Molecular Basis Of Transduction In Auditory Sensory Orga

该实验室使用先进的显微成像技术来研究听觉和前庭感觉器官机械转导的细胞和分子机制。三种非常规肌球蛋白：肌球蛋白VI、VIIA和XV在毛细胞立纤毛的形成和功能中起着至关重要的作用，因为它们在立纤毛内或周围特异性表达，其基因突变导致立纤毛畸形和耳聋。我们一直在使用显微镜和免疫细胞化学技术相结合来检查这些蛋白质在立体纤毛结构中的精确定位。我们也一直在使用酵母双杂交实验筛选小鼠Organ of Corti cDNA文库，寻找直接参与立体纤毛发育和功能的潜在蛋白质伴侣。到目前为止，已经鉴定出几个有希望的克隆具有Myosin XV和Espin的特定区域，Espin是另一种对立体纤毛功能至关重要的肌动蛋白结合蛋白。过去一年的实验也集中在确定prestin是否存在于前庭系统的毛细胞中，prestin是最近克隆的OHC质膜蛋白，被认为是负责电运动的。通过RT-PCR和原位杂交，我们发现prestin不仅在OHCs中表达，而且在前庭毛细胞（vhc）中也表达。利用肽特异性抗体的免疫定位研究证实，在小鼠、大鼠和沙鼠的囊、胞室和壶腹嵴的vhc中存在prestin。然而，在vhc中，在侧质膜或立体纤毛膜中未检测到prestin，但在细胞质中的囊泡细胞器中观察到prestin。这表明，与OHCs相反，vhc没有有效的机制将prestin靶向到质膜上。全细胞膜片钳记录显示，vhc不具有与电运动性相关的电压依赖性电容。我们得出结论，尽管prestin在vhc中表达，但它不太可能像OHCs那样支持躯体运动。在一项新启动的研究听力开始时Corti器官转录库的项目中，已经产生了一个高质量的小鼠Corti器官库，并对数千个克隆进行了测序。该EST数据库的初步分析显示了大量潜在的新基因和已知基因的新剪接形式。转录本先前显示是必不可少的听觉感觉功能以及候选人耳聋也被揭示。其中最丰富的转录本（0.4%）编码一种新的89个氨基酸蛋白，通过原位杂交证实在毛细胞内外表达。具有描述和附加注释（如老鼠和人类染色体位置）的无害环境技术的集合正在汇编成一个基于网络的资源供公众访问。

The laboratory uses advanced microscopy imaging to study cellular and molecular mechanisms that underlie mechano-transduction in auditory and vestibular sensory organs. Three unconventional myosins: myosins VI, VIIA and XV have a vital role in hair cell stereocilia formation and function because they are specifically expressed in or around the stereocilia and mutations in their genes lead to stereocilia malformation and deafness. We have been using a combination of microscopy and immunocytochemical techniques to examine the precise localization of these proteins within the structure of the stereocilia. We have also been using the yeast two-hybrid assay to screen a mouse Organ of Corti cDNA library for potential protein partners directly involved in stereocilia development and function. To date several promising clones have been identified to specific regions of Myosin XV and Espin, another actin binding protein essential for stereocilia function. Experiments over the past year have also focused on determining whether prestin, a recently cloned OHC plasma membrane protein postulated to be responsible for electromotility, is present in hair cells of the vestibular system. Using RT-PCR and in situ hybridization we have shown that prestin is not only expressed in OHCs but is also expressed in vestibular hair cells (VHCs). Immunolocalization studies using peptide specific antibodies confirmed the presence of prestin in VHCs in the mouse, rat and gerbil saccule, utricle, and cristae ampullaris. However, in the VHCs, prestin was not detected in the lateral plasma membrane or in the stereociliary membrane, but was observed in vesicular organelles in the cytoplasm. This suggests that contrary to OHCs, the VHCs do not have an efficient mechanism to target prestin to the plasma membrane. Whole-cell patch-clamp recordings showed that VHCs do not possess the voltage-dependent capacitance associated with electromotility. We conclude that although prestin is expressed in VHCs, it is unlikely that it supports somatic motility like that of OHCs. In a newly initiated project to study the transcriptional repertoire of the organ of Corti during the onset of hearing, a high quality mouse organ of Corti library has been produced and several thousand clones have been sequenced. Preliminary analysis of this EST database showed a large number of potentially novel genes and new splice forms of known genes. Transcripts previously shown to be essential for auditory sensory function as well candidates for deafness are also revealed. One of the most abundant transcripts (0.4%), encoding a novel 89 amino acid protein, was demonstrated to be expressed in inner and outer hair cells by in situ hybridization. The collection of ESTs with description and additional annotations (such as mouse and human chromosomal location) is being compiled as a web-based resource for public access.