Structural and Molecular Basis of Transduction in Auditory Sensory Organs

听觉感觉器官转导的结构和分子基础

• 批准号: 10250944
• 负责人: BECHARA KACHAR
• 金额: $ 142.24万
• 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/10250944
• 关键词: Actins; Action Potentials; Amino Acids; Aminoglycosides; Apical; Appearance; Auditory; Binding; Biological Assay; Biological Models; CD3 Antigens; Calcium and Integrin Binding Protein 2; Cations; Cells; Charge; Cilia; Co-Immunoprecipitations; Cochlea; Collaborations; Colorado; Complex; Data; Development; Dextrans; Dyes; Electrophysiology (science); Electrostatics; Epithelial; Epithelial Cells; Epithelium; Feedback; Filopodia; Genetic Transcription; Goals; Grant; Hair; Hair Cells; Hearing; Human; Hydrophobicity; Image; Inherited; Integral Membrane Protein; Knock-in Mouse; Knockout Mice; Laboratories; Lead; Length; Lipids; MYO7A gene; Maintenance; Methods; Molecular; Molecular Structure; Motor; Mus; Mutate; Myosin ATPase; National Institute of Neurological Disorders and Stroke; Organ; Organ of Corti; PCDH15 gene; Pathway interactions; Pattern; Permeability; Pharmacology; Play; Preventive Intervention; Probability; Prolate; Property; Protein Isoforms; Proteins; Proteomics; Regulation; Reporting; Research; Rest; Role; Sensory; Signal Transduction; Site; Structural Models; Structure; Supporting Cell; Supporting Cell of Organ of Corti; Surface; System; Testing; Therapeutic Intervention; Transgenic Mice; Translations; Universities; Variant; Vestibular Hair Cells; auditory pathway; base; calcium indicator; cell type; cellular microvillus; deafness; early onset; experimental study; fluorophore; gene product; genomic data; hearing impairment; insight; interest; intestinal epithelium; mechanotransduction; organizational structure; programs; promoter; self-renewal; sensory system; stoichiometry; ward

The hair cell mechanotransduction (MET) channel complex resides at the tips of the second and shorter rows of stereocilia in the hair bundle, which are both dynamic and prolate. In addition to TMC1 and TMC2, other transmembrane proteins have recently been identified as essential for normal MET. While TMC1 and TMC2 are candidates for the MET pore-forming channel proteins it is intriguing that they do not localize to stereocilia before P1-2 when other components of the MET apparatus is already in place. We hypothesized that there may be other TMCs that act as forerunners to TMC1 and TMC2 earlier in development. Reported proteomics and genomics data indicate that TMC4 and TMC5 are present in the organ of Corti. We generated knock in mice expressing both TMC4-GFP and TMC5-mCherry and made the striking discovery that TMC5 is present at tips of developing stereocilia in the organ of Corti between E17.5 and P2. After this point, TMC5 localization rapidly diminishes from stereocilia and TMC1 and TMC2 expression rises. After P2-3 TMC5 together with TMC4 is detected on the apical surface of supporting cells, and along and at the tip of their primary cilia. In vestibular hair cells we detected TMC4 and TMC5 in short, immature, hair bundles mostly at the periphery of the sensory epithelium. We also found that TMC4 and TMC5 interact with the MET proteins CIB2 and PCDH15 in a heterologous expression system. Together, these findings introduce TMC4 and TMC5 as potential precursors to TMC1 and TMC2 that likely play key roles in regulating stereocilia bundle development and/or function. Additionally, the function(s) of TMC4 and TMC5 in supporting cells and in their primary cilia remain to be elucidated. These findings provide new directions to investigate roles of TMCs in the formation of the MET complex and in other homeostatic functions in the developing organ of Corti. We are using single fluorophore counting methods to estimate the copy number of each of these fluorescently tagged proteins at the MET site to understand the compositional variation of the MET channel complex across stereocilia in a bundle, along the tonotopic gradient of the cochlea, and in different hair cell types. A putative variability in total number or stoichiometry of each of these proteins could provide insights into the extent of stochasticity in its assembly and regulation. Recent reports examining structural models of TMC1 based on the known structures of TMEM16 proteins, revealed the presence of a large cavity near the protein-lipid interface. This cavity harbors two residues that, when mutated, cause autosomal dominant hearing loss, suggesting that it could function as the elusive MET channel permeation pathway. To guide our experiments for probing the putative permeability properties of TMC4 and TMC5, we are collaborating with Angela Ballesteros (NINDS), to evaluate the potential structural and functional similarities between TMC1, 2, 4, and 5. Preliminary analyses indicate: 1) TMC4 and TMC5 share a common fold with TMC1, TMEM16, and TMEM63/OSCA proteins, consisting of 10 transmembrane helices and presenting a cavity that could function as a permeation pathway, 2) Differences in the amino acid composition of TMC1, 4 and 5 lead to changes in the electrostatic properties of the cavity; while the cavity of TMC1 is hydrophobic and anionic, the cavity in TMC5 is more cationic and in TMC4 is less charged. These differences in electrostatic properties of the cavity could grant ionic selectivity to the potential permeation pathways. We will be using early developing organ of Corti explants to test for permeability to aminoglycosides and 3kDa dextrans of different charges to evaluate the potential channel function of TMC4 and TMC5 in stereocilia and supporting cells. We have confirmed that TMC4 and TMC5 are also present in microvilli of intestinal epithelial cells and are using the intestinal epithelium as an accessible and robust model system to study TMC4 and TMC5 permeability, and for co-immunoprecipitation assays to identify candidate TMC binding partners. In collaboration with Jung-Bum Shin (UVA) we plan on examining the role of TMC4 and TMC5 in hair cells and supporting cells of the organ of Corti using TMC4 and TMC5 single and double KO mice. We previously showed that the appearance of the stereocilia cargo transporter MYO3A at stereocilia tips coincides with the onset of MET. MYO7A, another stereocilia myosin also shows a tip-to-base gradient of distribution consistent with tip-ward translocation and dynamic accumulation of this motor protein. Based on these observations, we hypothesize that MYO3A and MYO7A transport components of the MET complex to stereocilia tips with the potential for complementary function and/or redundancy. During the past year we refined our analyses of the ability of these two motor proteins to transport PCDH15 isoforms, which are components of the MET apparatus and involved in stereocilia bundle formation. Our new results show a cargo selectivity whereby MYO3A transports the PCDH15-CD2 isoform an integral component of the mature MET complex and MYO7A transports PCDH15-CD3 isoform previously localized at stereocilia tips in developing hair bundles. We also observed that in presence of the CD2 cargo there is a near two-fold increase in the enrichment of MYO3A at filopodia tips suggesting a cargo-dependent regulation of the myosin activity. In a collaboration with Jung-Bum Shin (UVA) and Anthony W. Peng (University of Colorado), we helped demonstrate that multiple MYO7A isoforms are expressed in the mouse cochlea. Two such isoforms MYO7A-C and MYO7A-S are generated by alternative transcription and translation start sites. MYO7A-C is expressed primarily in IHCs and in a tonotopic gradient in OHCs, with decreasing expression toward the cochlear base. These results are consistent with the observation of the expression levels of actin-GFP driven by a Myo7a-C promoter used in the Myo7a::Actin-GFP transgenic mouse. The Actin-GFP signal was predominantly observed in the IHCs and detected at low levels in the apical OHCs and decreased tonotopically toward the basal end of the cochlea. Myo7a-C mice show reduced levels of MYO7A at the UTLD and stereocilia base correlating with the observation that Myo7a-C hair cells show reduced MET resting channel open probability and slowed MET currents. Taken together, this collaborative study reveals unexpected isoform-specific differences in MYO7A expression in the cochlea and highlights the essential role of MYO7A in tensioning the hair cell MET complex. Spontaneous patterned action potential (AP) activity is considered important for the correct development of mammalian sensory systems. However, the relationship between Ca2+ AP activity in immature IHCs and the ATP-dependent intercellular Ca2+ signaling, which occurs spontaneously in cochlear non-sensory cells of the greater epithelial ridge, remains unclear. We performed Ca2+ imaging in organ of Corti explants from mice expressing genetically encoded calcium indicators and detected intrinsic spontaneous activity in IHCs. The independence of this spontaneous activity was corroborated by pharmacological experiments. These data have been combined with extensive imaging data using Ca2+ dyes and electrophysiology performed in the laboratories of Walter Marcotti (Uni. of Sheffield) and Fabio Mammano (Uni. of Padova) into a comprehensive study. Briefly, in this study we argue that a combination of intrinsic spontaneous Ca2+ activity and mutual influence between IHCs and non-sensory cells form an intricate feedback mechanism to control the level of AP synchronization in IHCs and generate the patterned activity implicated in the refinement of the auditory pathway before the onset of hearing.

毛细胞机械传导 (MET) 通道复合体位于发束中第二排和较短的静纤毛的尖端，这些静纤毛是动态的且呈长形。除了 TMC1 和 TMC2 之外，其他跨膜蛋白最近也被确定为正常 MET 所必需的。 虽然 TMC1 和 TMC2 是 MET 成孔通道蛋白的候选蛋白，但有趣的是，当 MET 装置的其他组件已经就位时，它们不会定位于 P1-2 之前的静纤毛。我们假设可能还有其他 TMC 在早期开发中充当 TMC1 和 TMC2 的先驱。 报道的蛋白质组学和基因组学数据表明 TMC4 和 TMC5 存在于 Corti 器官中。我们在表达 TMC4-GFP 和 TMC5-mCherry 的小鼠中进行了敲入，并惊人地发现 TMC5 存在于 E17.5 和 P2 之间的 Corti 器官中发育静纤毛的尖端。此后，TMC5 定位从静纤毛迅速减少，TMC1 和 TMC2 表达上升。 P2-3后，在支持细胞的顶端表面以及沿着其初级纤毛的顶端检测到TMC5和TMC4。 在前庭毛细胞中，我们在短的、未成熟的毛束中检测到 TMC4 和 TMC5，这些毛束主要位于感觉上皮的外围。我们还发现TMC4和TMC5在异源表达系统中与MET蛋白CIB2和PCDH15相互作用。总之，这些发现表明 TMC4 和 TMC5 作为 TMC1 和 TMC2 的潜在前体，可能在调节静纤毛束发育和/或功能中发挥关键作用。此外，TMC4 和 TMC5 在支持细胞及其初级纤毛中的功能仍有待阐明。 这些发现为研究 TMC 在 MET 复合物的形成以及 Corti 器官发育中的其他稳态功能中的作用提供了新的方向。 我们使用单一荧光团计数方法来估计 MET 位点上每种荧光标记蛋白的拷贝数，以了解跨静纤毛束、沿着耳蜗的音调梯度以及不同毛细胞类型的 MET 通道复合体的组成变化。这些蛋白质的总数或化学计量的假定变异性可以提供对其组装和调节的随机性程度的见解。 最近的报告根据 TMEM16 蛋白的已知结构检查了 TMC1 的结构模型，揭示了蛋白质-脂质界面附近存在一个大空腔。该空腔含有两个残基，当突变时，会导致常染色体显性听力损失，这表明它可能充当难以捉摸的 MET 通道渗透途径。为了指导我们探测 TMC4 和 TMC5 假定渗透性特性的实验，我们与 Angela Ballesteros (NINDS) 合作，评估 TMC1、2、4 和 5 之间潜在的结构和功能相似性。初步分析表明：1) TMC4 和 TMC5 与 TMC1、TMEM16 和 TMEM63/OSCA 具有共同的折叠 蛋白质，由10个跨膜螺旋组成，并呈现出可作为渗透途径的空腔，2）TMC1、4和5的氨基酸组成差异导致空腔静电特性的变化； TMC1 的空腔是疏水性和阴离子性的，而 TMC5 中的空腔是阳离子性更强的，而 TMC4 中的空穴则带电较少。空腔静电特性的这些差异可以赋予潜在渗透路径的离子选择性。我们将使用早期发育的 Corti 外植体器官来测试不同电荷的氨基糖苷类和 3kDa 葡聚糖的通透性，以评估 TMC4 和 TMC5 在静纤毛和支持细胞中的潜在通道功能。 我们已经证实 TMC4 和 TMC5 也存在于肠上皮细胞的微绒毛中，并使用肠上皮作为可访问且稳健的模型系统来研究 TMC4 和 TMC5 的渗透性，并进行免疫共沉淀测定来识别候选 TMC 结合伴侣。 我们计划与 Jung-Bum Shin (UVA) 合作，使用 TMC4 和 TMC5 单敲除和双敲除小鼠来检查 TMC4 和 TMC5 在 Corti 器官的毛细胞和支持细胞中的作用。 我们之前表明，静纤毛货物转运蛋白 MYO3A 在静纤毛尖端的出现与 MET 的发生一致。另一种静纤毛肌球蛋白 MYO7A 也显示出从尖端到基底的分布梯度，与该运动蛋白的向尖端易位和动态积累一致。基于这些观察，我们假设 MYO3A 和 MYO7A 将 MET 复合物的成分转运到静纤毛尖端，具有互补功能和/或冗余的潜力。在过去的一年中，我们完善了对这两种运动蛋白运输 PCDH15 同工型的能力的分析，PCDH15 同工型是 MET 装置的组成部分，参与静纤毛束的形成。 我们的新结果表明，MYO3A 具有货物选择性，其中 PCDH15-CD2 亚型是成熟 MET 复合物的组成部分，而 MYO7A 则运输 PCDH15-CD3 亚型，PCDH15-CD3 亚型先前位于发育中的毛束的静纤毛尖端。 我们还观察到，在存在 CD2 货物的情况下，丝状伪足尖端的 MYO3A 富集增加了近两倍，表明肌球蛋白活性存在货物依赖性调节。 在与 Jung-Bum Shin (UVA) 和 Anthony W. Peng（科罗拉多大学）的合作中，我们帮助证明了多种 MYO7A 亚型在小鼠耳蜗中表达。两种这样的亚型 MYO7A-C 和 MYO7A-S 是由替代转录和翻译起始位点产生的。 MYO7A-C 主要在 IHC 中表达，并在 OHC 中以音调梯度表达，并向耳蜗基底表达逐渐减少。这些结果与在 Myo7a::Actin-GFP 转基因小鼠中使用的 Myo7a-C 启动子驱动的肌动蛋白-GFP 表达水平的观察结果一致。 肌动蛋白-GFP 信号主要在 IHC 中观察到，在顶端 OHC 中检测到低水平，并向耳蜗基端音调减弱。 Myo7a-C 小鼠在 UTLD 和静纤毛基部表现出 MYO7A 水平降低，这与观察到的 Myo7a-C 毛细胞表现出 MET 静息通道开放概率降低和 MET 电流减慢相关。 总而言之，这项合作研究揭示了耳蜗中 MYO7A 表达的意想不到的亚型特异性差异，并强调了 MYO7A 在拉紧毛细胞 MET 复合体中的重要作用。 自发模式动作电位（AP）活动被认为对于哺乳动物感觉系统的正确发育很重要。然而，未成熟 IHC 中的 Ca2+ AP 活性与 ATP 依赖性细胞间 Ca2+ 信号传导（在大上皮嵴的耳蜗非感觉细胞中自发发生）之间的关系仍不清楚。我们对表达基因编码钙指示剂的小鼠 Corti 外植体器官进行了 Ca2+ 成像，并检测了 IHC 的内在自发活性。药理学实验证实了这种自发活动的独立性。这些数据已与 Walter Marcotti（谢菲尔德大学）和 Fabio Mammano（帕多瓦大学）实验室使用 Ca2+ 染料和电生理学进行的广泛成像数据相结合，形成一项综合研究。简而言之，在这项研究中，我们认为内在的自发 Ca2+ 活性以及 IHC 和非感觉细胞之间的相互影响相结合，形成了一种复杂的反馈机制，以控制 IHC 中的 AP 同步水平，并产生与听力开始前听觉通路细化相关的模式化活动。