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: 9354093
• 负责人: BECHARA KACHAR
• 金额: $ 142.1万
• 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/9354093
• 关键词: ATP phosphohydrolase; Actins; Adult; Affect; Affinity; Animals; Auditory; Bilateral; Binding; Biochemical; Brazil; Calcium; Cations; Cell membrane; Cells; Chimera organism; Collaborations; Complex; Development; Dimensions; Dominant-Negative Mutation; Drosophila genus; Electrophysiology (science); Elements; Excision; Exhibits; Family; Filopodia; Financial compensation; Generations; Goals; Hair; Hair Cells; Hearing; Human; Image; Individual; Inherited; Inner Hair Cells; Invertebrates; Knock-out; Knockout Mice; Laboratories; Labyrinth; Length; Light; Liquid substance; Location; Maintenance; Mechanics; Mediating; Molecular; Motor; Motor Activity; Mus; Mutation; Myosin ATPase; Myosin III; Nature; Organ; Organ of Corti; Outer Hair Cells; Pattern; Phenotype; Physiologic pulse; Preventive Intervention; Process; Property; Protein Isoforms; Proteins; Protocols documentation; Regulation; Reporting; Research; Resolution; Role; Sampling; Sensory; Series; Stereocilium; Structure; System; Tail; Testing; Therapeutic Intervention; Tissues; Transgenic Mice; Transport Process; Utricle structure; Vertebrates; Visual; base; calcium indicator; cell motility; channel blockers; deafness; early onset; exome sequencing; gene product; hearing impairment; interest; knockout animal; member; mutant; novel; paralogous gene; programs; protein complex; research study; response; segregation; self-renewal; sensory system; spatiotemporal

Myosin III (MYO3) proteins are associated with the auditory and visual sensory systems in both invertebrates and vertebrates. In Drosophila, MYO3 (NINAC) is responsible for the transport and localization of the photo-transduction machinery. In humans, mutations in MYO3A cause non-syndromic progressive hearing loss. Our lab focuses on studying the interrelated functions of two MYO3 paralogs, MYO3A and MYO3B and their associated cargoes in regulating structure and mechanosensitivity of the actin-based stereocilia. MYO3A differs from MYO3B in that it contains an extended tail domain with an additional actin binding motif. In collaboration with Chris Yengo's lab (PSU) we examined how the motor and tail domains of these two myosins influence the formation and elongation of actin protrusions. MYO3A is a two-fold faster motor with enhanced ATPase activity and actin affinity. A chimera in which the MYO3A tail was fused to the MYO3B motor demonstrated that motor activity correlates with formation and elongation of actin protrusions. Removal of MYO3A tail abolishes the ability to enhance actin protrusion formation and elongation. MYO3A also slows filopodia dynamics and enhances filopodia lifetime. We conclude that the unique enhanced motor activity in combination with its extended tail actin-binding motif favors MYO3A in its role in elongation and maintenance of actin protrusions. Whole-exome sequencing of samples from affected members of a Brazilian family with bilateral late-onset non-syndromic hearing-loss performed by the laboratory of Regina C. M. Neto (USP, Brazil), revealed a novel mutation (T2090G) in MYO3A. Segregation studies confirmed the presence of the mutation in 25 affected individuals from seven-generations of the same family, showing autosomal dominant inheritance. The mutation alters a conserved residue (L697W) in MYO3A motor domain. Biochemical studies from the Yengo's lab shows that the mutation causes a reduction in motor ATPase and motility of MYO3A. We further demonstrate that the mutant MYO3A causes reduced actin protrusion formation in COS7 cells. Coexpression of L697W and wild-type MYO3A in hair cells and COS7 cells in the presence of the MYO3A cargo ESPN1, revealed a predominant tipward accumulation of L697W at the stereocilia and filopodia. We argue that the higher duty ratio of L697W may mediate a dominant negative effect caused by the enhanced stability of a slower motor (L697W) at actin protrusion tips. We propose that L697W with reduced motor activity may impact stereocilia length regulation, cargo transport, and mechanotransduction. Our results shed light on the dominant phenotype of a novel, late onset-deafness causing, MYO3A mutation. Hair cells tightly control the dimensions of stereocilia to form a precisely regulated staircase pattern that is essential for normal mechanotransduction. We observed that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, in collaboration with Peter Barr-Gillespie's lab (OHSU) we performed a series of experiments that show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell. To determine the compensation between ESPN-1 and ESPNL on stereocilia length regulation, we made an ESPN-1/ ESPNL double knockout mouse. In this mouse the stereocilia bundles in the utricle are normal, but those in the organ of Corti have very short lower two rows of stereocilia. This phenotype is most severe in outer hair cells. These shortened stereocilia are present at P6 or before and persist into adulthood. ESPN-1 knockouts have similar mechanotransduction (MET) currents to wildtype while the ESPNL knockout had 40% and double knockout had 12% of control MET. While the stereocilia length regulation in the double knockout is compromised, the MET machinery appears intact. The differences in phenotype between the utricle, outer hair cells and inner hair cells suggest that stereocilia length regulation is a complex process that differs between type of hair cell and includes but is not limited to the MYO3/ESPN/ESPNL system. We continue to investigate the localization and molecular nature of the mechanosensitive channel complexes in hair cells. Previous reports and results from our laboratory have shown that there are multiple proteins expressed in hair cells that are essential for mechanotransduction (MET). It has been shown that deletion of TMIE and TMC proteins completely eliminates conventional MET in hair cells. However, even in the absence of conventional MET, hair cells are still capable of reverse polarity mechanotransduction. The molecular nature of the MET channel has long been elusive and it is still not clear whether TMIE or TMCs can form the actual channel pore. Therefore, we developed a stimulation and recording electrophysiology protocol to detect mechanically activated currents in HEK293T cells transfected with the candidate proteins. As positive control, we used cells transfected with Piezo 1, a protein that has been reported to be important in mechanosensitivity. We show that cells transfected with Piezo 1 respond with large, mechanically activated currents upon mechanical perturbation/stimulation of the cell membrane. However, we were unable to evoke mechanically activated currents in cells transfected with TMIE, TMC1, TMC2, TMC4, or combinations of these proteins. Further examining the role of TMIE in MET, we have shown that full deletion of this protein completely eliminates conventional MET from hair cells. However, all tested hair cells exhibited reverse polarity MET upon sine wave stimulation or square pulse stimulation with fluid jet. These results confirm that, like TMC1, TMIE is absolutely necessary for conventional MET, but it is not clear if it is part of the pore forming element of the MET channel, or if it interacts with TMC and/or other proteins to form the MET complex as previously suggested. Investigating the developmental regulation of the reverse polarity MET in TMIE knockout animals, we discovered that these reverse polarity responses can be evoked in hair cells before the onset of hearing (P2-P9). However, they significantly decline and are almost entirely absent or very difficult to evoke in animals older than P7. We are currently examining the spatiotemporal localization of TMIE, P2X2, Piezo 1, Piezo 2, TMC4 and TMC5 using transgenic mice expressing GFP and mCherry -tagged versions of these candidate MET channel complex proteins. The MET channel is a large conductance, non-specific cation channel highly permeable to calcium. Using high spatiotemporal resolution calcium imaging in cultured inner ear tissue from mice expressing the calcium indicator GCaMP3 we observe widespread spontaneous calcium transients in stereocilia in both pre-hearing (P3-P10) and mature (P17-P21) hair cells. Application of MET channel blocker dihydrostreptomycin eliminated all calcium transients observed in both auditory and vestibular stereocilia. More importantly, in the organ of Corti, this treatment also eliminated calcium transients from the tallest rows of stereocilia, confirming our earlier findings that MET-like channels are present in previously unrecognized locations within the hair bundles.

肌球蛋白III （MYO3）蛋白与无脊椎动物和脊椎动物的听觉和视觉感觉系统有关。在果蝇中，MYO3 （NINAC）负责光转导机制的运输和定位。在人类中，MYO3A突变导致非综合征性进行性听力损失。本实验室主要研究两种MYO3类似物MYO3A和MYO3B及其相关物质在调节肌动蛋白基立体纤毛结构和机械敏感性中的相关功能。MYO3A与MYO3B的不同之处在于，它包含一个带有额外肌动蛋白结合基序的扩展尾结构域。在与Chris Yengo实验室（PSU）的合作中，我们研究了这两种肌凝蛋白的运动和尾部结构域如何影响肌动蛋白突起的形成和延伸。MYO3A是一种速度快两倍的马达，具有增强的atp酶活性和肌动蛋白亲和力。MYO3A尾巴与MYO3B马达融合的嵌合体表明，运动活动与肌动蛋白突起的形成和延伸相关。MYO3A尾部的去除消除了增强肌动蛋白突起形成和延伸的能力。MYO3A还能减缓丝状足的动力学并延长丝状足的寿命。我们得出的结论是，独特的增强运动活性及其延伸的尾部肌动蛋白结合基序有利于MYO3A在肌动蛋白突起的延伸和维持中的作用。