MET分子机制的认识。了解被动毛束刚度的关键组成部分，将为理解毛束特性的关键调控点和声损伤对立体纤毛的影响奠定基础。所开发的技术将极大地加强听觉研究，并可能在听觉领域和其他领域有更广泛的力学应用。

DESCRIPTION (provided by applicant): Sound vibrations enter the outer ear through the ear canal and are converted into pressure waves by the middle ear. Pressure waves in the inner ear are converted to an electrical signal via the mechano-electrical transduction (MET) process in the hair bundle of sensory hair cells; this electrical signal drives synaptic transmission resultin in information traveling to the brain. Failures in this process lead to hearing loss and deafness. Multiple human genetic mutations exhibit deficits in the MET process. Understanding the basic properties of MET will lead to a better understanding of genetic deafness, leading to targeted treatments and therapies. A growing body of data on mammalian cochlear hair cell MET properties is incompatible with existing molecular models of MET. Specifically, adaptation, a key process of MET universally accepted to be signaled by calcium, does not appear to be driven by calcium ion entry, thus challenging current models of adaptation. To better understand the underlying mechanisms responsible for cochlear MET, mechanical changes in the hair bundle need to be measured at rates that match the fast rates of MET processes in cochlear hair cells. In this proposal, to overcome current technological limitations, new micro-electro-mechanical systems (MEMS) devices are developed to specifically measure cochlear hair bundle mechanics. Using whole-cell voltage clamp recordings of mammalian cochlear hair cells along with new MEMS devices, kinetics and mechanics of fast cochlear MET processes will be measured. This data will be used to generate new models of cochlear MET. Myosin motors localized to the upper tip-link region have been proposed to be important to MET. New experiments in the cochlea will be performed using these novel MEMS devices to characterize mechanics of the hair bundle when modifying motor activity. From these experiments, the role of molecular motors as well as the upper tip-link region in cochlear hair cells in MET processes will be determined. During acoustic trauma, hair bundles are stressed from overstimulation resulting in stiffness changes to the hair bundle. To characterize mechanical properties of the mammalian hair bundle, this proposal aims to quantify the contribution of stereocilia links and the stereocila rootlet to passive hair bundle stiffness using drug application and genetic mouse models lacking specific structures. The experiments in this proposal will further our understanding of the molecular mechanisms of mammalian cochlear MET. Understanding the crucial components in passive hair bundle stiffness will lay groundwork for understanding the key regulation points of hair bundle properties and the effects of acoustic trauma on stereocilia. The technology developed will greatly enhance auditory research and likely have broader mechanics applications in the auditory field and beyond.