Molecular Basis of Transduction in Auditory Sensory Organs

细胞肌动蛋白突起（如丝状伪足、微绒毛和静纤毛）显示出与其特定细胞功能密切相关的广泛长度和寿命。静纤毛是毛细胞的机械感觉细胞器，是一类独特的基于肌动蛋白的细胞突起，具有无与伦比的随时间调节其长度的能力。我们的实验室在阐明静纤毛的形成、调节、更新和寿命的机制方面取得了重大进展。 对静纤毛中肌动蛋白周转率的研究以及对几种静纤毛结构和功能所必需的稳定性相关蛋白的鉴定为静纤毛长度调节、长期维持以及过度刺激或声损伤后的修复潜力的机制和分子提供了新的见解。肌球蛋白和它们的货物已被牵连在肌动蛋白突起的形成和延长，但它们影响F-肌动蛋白延长的机制是多种多样的，并没有完全理解。 在研究肌球蛋白IIIa的一种亚型肌球蛋白IIIb是否可以补偿肌球蛋白IIIa的功能并解释DFNB 30迟发性听力损失的过程中，我们发现了一种全新形式的货物依赖性肌球蛋白运动。推测肌球蛋白IIIa以尺蠖的方式沿着沿着肌动蛋白丝移位，其中尾部的保守结构域（THDII）提供了第二个肌动蛋白结合位点，防止肌球蛋白在每次肌球蛋白头部动力冲程时从肌动蛋白上完全解离。有趣的是，肌球蛋白IIIb被认为缺乏运动活性，因为它不包含THDII结构域。然而，我们观察到，当肌球蛋白IIIb和espin 1在COS 7细胞中共转染时，肌球蛋白IIIb的行为几乎与肌球蛋白IIIa相同。使用Co-IP和共转染试验，我们发现肌球蛋白IIIb通过其THDI与espin 1结合，并使用espin 1的肌动蛋白结合结构域作为缺失THDII的拐杖或替代物，以易位到肌动蛋白突起的尖端。免疫定位和转染试验表明，肌球蛋白IIIb形成相同的模式定位肌球蛋白IIIa的静纤毛的尖端。这些研究表明，肌球蛋白IIIb可以补偿肌球蛋白IIIa的功能，并提供了一个框架，以进一步研究DFNB 30迟发性耳聋。 在与Christopher Yengo（宾夕法尼亚州立大学）的合作中，我们发现分子间的自磷酸化调节肌球蛋白IIIa的活性和静纤毛和其他肌动蛋白突起的定位。肌球蛋白IIIa有一个激酶结构域，被认为可以自动调节其活性。由于肌球蛋白IIIa倾向于聚集在肌动蛋白突起的尖端，我们研究了分子间磷酸化是否可以调节其生化活性，细胞定位和细胞功能。虽然点突变K50 R的肌球蛋白IIIa激酶结构域的失活并没有改变最大ATP酶活性，但肌球蛋白IIIa的磷酸化导致最大ATP酶活性和肌动蛋白亲和力降低。 肌动蛋白的存在下，肌球蛋白IIIa自磷酸化的速率和程度是不变的，但发现依赖于肌球蛋白IIIa浓度范围内的0.1-1.2 M，表明分子间自磷酸化。在培养的细胞中，我们观察到缺乏激酶结构域的肌球蛋白IIIa的丝状伪足尖端定位减少时，与激酶活性，全长肌球蛋白IIIa共表达。肌球蛋白IIIa尖端定位减少的细胞后果是沿着细胞周边的丝状伪足密度沿着降低，确定了肌球蛋白IIIa在介导肌动蛋白基突起的形成和稳定性中的新细胞功能。这些结果表明，肌球蛋白IIIa运动活性的调节，通过一种新的机制，涉及浓度依赖性自磷酸化 在过去的一年中，在与亨里克冯Gersdorff（OHSU）合作，我们使用连续切片和电子断层扫描重建库存模型毛细胞中的所有带状突触。通过将我们全面的超微结构数据（精确的空间关系，囊泡池和突触几何形状）与Henriques实验室中进行的生理学配对，我们探索了这些带状突触的设计特征如何使它们能够在宽范围的刺激强度下将毛细胞分级电位转换为多泡释放。通过维持具有释放阈值梯度的对接突触囊泡的群体，突触似乎能够使囊泡释放的功效适应刺激的强度。

Cellular actin protrusions (e.g. filopodia, microvilli, and stereocilia) display a broad range of lengths and lifetimes critically related to their specific cellular function. Stereocilia, the mechanosensory organelles of hair cells, are a distinctive class of actin-based cellular protrusions with an unparalleled ability to regulate their lengths over time. Our laboratory has made significant advances towards elucidating the mechanisms that underlie the formation, regulation, renewal, and life span of stereocilia. Studies on actin turnover in stereocilia as well as the identification of several deafness-related proteins essential for proper stereocilia structure and function provide new insights into the mechanisms and molecules involved in stereocilia length regulation, long-term maintenance, and potetnial for repair following overstimulation or acoustic trauma. Myosins and their cargo have been implicated in formation and elongation of actin protrusions, but the mechanisms by which they influence F-actin elongation are diverse and not fully understood. In the process of investigating whether myosin IIIb, a paralog of myosin IIIa, could compensate for myosin IIIa function and explain the DFNB30 late onset hearing loss, we have discovered an entirely novel form of cargo-dependent myosin motility. Myosin IIIa is presumed to translocate along actin filaments in an inchworm fashion, where a conserved domain in the tail (THDII) provides a second actin-binding site that prevents complete dissociation of the myosin from the actin with each myosin head power-stroke. Intriguingly, myosin IIIb has been presumed to lack motile activity because it does not contain a THDII domain. We observed, however, that when myosin IIIb and espin 1 are co-transfected in COS7 cells, the behavior of myosin IIIb is nearly identical to myosin IIIa. Using Co-IP and co-transfection assays we showed that myosin IIIb binds to espin 1 through its THDI and uses the actin-binding domain of espin 1 as a crutch or replacement for the missing THDII to translocate to the tips of actin protrusions. Immunolocalization and transfection assays showed that myosin IIIb forms the same pattern of localization as myosin IIIa at the tips of stereocilia. These studies demonstrate that myosin IIIb can compensate for myosin IIIa function and provide a framework to further investigate DFNB30 late onset deafness. In collaboration with Christopher Yengo (Penn State University), we showed that intermolecular auto-phosphorylation regulates myosin IIIa activity and localization in stereocilia and other actin protrusions. Myosin IIIa has a kinase domain that is thought to auto-regulate its activity. Because myosin IIIa tends to cluster at the tips of actin protrusions, we investigated whether intermolecular phosphorylation could regulate its biochemical activity, cellular localization, and cellular function. While inactivation of the myosin IIIa kinase domain with the point mutation K50R did not alter maximal ATPase activity, phosphorylation of myosin IIIa resulted in reduced maximal ATPase activity and actin affinity. The rate and degree of myosin IIIa autophosphorylation was unchanged by the presence of actin but found to be dependent upon myosin IIIa concentration within the range of 0.1-1.2 M, indicating intermolecular autophosphorylation. In cultured cells, we observed that the filopodial tip localization of myosin IIIa lacking the kinase domain decreases when co-expressed with kinase-active, full-length myosin IIIa. The cellular consequence of reduced myosin IIIa tip localization was a decreased filopodial density along the cell periphery, identifying a novel cellular function for myosin IIIa in mediating the formation and stability of actin-based protrusions. These results suggest that myosin IIIa motor activity is regulated through a novel mechanism involving concentration-dependent autophosphorylation In the past year, in collaborations with Henrique von Gersdorff (OHSU) we used serial section and electron tomographic reconstructions to inventory all ribbon synapses in a model hair cell. By pairing our comprehensive ultrastructural data (precise spatial relationships, vesicle pools, and synaptic geometry) with physiology performed in Henriques lab, we explored how the design features of these ribbon synapses might allow them to convert the hair cells graded electrical potentials into multivesicular release over a wide range of stimulus intensities. By maintaining a population of docked synaptic vesicles with a gradient of release thresholds, the synapse appears able to adapt the efficacy of vesicle release to the intensity of stimulation.