Myosin 6 role in hair cells' ribbon synapse formation and vesicle trafficking

肌球蛋白 6 在毛细胞带状突触形成和囊泡运输中的作用

• 批准号: 8688575
• 负责人: Felipe Salles
• 金额: $ 16.31万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8688575
• 关键词: Acute; Binding; Biological Assay; Brain; Bypass; Calcium; Cell Shape; Cells; Cochlea; Collaborations; Complex; Coupling; Data; Data Analyses; Development; Docking; Electric Capacitance; Electrophysiology (science); Elements; Environment; Exhibits; Exocytosis; Experimental Designs; Fatigue; Fiber; Foundations; Functional disorder; Future; Ganglia; Glutamates; Goals; Hair; Hair Cells; Hearing; Image; In Vitro; Inner Hair Cells; Intracellular Transport; Knock-out; Knockout Mice; Label; Labyrinth; Lead; Learning; Life; Maintenance; Measurement; Mechanics; Mediating; Microfilaments; Microscopy; Minus End of the Actin Filament; Modeling; Molecular; Molecular Biology; Molecular Motors; Motor; Movement; Mus; Mutant Strains Mice; Myosin ATPase; Neurons; OTOF gene; Optics; Organelles; Peptides; Phase; Phenotype; Play; Potassium; Preparation; Process; Property; Proteins; Protocols documentation; Qualifying; Reporter; Research; Resolution; Role; Sensory; Signal Transduction; Solid; Speed; Stimulus; Structure; Synapses; Synaptic Transmission; Synaptic Vesicles; Techniques; Testing; Time; Translational Research; Transport Vesicles; Vesicle; career development; cell type; cellular imaging; deafness; diagnosis design; experience; inhibitor/antagonist; knockout animal; neuronal cell body; neurotransmitter release; novel; protein function; public health relevance; receptor; research study; ribbon synapse; scaffold; sensor; structural biology; synaptogenesis; tool; trafficking; transmission process; treatment strategy

DESCRIPTION (provided by applicant): The sensory units of the mammalian inner ear, the hair cells, convert mechanical stimuli into electric signals that lead to the perception of sound. The specialized ribbon synapses in the inner hair cells (IHC) are responsible for signal transmission to ganglia neurons. These ribbons have the unique ability to release transmitters into the synaptic space at high rates with no signs of fatigue after Ca2+ stimulation. Despite being small and compact, the pre-synaptic complex comprises a large number of proteins, involved in transport, signaling, scaffolding, and vesicle fusion. In hair cells, unconventional myosins play a critical role in hearing, in processes including mechanotransduction, scaffolding, intracellular transport, maintenance of cell shape, and, more recently, have been associated with the synapses maturation and function. Myosin 6, the sole myosin to move towards the minus ends of actin filaments, is hypothesized as necessary for ribbon maturation, and its interaction with the vesicle protein otoferlin makes it a candidate for a role in vesicle traffickig. It also transports vesicles in various cell types, independently of otoferlin. Myosin 6's step sizeis small and not suitable for long distance intra cellular transport but may be suitable for short-distance movements. With our imaging and electrophysiological capabilities, along with molecular biology tools and animal knockout models (Snell's Waltzer, sv), we plan to expand what is known regarding the proper role of myosin 6 in the structure and function of inner hair cell ribbon synapses. The main question to be tested is whether or not myosin 6 has an active role in synaptic vesicle trafficking, and/or if it acts as a scaffolding molecule, contributing to he assembly and long term maintenance of the ribbon complex. By using a myosin 6 knockout model, proteins involved in the ribbon structure and synaptic activity will be analyzed under high resolution confocal and super-resolution (STED) microscopy. Electrophysiological measurements of synaptic activity will be used to assess the functional consequence of myosin 6 loss. In addition, we will treat acutely dissected cochleae with a recently described myosin 6 inhibitor (2,4,6-triiodophenol, or TIP) and compare release properties with those of the knockout animals. This would reveal whether a synaptic phenotype is a primary or secondary effect on sv mice, perhaps due to a developmental abnormality. Coupling the imaging and electrophysiological approaches is fundamental for understanding how myosin 6 influences synaptic structure and activity in the inner hair cells. We expect to unveil more details of this crucial step in the hearing process, the synaptic transmission mediated by the unique ribbons.

描述（由申请人提供）：哺乳动物内耳的感觉单位，毛细胞，将机械刺激转化为电信号，导致声音的感知。内毛细胞（IHC）中的特化带状突触负责将信号传递到神经节神经元。这些丝带具有独特的能力，以高速率将递质释放到突触空间中，在Ca 2+刺激后没有疲劳的迹象。尽管突触前复合体小而紧凑，但它包含大量蛋白质，参与运输、信号传导、支架和囊泡融合。在毛细胞中，非常规肌球蛋白在听力中起关键作用，在包括机械传导、支架、细胞内运输、细胞形状维持的过程中，并且最近，与突触成熟和功能相关。肌球蛋白6，唯一的肌球蛋白向肌动蛋白丝的负端移动，被假设为带状成熟所必需的，它与囊泡蛋白otoferlin的相互作用使其成为囊泡traffickig中的一个角色的候选者。它还在各种细胞类型中转运囊泡，独立于otoferlin。肌球蛋白6的步长小，不适合长距离的细胞内运输，但可能适合短距离的运动。利用我们的成像和电生理学能力，沿着分子生物学工具和动物敲除模型（Snell's Waltzer，sv），我们计划扩展已知的关于肌球蛋白6在内毛细胞带状突触的结构和功能中的适当作用。要测试的主要问题是肌球蛋白6是否在突触囊泡运输中具有积极作用，和/或它是否充当支架分子，有助于带状复合物的组装和长期维持。通过使用肌球蛋白6敲除模型，将在高分辨率共聚焦和超分辨率（STED）显微镜下分析参与带状结构和突触活性的蛋白质。突触活动的电生理学测量将用于评估肌球蛋白6损失的功能后果。此外，我们将治疗急性解剖耳蜗与最近描述的肌球蛋白6抑制剂（2，4，6-triiodophenol，或TIP）和比较释放特性与敲除动物。这将揭示突触表型对sv小鼠的影响是主要的还是次要的，也许是由于发育异常。耦合成像和电生理方法是了解肌球蛋白6如何影响内毛细胞突触结构和活动的基础。我们期待着揭开听觉过程中这一关键步骤的更多细节，即由独特的丝带介导的突触传递。