Mechanosensor Development, Function and Dysfunction

描述（由申请人提供）：我的实验室的长期目标是阐明控制毛细胞机电转导（MET）的机制，以及这一过程中导致耳聋的缺陷。本文拟研究细胞粘附分子cadherin 23 （CDH23）和原cadherin 15 （PCDH15）在毛细胞功能和疾病中的作用。先前的研究表明，CDH23和PCDH15是发束发育所必需的。最近的研究表明，CDH23和PCDH15不仅在发育中，而且在功能成熟的毛细胞中也是连接立体纤毛的细胞外细丝的组成部分。基于这些发现和新的初步数据，我们假设CDH23和PCDH15是毛细胞中更大的跨膜信号复合物的组成部分，不仅控制毛束的形态发生，而且控制MET。我们预测，选择性剪接调节这些蛋白质复合物的组装和功能。为了验证我们的假设，我们将：(i)使用转基因小鼠来确定CDH23对MET的功能，这些小鼠被设计为绕过与CDH23空等位基因相关的发育性毛细胞缺陷；（ii）确定PCDH15剪接变异体对毛细胞发育和MET的功能；（iii）通过酵母-双杂交法分离毛细胞中CDH23和PCDH15依赖性粘附复合物的新组分；（iv）分析CDH23突变的小鼠系，这些突变模拟Usher综合征1D和常染色体隐性耳聋DFNB12患者的突变。我们预计，与不同疾病表型相关的突变会影响毛细胞中CDH23功能的不同方面。耳聋是一个主要的健康问题。每1000名儿童中就有1名患有听力障碍，大部分老龄人口患有与年龄有关的听力损失。近年来，在识别导致耳聋的基因突变方面取得了巨大进展，但我们对受影响基因突变导致耳聋的机制知之甚少。我们在此建议研究毛细胞中与耳聋相关的两个基因Cdh23和Pcdh15的功能。根据已发表的和初步的数据，我们预计这两个基因对于机械感觉毛细胞的发育和功能都是必需的，并且导致综合征和非综合征型耳聋的两个基因的不同突变影响毛细胞中基因功能的不同方面。

DESCRIPTION (provided by applicant): The long-term goal of my laboratory is to elucidate the mechanisms that control mechanoelectrical transduction (MET) in hair cells, and the defects in this process that cause deafness. We propose here to study the function of the cell adhesion molecules cadherin 23 (CDH23) and protocadherin 15 (PCDH15) in hair cell function and disease. Previous studies have shown that CDH23 and PCDH15 are required for hair bundle development. Recent findings show that CDH23 and PCDH15 are components of extracellular filaments that connect stereocilia not only in developing but also in functionally mature hair cells. Based on these findings and new preliminary data, we hypothesis that CDH23 and PCDH15 are components of larger transmembrane signaling complexes in hair cells that control not only hair bundle morphogenesis but also MET. We predict that alternative splicing regulates the assembly and function of these protein complexes. To test our hypothesis, we will: (i) Determine the function of CDH23 for MET using genetically modified mice that were designed to circumvent developmental hair cell defects that are associated with CDH23 null alleles; (ii) define the function of PCDH15 splice variants for hair cell development and MET; (iii) isolate by yeast-two-hybrid assays novel components of CDH23 and PCDH15 dependent adhesion complexes in hair cells; (iv) analyze mouse lines with mutations in CDH23 that mimic mutations in patients suffering from Usher Syndrome 1D and autosomal recessive deafness DFNB12. We anticipate that mutations that are associated with different disease phenotypes affects distinct aspects of CDH23 function in hair cells. Deafness is a major health problem. 1 in 1000 children is born with hearing impairment and large parts of the aging population are afflicted by age-related hearing loss. In recent years, dramatic progress has been made in identifying gene mutations that cause deafness, but we know comparatively little about the mechanisms by which mutations in the affected genes cause deafness. We propose here to study the function of two genes that have been linked to deafness, Cdh23 and Pcdh15, in hair cells. Based on published and preliminary data, we anticipate that the two genes are required both for the development and function of mechanosensory hair cells, and that different mutations in the two genes that cause syndromic and non-syndromic forms of deafness affect distinct aspects of gene function in hair cells.