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

描述(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，导致对声音的感知。内毛细胞(IHC)中的特化带状突触负责向神经节神经元传递信号。这些带具有独特的能力，可以高速地将递质释放到突触间隙中，并且在钙离子刺激后没有疲劳的迹象。尽管小而紧凑，突触前复合体包含大量的蛋白质，参与运输、信号、支架和囊泡融合。在毛细胞中，非常规肌球蛋白在听力过程中发挥着关键作用，包括机械转导、支架、细胞内运输、细胞形状的维持，最近还与突触的成熟和功能有关。肌球蛋白6是唯一一种向肌动蛋白细丝负端移动的肌球蛋白，被认为是肌带成熟所必需的，它与囊泡蛋白Otoferlin的相互作用使其成为囊泡运输中的候选角色。它还运输不同细胞类型的囊泡，不依赖于耳铁蛋白。肌球蛋白6‘S步长较小，不适合长距离细胞内转运，但可能适合短距离运动。凭借我们的成像和电生理能力，以及分子生物学工具和动物基因敲除模型(Snell‘s Waltzer，SV)，我们计划扩大有关肌球蛋白6在内毛细胞带状突触的结构和功能中的适当作用的已知内容。要测试的主要问题是，肌球蛋白6是否在突触小泡运输中发挥积极作用，和/或它是否作为支架分子，促进带状复合体的组装和长期维持。通过使用肌球蛋白6基因敲除模型，参与条带结构和突触活动的蛋白质将在高分辨率共聚焦和超分辨率(STED)显微镜下进行分析。突触活性的电生理测量将用于评估肌球蛋白6丢失的功能后果。此外，我们将用最近描述的肌球蛋白6抑制剂(2，4，6-三碘苯酚，或TIP)处理急性解剖的耳蜗组织，并将其释放特性与基因敲除动物的释放特性进行比较。这将揭示突触表型是对SV小鼠的主要影响还是继发性影响，可能是由于发育异常。结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。我们预计将公布听力过程中这一关键步骤的更多细节，即由独特的条带介导的突触传递。

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.