Molecular Basis of Transduction in Auditory Sensory Organs

听觉感觉器官转导的分子基础

• 批准号: 7966951
• 负责人: BECHARA KACHAR
• 金额: $ 168.21万
• 依托单位: NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7966951
• 关键词: Acoustic Trauma; Actins; Affect; Age; Ankyrin Repeat; Auditory; Back; Binding; Biochemical; Bundling; C-terminal; Cadherins; Cell physiology; Cells; Collaborations; Complex; Coupling; Data; Defect; Development; Diamond; Diffusion; Distal; Electron Microscopy; Elements; Extracellular Domain; F-Actin; Filopodia; Freeze Fracturing; Genes; Goals; Hair Cells; Health Sciences; Hearing; Hearing Impaired Persons; Height; Immunofluorescence Immunologic; Individual; Inherited; Inhibitory Synapse; Institutes; Laboratories; Labyrinth; Length; Light; Link; Longevity; Maintenance; Measurement; Methods; Microfilaments; Missense Mutation; Molecular; Molecular Motors; Motion; Motor; Mutation; Myosin ATPase; Myosin III; National Institute of Neurological Disorders and Stroke; Oregon; Organ; Organelles; Patients; Pattern; Phenotype; Physiological; Play; Plus End of the Actin Filament; Protein Isoforms; Proteins; Regulation; Retina; Role; Sensory; Sensory Hair; Shapes; Stereocilium; Structure; Synapses; System; Tail; Testing; Time; Vesicle; Vestibular Hair Cells; base; cellular microvillus; deafness; depolymerization; electron tomography; follow-up; hearing impairment; human CDH23 protein; insight; monomer; mouse model; overexpression; physical model; polymerization; postsynaptic; presynaptic; reconstruction; repaired; retinal rods; ribbon synapse; self-renewal

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. Two proteins implicated in inherited deafness, myosin IIIa, a plus end directed motor, and espin1, an actin bundling protein containing an actin-monomer-binding WH2 (WASP homology 2) motif, have been shown to influence the length and shape of mechanosensory stereocilia of the inner ear. Ongoing studies in our lab demonstrate that espin 1, the only isoform of espin that contains ankyrin repeats, shows a spatial and temporal pattern of localization at the tips of stereocilia similar to that described for myosin IIIa, and that the espin 1 ankyrin repeats domain (ARD) interacts with a unique conserved domain in the myosin IIIa carboxyl-terminal tail region. We show that, like myosin IIIa, espin 1 causes stereocilia elongation when overexpressed in cultured hair cells. Using a heterologous expression system we showed an extraordinary elongation of filopodia resulting from the transport of espin 1 to the plus ends of filopodial F-actin by myosin IIIa, and that this elongation is dependent on espin 1 WH2 activity. This study provides the basis for understanding the role myosin IIIa and espin 1 play in regulating stereocilia length, presenting a physiological example where myosins can boost elongation of actin protrusions by transporting actin regulatory factors to the plus ends of actin filaments. This system also demonstrates that the counter action of diffusion by molecular motors may be a general mechanism that can be used by a cell to localize certain components at the distal end of actin protrusions. We are currently also studying myosin IIIb, a shorter myosin III isoform without the C-terminal actin-binding domain. Since there is no explanation why DFNB30 patients with a homozygous mutation in the MYO3A gene have a normal ability to hear for the first twenty years and progressively become deaf from that point and on, we sought answers to whether myosin IIIb compensates for loss of myosin IIIa function. We observed that myosin IIIb is also localized to the same compartment as myosin IIIa and espin 1 in hair cell stereocilia. However, we found that the temporal expression of myosin IIIb differs. Myosin IIIb showed a peak of immunofluorescence around age P2, while myosin IIIa and espin 1 have the peak at age P6. Furthermore, we found that myosin IIIb cannot self-localize to the tips of filopodia due the lack of actin-binding 3THDII in the tail. Myosin IIIb rather depends on espin 1 for the tip localization. Myosin IIIb is also found to down regulate the localization of myosin IIIa to the stereocilia tips of vestibular hair cells but not to the cochlear hair cells. The localization and interplay of myosins IIIa, IIIb, and espin 1 and their influence on stereocilia length shed light on a previously unrecognized molecular complex at the polymerizing end of actin filaments. We postulate that myosin IIIb compensates for myosin IIIa in the function during the development and maturation of stereocilia thus explaining the late onset hearing loss in DFNB30 patients. We also collaborated with Nir Gov from the Weizmann Institute to produce a physical model that describes the active localization of actin-regulating proteins inside stereocilia during steady-state conditions. The mechanism of localization is through the interplay of free diusion and di-rected motion, which is driven by coupling to the treadmilling actin filaments and to myosin motors that move along the actin laments. The resulting localization of both the molecular motors and their cargo is calculated, and is found to have an exponential (or steeper) profile. This localization can be at the base (driven by actin retrograde flow and minus-end myosin motors), or at the stereocilia tip (driven by plus-end myosin motors). The localization of proteins that infuence the actin depolymerization and polymerization rates allow us to describe the narrow shape of the stereocilia base, and the observed increase of the actin polymerization rate with the stereocilia height. Tip links are thought to be an essential element of the mechanoelectrical transduction apparatus in sensory hair cells of the inner ear. In previous structural, histological and biochemical studies in collaboration with Ulrich Mueller (Scripps Institute) we showed that the extracellular domains of two deafness-associated cadherins, cadherin 23 (CDH23) and protocadherin 15 (PCDH15), interact in trans to form the upper and lower part of each tip link, respectively. In a continuation of these collaborative studies using a mouse model for nonsyndromic deafness (DFNB12) called salsa, we were able to show that hearing loss is related to defects in tip links. The phenotype of salsa suggests that DFNB12 is a new class of deafness caused by the loss of the tip links due to an unstable interaction between CDH23 and PCDH15. A DFNB23-causative missense mutation in PCDH15-ECD is also known to affect the interaction between CDH23 and PCDH15 1, suggesting a common athogenesis underlying both DFNB12 and DFNB23. Establishing mouse models for DFNB23 will be important for testing this hypothesis. In the past year, we have also initiated new collaborations with the laboratories of Henrique von Gersdorff of Oregon Health Sciences Universitys Vollum Institute and Jeffrey Diamond of the National Institute of Neurological Disorders and Stroke (NINDS). These collaborations have allowed us to explore the presynaptic distributions of ribbon synapses and their structural relationships with postsynaptic contacts in both hair cells (von Gersdorff collaboration) and bipolar cells of the retina. For the hair cell project, we have been using serial section electron microscopy to generate detailed anatomical measurements of ribbon synapse sizes, distributions, and vesicle pools which will be combined with electrophysiological recordings from the von Gersdorff lab. The bipolar cell project takes a similar approach, by using serial section reconstructions to characterize the synaptic relationship between rod bipolar cells and A17amacrine cells, which contain reciprocal inhibitory synapses back on to the rod bipolar cell terminal, and combining it with functional data from the Diamond lab. Currently, the large-scale reconstruction projects are nearing completion. As a follow-up approach, we plan to use electron tomography and freeze-fracture methods to investigate the structure and composition of individual ribbon synapses.

细胞肌动蛋白突起（例如丝状伪足、微绒毛和静纤毛）显示出广泛的长度和寿命，与其特定的细胞功能密切相关。立体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的研究以及对静纤毛正确结构和功能所必需的几种耳聋相关蛋白的鉴定，为静纤毛长度调节、长期维持以及过度刺激或声损伤后修复潜力所涉及的机制和分子提供了新的见解。肌球蛋白及其货物与肌动蛋白突起的形成和伸长有关，但它们影响 F-肌动蛋白伸长的机制多种多样，且尚未完全了解。 与遗传性耳聋有关的两种蛋白质，即肌球蛋白 IIIa（一种正端定向运动）和 espin1（一种含有肌动蛋白单体结合 WH2（WASP 同源性 2）基序的肌动蛋白捆绑蛋白）已被证明会影响内耳机械感觉静纤毛的长度和形状。 我们实验室正在进行的研究表明，espin 1（唯一包含锚蛋白重复的 espin 亚型）显示出与肌球蛋白 IIIa 相似的静纤毛尖端定位的空间和时间模式，并且 espin 1 锚蛋白重复结构域 (ARD) 与肌球蛋白 IIIa 羧基末端尾部区域中的独特保守结构域相互作用。我们发现，与肌球蛋白 IIIa 一样，espin 1 在培养的毛细胞中过度表达时会导致静纤毛伸长。使用异源表达系统，我们展示了丝状伪足的非凡伸长，这是由于肌球蛋白 IIIa 将 espin 1 转运到丝状伪足 F-肌动蛋白的正端而引起的，并且这种伸长依赖于 espin 1 WH2 活性。这项研究为理解肌球蛋白 IIIa 和 espin 1 在调节静纤毛长度中的作用提供了基础，并提出了一个生理学例子，其中肌球蛋白可以通过将肌动蛋白调节因子转运到肌动蛋白丝的正端来促进肌动蛋白突起的伸长。该系统还证明，分子马达扩散的反作用可能是一种通用机制，细胞可以使用它来将某些组件定位在肌动蛋白突起的远端。 我们目前还在研究肌球蛋白 IIIb，这是一种较短的肌球蛋白 III 亚型，没有 C 端肌动蛋白结合结构域。 由于无法解释为什么 MYO3A 基因纯合突变的 DFNB30 患者在前 20 年听力正常，并从那时起逐渐耳聋，因此我们寻找肌球蛋白 IIIb 是否可以补偿肌球蛋白 IIIa 功能丧失的答案。 我们观察到肌球蛋白 IIIb 也位于毛细胞静纤毛中与肌球蛋白 IIIa 和 espin 1 相同的区室。 然而，我们发现肌球蛋白 IIIb 的时间表达有所不同。 肌球蛋白 IIIb 在 P2 龄左右出现免疫荧光峰值，而肌球蛋白 IIIa 和 espin 1 在 P6 龄时出现免疫荧光峰值。 此外，我们发现肌球蛋白 IIIb 不能自我定位到丝状伪足的尖端，因为尾部缺乏肌动蛋白结合 3THDII。 肌球蛋白 IIIb 的尖端定位更依赖于 espin 1。 肌球蛋白 IIIb 还被发现下调肌球蛋白 IIIa 到前庭毛细胞静纤毛尖端的定位，但不下调到耳蜗毛细胞的定位。 肌球蛋白 IIIa、IIIb 和 espin 1 的定位和相互作用及其对静纤毛长度的影响揭示了肌动蛋白丝聚合端先前未识别的分子复合物。我们假设肌球蛋白 IIIb 在静纤毛的发育和成熟过程中补偿了肌球蛋白 IIIa 的功能，从而解释了 DFNB30 患者迟发性听力损失。 我们还与魏茨曼研究所的 Nir ​​Gov 合作创建了一个物理模型，该模型描述了稳态条件下静纤毛内肌动蛋白调节蛋白的主动定位。定位机制是通过自由扩散和定向运动的相互作用，通过与跑步肌动蛋白丝和沿着肌动蛋白丝移动的肌球蛋白马达的耦合来驱动。计算出分子马达及其货物的最终定位，并发现其具有指数（或更陡峭）的分布。这种定位可以位于基部（由肌动蛋白逆行流和负端肌球蛋白马达驱动），或位于静纤毛尖端（由正端肌球蛋白马达驱动）。影响肌动蛋白解聚和聚合速率的蛋白质的定位使我们能够描述静纤毛基底的狭窄形状，以及观察到的肌动蛋白聚合速率随静纤毛高度的增加。 尖端连杆被认为是内耳感觉毛细胞中机电传导装置的基本元件。 在之前与 Ulrich Mueller（斯克里普斯研究所）合作进行的结构、组织学和生化研究中，我们发现两种与耳聋相关的钙粘蛋白钙粘蛋白 23 (CDH23) 和原钙粘蛋白 15 (PCDH15) 的细胞外结构域以反式相互作用，分别形成每个尖端链接的上部和下部。 在使用名为 Salsa 的非综合征性耳聋小鼠模型 (DFNB12) 的后续合作研究中，我们能够证明听力损失与耳尖连接缺陷有关。 salsa 的表型表明 DFNB12 是一种新的耳聋类型，是由于 CDH23 和 PCDH15 之间不稳定的相互作用导致耳尖连接丢失而引起的。 PCDH15-ECD 中的 DFNB23 致病性错义突变也已知会影响 CDH23 和 PCDH15 1 之间的相互作用，表明 DFNB12 和 DFNB23 存在共同的同源性。建立 DFNB23 小鼠模型对于检验这一假设非常重要。 去年，我们还与俄勒冈健康科学大学 Vollum 研究所的 Henrique von Gersdorff 实验室和国家神经疾病和中风研究所 (NINDS) 的 Jeffrey Diamond 实验室启动了新的合作。这些合作使我们能够探索带状突触的突触前分布及其与毛细胞（von Gersdorff 合作）和视网膜双极细胞中突触后接触的结构关系。对于毛细胞项目，我们一直使用连续切片电子显微镜来生成带状突触大小、分布和囊泡池的详细解剖测量结果，这些测量结果将与冯·格斯多夫实验室的电生理记录相结合。双极细胞项目采用了类似的方法，通过使用连续切片重建来表征杆双极细胞和 A17 无长突细胞之间的突触关系，其中包含返回到杆双极细胞末端的相互抑制性突触，并将其与 Diamond 实验室的功能数据相结合。目前，大型重建工程已接近尾声。作为后续方法，我们计划使用电子断层扫描和冷冻断裂方法来研究单个带状突触的结构和组成。