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

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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.