Microtubule-mediated mechanisms underlying hair cell development and deafness

描述（由申请人提供）：听觉毛细胞顶端的基于肌动蛋白的静纤毛束（或发束）发挥着将声能转换为电信号的关键功能。其 V 形阶梯结构使束对机械刺激定向敏感。因此，听觉毛束必须均匀定向才能正确传导声音。发束极性或方向异常会导致耳聋和听力障碍。这项工作的长期目标是详细了解发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音神经性耳聋。特别是，GPSM2 基因的突变会导致人类遗传性耳聋 DFNB82 和查德利-麦卡洛综合症。然而，潜在的疾病机制仍然完全未知。我们最近对毛细胞中微管介导的毛束极性和方向通路的见解提出了关于 GPSM2 在该通路中的作用的新的可检验假设。具体来说，我们发现了毛细胞微管和基于微管的分子马达在基底体定位中的一个关键且以前未被认识到的功能，这对于发束的极性和方向都至关重要。我们发现，缺乏驱动蛋白-II 亚基 Kif3a 或动力蛋白调节因子 Lis1 的毛细胞具有基底体定位缺陷。因此，发束的偏光 V 形和方向都被破坏。我们进一步证明，这些微管马达调节毛细胞皮层上 Rac GTPase-PAK 信号传导的不对称结构域，以介导基底体定位。这项研究的主要目标是进一步剖析这种微管介导途径的分子成分，包括耳聋基因 GPSM2，并获得对发束极性的微管调节的机制见解。我们的目标将通过以下具体目标来实现。目标 1 将检验以下假设：细胞极性蛋白 Par 3 和 GPSM2 作为皮质地标，将动力蛋白拴在皮质上，以拉动微管并定向基体，类似于不对称细胞分裂期间定向有丝分裂纺锤体的机制。目标 2 将检验以下假设：驱动蛋白-II 介导的 Par3 和 Rac 激活剂 Tiam1 向细胞皮层的靶向递送对于 Rac 信号传导和基础体定位的空间调节至关重要。 Aim 3 将使用创新的实时成像来测试以下假设：PAK 信号传导调节微管稳定性和皮质蛋白，以稳定细胞皮质上的微管正端附着。这项研究将为毛细胞发育的研究提供新途径，并阐明人们知之甚少的人类耳聋基因的功能。更深入地了解发束形态发生程序对于设计合理的疗法来刺激损伤后的发束修复、治疗遗传性人类耳聋以及通过干细胞技术再生听毛细胞至关重要。

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.