Microtubule-mediated mechanisms underlying hair cell development and deafness

描述（由申请人提供）：听觉毛细胞顶端的肌动蛋白基静纤毛束（或毛束）具有将声能转换为电信号的关键功能。它的V形阶梯结构使束对机械刺激方向敏感。因此，听觉毛束必须均匀定向以进行正确的声音传导。发束极性或方向的异常会导致耳聋和听力障碍。这项工作的一个长期目标是详细了解毛束形态发生程序以及破坏这些程序的基因突变如何导致感觉神经性耳聋。特别是，名为GPSM 2的基因突变导致人类遗传性耳聋DFNB 82和Chudley-McCullough综合征。然而，潜在的疾病机制仍然完全未知。我们最近关于毛细胞中微管介导的毛束极性和方向的通路的见解提出了关于GPSM 2在该通路中的作用的新的可检验的假说。具体来说，我们已经发现了一个关键的和以前未被重视的功能的毛细胞微管和微管为基础的分子马达在基体定位，这是至关重要的毛束极性和方向。我们发现，无论是驱动蛋白-II亚基Kif 3a或动力蛋白调节器Lis 1缺陷的毛细胞有基体定位缺陷。因此，毛束的偏振V形和取向都被破坏。我们进一步证明，这些微管马达调节毛细胞皮层上的Rac GTPase-PAK信号传导的不对称结构域，以介导基底体定位。本研究的主要目标是进一步剖析这种微管介导的途径的分子组成部分，包括耳聋基因GPSM 2，并获得微管调控毛束极性的机制见解。我们将通过以下具体目标来实现这一目标。目的1将测试的假设，即细胞极性蛋白Par 3和GPSM 2作为皮质的地标栓动力蛋白在皮质上拉动微管和定向基体，类似的机制，定向有丝分裂纺锤体在不对称的细胞分裂。目的2将测试驱动蛋白-II介导的Par 3和Rac激活剂Tiam 1靶向递送至细胞皮层对于Rac信号传导和基底体定位的空间调节是至关重要的这一假设。目标3将使用创新的实时成像来测试PAK信号调节微管稳定性和皮质蛋白以稳定微管加端附着在细胞皮质的假设。这项研究将为毛细胞发育的研究提供新的途径，并阐明对人类耳聋基因知之甚少的功能。获得更深入的了解毛束形态程序将是至关重要的设计合理的疗法，刺激损伤后的毛束修复，治疗遗传性人类耳聋和再生听觉毛细胞通过干细胞技术。

DESCRIPTION (provided by applicant): The actin-based stereociliary bundle (or hair bundle) on the apex of auditory hair cells serves the critical function of converting sound energy to electric signals. Its V-shaped staircase structure renders the bundle directionally sensitive to mechanical stimuli. As such, auditory hair bundles must be uniformly oriented for correct sound transduction. Abnormalities in hair bundle polarity or orientation cause deafness and hearing impairment. A long-term objective of this work is to gain a detailed understanding of the hair bundle morphogenesis programs and how genetic mutations that disrupt these programs cause sensorineural deafness. In particular, mutations in a gene named GPSM2 cause the human hereditary deafness DFNB82 and Chudley-McCullough Syndrome. However, the underlying disease mechanisms remain completely unknown. Our recent insights about a microtubule-mediated pathway in hair cells for hair bundle polarity and orientation suggest a novel testable hypothesis about GPSM2's role in this pathway. Specifically, we have uncovered a critical and previously unappreciated function of the hair cell microtubules and microtubule-based molecular motors in basal body positioning, which is critical for both hair bundle polarity and orientation. We found that hair cells deficient in either the kinesin-II subunit Kif3a or the dynein regulator Lis1 have basal body positioning defects. Consequently, both the polarized V-shape and orientation of the hair bundles are disrupted. We further demonstrate that these microtubule motors regulate an asymmetric domain of Rac GTPase-PAK signaling on the hair cell cortex to mediate basal body positioning. The major goal of this research is to further dissect the molecular components of this microtubule-mediated pathway, including the deafness gene GPSM2, and gain mechanistic insights into microtubule regulation of hair bundle polarity. Our goal will be pursued through the following specific aims. Aim 1 will test the hypothesis that the cell polarity proteins Par 3 and GPSM2 serve as cortical landmarks to tether dynein at the cortex to pull on microtubules and orient the basal body, similar to mechanisms that orient the mitotic spindle during asymmetric cell division. Aim 2 will test the hypothesis that kinesin-II mediated targeted delivery of Par3 and the Rac activator Tiam1 to the cell cortex is critical for spatial regulation of Rac signaling and basal body positioning. Aim 3 will use innovative live imaging to test the hypothesis that PAK signaling regulates both microtubule stability and cortical proteins to stabilize microtubule plus-end attachment at the cell cortex. This research will provide new avenues of investigation into hair cell development and elucidate the function of poorly understood human deafness genes. Gaining a deeper understanding of the hair bundle morphogenesis program will be essential for devising rational therapies to stimulate hair bundle repair following injury, to treat hereditary human deafness and to regenerate auditory hair cells through stem cell technologies.