Dynamic interaction among proteins in hair cells

项目摘要 耳蜗将声波转换成电信号，将信息传递给中枢神经 系统高度特化的细胞包括毛细胞、支持细胞和螺旋神经节神经元介导了这一过程 操作由于个体化需要复杂和精密的微管（MT）网络， 细胞的形状和结构所需的外周信号编码，揭示蛋白质网络，调节 MT组织将丰富我们对正常和受损耳蜗生理的理解。的目标 这项建议是研究三种基本上未被探索的蛋白质，它们的功能可能对人类的免疫系统很重要。 内耳中MT的调节：CAMSAP 3、CDH 23-C和WDR 47。CAMSAP 3是一个MT负端结合蛋白 蛋白，而CDH 23-C，CDH 23的细胞质同种型，和WDR 47，一种功能未知的蛋白， 与CAMSAP 3交互。这些基因的缺失或突变与人类的听力障碍有关， 小鼠模型。因此，它们可能是导致遗传性听力损失的潜在原因。在AIM I中，我们 使用缺乏CAMSAP 3的条件性敲除小鼠模型研究CAMSAP 3在个体中的作用。 耳蜗中的细胞类型包括毛细胞、支持细胞和螺旋神经节神经元。我们将研究 这些小鼠使用各种解剖学和生理学方法来确定CAMSAP 3在 听证会在AIM II中，我们研究了CDH 23-C和CDH 23-C之间相互作用受损的功能后果。 CAMSAP 3.我们将产生一个CDH 23-C突变小鼠模型，模拟人类Usher综合征1D 突变的负面影响这种相互作用，并评估的重要性，作用的CDH 23-C在MT 通过与CAMSAP 3的相互作用进行调节。这些实验旨在揭示一种新的分子 这种机制涉及耳蜗中的MT调节，并与Usher综合征1D有关。在Aim中 第三，我们通过体内和体外两种方法研究了WDR 47在听力中的功能。我们将 确定WDR 47在耳蜗中的详细表达模式，并研究WDR 47对 使用WDR 47敲除小鼠进行听力测试。我们还将剖析WDR 47之间相互作用的分子基础。 和CAMSAP 3，并通过生物化学和蛋白质组学揭示WDR 47相关蛋白。采取 总之，拟议的研究旨在提供有关调节蛋白质网络的关键信息， MT组织在耳蜗通过专注于三种蛋白质。我们的综合方法利用各种 方法包括分子和细胞生物学、生物物理学、电生理学、体内生理学和质量 光谱，从而使我们能够实现我们的目标。从拟议的实验中获得的数据将 提供有关听力损失病理学基础的分子机制的新知识，并丰富 我们对蛋白质网络的理解对听力和耳聋很重要。这些信息将指导我们的 当前和未来在指导治疗和/或预防听力损失方面的努力。

PROJECT SUMMARY The cochlea converts sound waves into electrical signals to convey information to the central nervous system. Highly specialized cells including hair cells, supporting cells and spiral ganglion neurons mediate this operation. Because complex and sophisticated microtubule (MT) networks are needed for the individualized cell shapes and structures required for peripheral signal coding, uncovering the protein networks that modulate MT organization will enrich our understanding of both normal and impaired cochlear physiology. The goal of this proposal is to investigate three largely unexplored proteins, whose functions may be important for the regulation of MTs in the inner ear: CAMSAP3, CDH23-C, and WDR47. CAMSAP3 is a MT minus-end binding protein, while CDH23-C, a cytoplasmic isoform of CDH23, and WDR47, a protein of unknown function, both interact with CAMSAP3. Deletion or mutation of these genes is linked to hearing impairment in humans and in mouse models. Thus, they may potentially be responsible for causing hereditary hearing loss. In AIM I, we investigate the role of CAMSAP3 using conditional knockout mouse models that lack CAMSAP3 in individual cell types in the cochlea including hair cells, supporting cells, and spiral ganglion neurons. We will examine these mice using various anatomical and physiological methods to determine the function of CAMSAP3 in hearing. In AIM II, we investigate the functional consequences of impaired interaction between CDH23-C and CAMSAP3. We will generate a CDH23-C mutant mouse model that mimics the human Usher Syndrome 1D mutation that negatively affects this interaction, and evaluate the importance of the role of CDH23-C in MT regulation via its interaction with CAMSAP3. The experiments are designed to reveal a novel molecular mechanism that involves MT modulation in the cochlea and that is implicated in Usher Syndrome 1D. In Aim III, we investigate the function of WDR47 in hearing through both in vivo and in vitro approaches. We will determine the detailed expression patterns of WDR47 in the cochlea and investigate WDR47's contribution to hearing using WDR47 knockout mice. We will also dissect the molecular basis of interactions between WDR47 and CAMSAP3, and uncover WDR47-associated proteins through both biochemistry and proteomics. Taken together, the proposed studies aim to provide critical information regarding the protein networks that regulate MT organization in the cochlea by focusing on three proteins. Our comprehensive approach utilizes a variety of methods including molecular and cell biology, biophysics, electrophysiology, in vivo physiology and mass spectrometry, thereby allowing us to achieve our goals. The data obtained from the proposed experiments will provide new knowledge about the molecular mechanisms underlying the pathology of hearing loss, and enrich our understanding of the protein networks important for hearing and deafness. This information will guide our current and future efforts in directing treatments and/or in preventing hearing loss.