Investigating the role of tip link biophysics on MET function

Project Summary: Broad Impact: Hair cells (HCs) are the sensory transduction cells of the auditory and vestibular systems. When a mechanical stimulus such as sound or linear, gravitational, or angular accelerations

are applied to these hair cells, small microvillus-like, actin-filled protrusions called stereocilia from the apical pole of the HCs are deflected. This causes filamentous tip link protein complexes at the tips of hair cell called stereocilia to pull upon and lead to the opening of mechanoelectrical transduction (MET) channel complex. The

opening of MET channels causes an influx of positively charged ions that depolarize the HCs, resulting in a variety of physiological processes from synaptic transmission to nerve fibers to physical changes in HC length by a phenomenon known as electromotility. However, there is a reigning question of the roles of the protein constituent of the tip links and the MET channel

complex is unclear. For example, whether tip links modulate the mechanical stimuli in HCs by acting as dampers, extra masses, or springs is yet to be discovered. These tip links are composed of four large extracellular peptide chains – two of protocadherin 15 (PCDH15) and two of cadherin 23 (CDH23). Due to limitations in heterologous

expression of MET channel complex proteins, MET must be studied in animal models. Recent gene therapy developments provide a great tool to study one component of tip links: PCDH15. Here, this project seeks to exploit this novel protein manipulation technique to study the biophysical properties of tip links and the influence

of these properties on the MET channel function. Aim 1: To uncover whether the length of the tip links is important for MET, MET function of miniaturized PCDH15s with shortened lengths will be compared to that of wild-type- like tip links. Aim 2: To reveal the role of Ca2+-binding site-mediated flexibility on the hair-cell MET function, tip

links formed by mutant PCDH15s will be compared against wild-type-like tip links using single-cell electrophysiology. Aim 2.1: Computational simulations that are used to predict protein dynamics will be used to estimate the flexibility of the tip links when specific Ca2+ binding sites are perturbed. Aim 2.2: MET currents of

different Ca2+-binding-site-perturbed mutant PCDH15s will be measured to reveal the effect of Ca2+-binding at specific sites on MET. Training: This project aims to forge the applicant into well-rounded auditory neuroscientist. In pursuit of this goal, the project will teach the applicant inner ear whole-cell hair-cell electrophysiology,

stereocilia bundle deflection, confocal microscopy imaging, animal handling, and in silico methods. Additional training will provide dedicated time to allow the applicant to grow his network with collaborations across many different laboratories. Together, this project seeks not only to develop a strong scientist, but also to increase our

collective knowledge on the biophysics underlying the hair-cell mechanotransduction function.