Hair Bundle Structure and Dynamics

描述（由申请人提供）：我们使用质谱法同时测量约300种最丰富的毛束蛋白质的身份和浓度，这些蛋白质共同构成了感觉毛细胞的特殊机械敏感结构。利用我们纯化鸡前庭系统毛束的高产量和高纯度的能力，我们将应用系统生物学中使用的技术，包括蛋白质组学和微阵列并行分析许多分子，以了解毛束发育和转导装置组装的机制。在Aim 1中，我们将使用新的质谱实验进一步表征已经鉴定的束蛋白的浓度和磷酸化。作为一个测试案例，我们将提高cAMP水平在束来检查蛋白质的再分配和磷酸化。为此，我们还将系统地研究在束中鉴定的新蛋白质的位置。在Aim 2中，我们将监测参与毛束形成的mrna和蛋白质的表达，分析由Lew Tilney发现的鸡基底乳头发育阶段。通过确定参与束组装的分子何时被表达或调节，我们将对束的构建有更好的机制理解。最后，在目标3中，我们将检查在钙螯合剂消融尖端链接后，哪些蛋白质在毛束中丰度增加或减少。这些实验将使我们能够探索转导装置的组装机制。实现这三个目标将使我们能够对头发束采取系统级视图。此外，将系统生物学方法应用于毛发束比应用于整个细胞有明显的优势：毛发束比整个细胞或组织要简单得多，因此要分析的分子数量相对较少，其机制和模型可能比其他系统要简单得多。虽然这种简单的断言可能并不完全准确，但我们将能够看到数百种蛋白质在束和转导装置组装的关键阶段的协调反应。通过走向相对公正的实验方法，我们获得了对听力和平衡至关重要的结构的新见解。

DESCRIPTION (provided by applicant): We use mass spectrometry to simultaneously measure the identities and concentrations of the ~300 most abundant hair-bundle proteins, which together make up the specialized mechanically sensitive structure of sensory hair cells. Taking advantage of our ability to purify hair bundles of the chicken vestibular system with high yield and excellent purity, we will apply techniques used in systems biology, including proteomic and microarray analysis of many molecules in parallel, to understand the mechanisms of bundle development and transduction-apparatus assembly. In Aim 1, we will further characterize the concentrations and phosphorylation of already-identified bundle proteins using new mass-spectrometry experiments. As a test case, we will elevate cAMP levels in bundles to examine protein redistribution and phosphorylation. In this aim, we will also systematically study the locations of novel proteins identified in bundles. In Aim 2, we will monitor the expression of mRNAs and proteins that participate in formation of the hair bundle, analyzing chicken basilar papilla during the stages of development identified by Lew Tilney. By determining when molecules involved in bundle assembly are expressed or regulated, we will gain better mechanistic understanding of construction of the bundle. Finally, in Aim 3, we will examine which proteins increase or decrease in abundance in hair bundles following tip-link ablation with calcium chelators. These experiments will allow us to probe the mechanism of assembly of the transduction apparatus. Carrying out these three aims will allow us to take a systems-level view of the hair bundle. Moreover, application of systems-biology methodology to the bundle has a significant advantage over application to whole cells: the hair bundle is significantly less complex than a whole cell or tissue, and thus the number of molecules to be analyzed is relatively small and the mechanism and models may be significantly less complicated than those of other systems. While this assertion of simplicity may not be entirely accurate, we will be able to see the coordinated response of hundreds of proteins during critical phases of bundle and transduction-apparatus assembly. By moving towards relatively unbiased experimental approaches, we gain novel insights into structures critical for hearing and balance. PUBLIC HEALTH RELEVANCE: We propose here to study what the business end of the inner ear, the hair bundle, is made out of and how it is constructed during development of an organism. Moreover, we aim to determine what proteins make up the specialized molecular machine that actually detects sound. Success in these aims will allow us to identify additional genes that, when disrupted, lead to hearing loss. More significantly, these experiments will allow us to design rational approaches to detecting and ameliorating hearing loss and disrupted balance.