Localization of the mechanotransducer channel and its accessory proteins during development of cochlear hair cells.

我们的内耳由螺旋形的耳蜗和前庭系统组成，赋予我们听觉和平衡感。这些感觉的分子机制仍然是许多争论的主题。此外，听力损失是人类随着年龄增长而遭受的最常见的感觉剥夺形式。根据BBSRC关于老龄化的战略优先事项，我们的项目旨在了解分子水平上的听力基本机制，以便为保护我们的听力和防止年龄损害的战略提供信息。除了衰老的过程之外，在细胞和分子水平上对听力的发展仍有许多疑问。听力和平衡的刺激都是机械性的：在听力中，声波最初是空气的运动，在耳蜗中转化为流体运动;在平衡中，头部的运动导致前庭结构内的流体运动。在任何一种情况下，流体运动直接或间接通过其他结构刺激毛细胞。这些专门的感觉细胞的特征是一束微小的毛发（静纤毛）从它们的顶部伸出。使静纤毛向一个（兴奋性）方向偏转在毛细胞中产生机械电传导（MET），其中机械刺激转变为电反应。这种MET装置的关键组成部分是：静纤毛本身;将静纤毛连接在一起的微小细丝，称为尖端连接和侧向连接;以及与连接相关的蛋白质，如MET离子通道（嵌入细胞外层或膜中的蛋白质，可以形成孔）和连接尖端连接到MET通道的辅助蛋白质。已知所有这些蛋白质的候选突变都会导致进行性听力损失。MET反应的流行观点是，束偏转拉伸了尖端链接，使它们能够拉动附着在MET通道上的辅助蛋白，从而导致后者打开。打开通道允许正离子（钾和钙）进入细胞，引起电反应。对哺乳动物耳蜗通道的研究也表明，它们的特性沿着这个器官的长度沿着变化，但这种变化的基础尚不清楚。某些影响尖端连接的突变，以及对它们的实验性破坏，会导致这种反应逆转，这是一种迄今无法解释的现象。体外损伤后，已知头端连接件可随MET沿着恢复。重要的是要解释的逆转反应，并了解该机制如何发展或损害后恢复。本项目的主要目标是：1和2。定位被认为是毛细胞检测听觉（和平衡）中的机械刺激所需的候选MET通道和辅助蛋白。这些蛋白质是否出现在尖端链接的末端？3.为了确定这些蛋白质在毛细胞反应逆转的情况下的分布。蛋白质是否处于异常位置，从而导致逆转？4.目的：研究哺乳动物毛细胞发育过程中蛋白质分布的变化。这些蛋白质何时以及如何与尖端链接相关联？5.跟踪头端连接恢复和重塑过程中的蛋白质分布。头端链接的修复和重组是否确保其正确定位？6.以量化这些蛋白质在不同位置的数量。它们的组成是否发生了变化，并因此赋予了沿着耳蜗的MET通道特性的已知变化？确定允许毛细胞做出反应的装置的分子组织将使我们能够填补我们在发育和衰老期间对听力和平衡的知识中的现有空白。此外，它还将为我们提供新的机会，以找到可以改善随着年龄增长而发生的退行性变化的战略。

Our inner ear, comprised of the spiral shaped cochlea and the vestibular system, gives us our senses of hearing and balance. The molecular mechanisms involved in these senses are still the subject of much debate. Moreover, hearing loss is the commonest form of sensory deprivation suffered by humans as they age. In accordance with the BBSRC strategic priority on ageing, our project aims to understand basic mechanisms of hearing at the molecular level in order to provide information for strategies to protect our hearing and prevent damage with age. As well as the ageing process, there are still many questions about the development of hearing at the cellular and molecular level.The stimulus in both hearing and balance is mechanical: in hearing, sound waves, which start as movements of air, are transformed into fluid movements in the cochlea; in balance, movements of the head cause fluid motion within the vestibular structures. In either case, the fluid motion directly, or indirectly via other structures, stimulates hair cells. These specialised sensory cells are characterised by a bundle of tiny hairs (stereocilia) sticking out of their top. Deflecting the stereocilia in one (excitatory) direction produces mechaonelectrocial transduction (MET) in the hair cell where the mechanical stimulius is turned into an electrical response. Key components of this MET apparatus are: the stereocilia themselves; tiny filaments that link the stereocilia together, called tip links and lateral links; and proteins associated with the links such as the MET ion channels (proteins embedded in the cell's outer layer or membrane that can form pores) and accessory proteins connecting the tip links to the MET channels. Mutations in candidates for all of these proteins are known to cause progressive hearing loss.The prevailing view of the MET response is that bundle deflection stretches the tip links, allowing them to pull on the accessory proteins attached to the MET channels, and so cause the latter to open. Opening the channels allows positive ions (potassium and calcium) to enter the cell causing the electrical response. Studies of the channels in the mammalian cochlea also show that their properties vary along the length of this organ, but what underlies this variation is not known. Certain mutations that affect the tip links, and experimental destruction of them, cause this response to be reversed, a phenomanon so far unexplained. After damage in vitro the tip links are known to recover along with MET. It is important to explain the reversed responses and understand how the mechanism develops or recovers after damage.The key objectives of this project are:1 and 2. To localize candidate MET channels and accessory proteins that are thought to be required by the hair cell to detect the mechanical stimulus in hearing (and balance). Do these proteins occur at the end of the tip links?3. To determine the distribution of these proteins in situations where hair cell responses are reversed. Are the proteins in abnormal locations, thus causing the reversal? 4. To follow changes in distribution of the proteins during mammalian hair cell development. When and how do these proteins become associated with the tip links?5. To follow the protein distributions through the process of tip link recovery and remodeling. Does repair and restructuring of the tip link ensure they become correctly localised?6. To quantify the amount of these proteins in different locations. Do they change in composition, and so confer the known changes in MET channel properties along the cochlea?Determining the molecular organization of the apparatus that allows the hair cell to respond will enable us to fill in existing gaps in our knowledge of hearing and balance during development and ageing. It will furthermore give us new opportunities to find strategies that could ameliorate degenerative changes that occur with age.