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

我们的内耳，由螺旋状的耳蜗和前庭系统组成，赋予我们听觉和平衡感。涉及这些感官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在衰老方面的战略重点，我们的项目旨在从分子水平上了解听力的基本机制，从而为保护我们的听力和防止年龄损害的策略提供信息。除了衰老的过程，在细胞和分子水平上，关于听力的发展还有许多问题。听觉和平衡中的刺激都是机械的：在听觉中，声波以空气的运动开始，在耳蜗中转化为流体运动；在平衡中，头部的运动引起前庭结构内的流体运动。在这两种情况下，液体的运动直接或间接地通过其他结构刺激毛细胞。这些特殊的感觉细胞的特征是一束微小的毛发（立体纤毛）从它们的顶部伸出来。将立体纤毛向一个（兴奋性）方向偏转，在毛细胞中产生机电转导（MET），机械刺激在此转化为电反应。MET装置的关键部件是：纤毛本身；将直立纤毛连接在一起的细丝，称为尖端环和侧环；以及与链接相关的蛋白质，如MET离子通道（嵌入细胞外层或膜中可以形成毛孔的蛋白质）和连接尖端链接到MET通道的辅助蛋白质。已知所有这些蛋白质候选基因的突变会导致进行性听力损失。关于MET反应的主流观点是，束的偏转拉伸了尖端连接，使它们能够拉动附着在MET通道上的附属蛋白质，从而导致后者打开。打开通道允许正离子（钾离子和钙离子）进入细胞，引起电反应。对哺乳动物耳蜗通道的研究也表明，它们的特性随着这个器官的长度而变化，但这种变化的原因尚不清楚。影响尖端连接的某些突变，以及对它们的实验破坏，会导致这种反应被逆转，这是一种迄今尚未解释的现象。在体外损伤后，已知尖端连接与MET一起恢复。解释这种反向反应和理解损伤后的机制如何发展或恢复是很重要的。这个项目的主要目标是：1和2。定位被认为是毛细胞检测听力（和平衡）机械刺激所需的候选MET通道和辅助蛋白。这些蛋白质发生在尖端连接的末端吗？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. 追踪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联系在一起？通过尖端连接恢复和重塑过程跟踪蛋白质分布。尖端连接的修复和重组是否能确保它们正确定位？来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，从而导致耳蜗上已知的MET通道性质的变化？确定让毛细胞做出反应的器官的分子组织将使我们能够填补我们在发育和衰老过程中对听力和平衡知识的现有空白。这将进一步给我们提供新的机会，找到可以改善随着年龄增长而发生的退行性变化的策略。

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.