UNIVERSITY OF KENTUCKY
The goal of this ARRA supplement is to obtain resources that would allow us to follow regeneration of tip links in a live mammalian hair cell in real time. The parent grant (R01 DC008861) proposed this study as a part of Specific Aim #3, but using scanning electron microscopy, i.e. in dead (fixed and dried) cells. Now we have developed a technique that allows visualization of stereocilia links in physiological conditions (Novak et al., Nat. Methods, 6(4): 279-281; 2009). The requested resources under this supplement will be used to upgrade the existing hardware, which would allow time-lapse imaging of stereocilia links in live hair cells. The resources will be also used to hire temporal personnel for development of custom image acquisition and processing software, which would optimize stereocilia link imaging. Disruption of tip links during intense acoustic stimulation is thought to be one of the early events underlying temporal shift of hearing thresho lds. This temporal shift often progresses into the permanent noise-induced hearing loss. Therefore, our study may provide an insight into the mechanisms of this very common hearing impairment. Overall, the requested resources would allow achieving the goals of the parent grant, but at a new technological level. In the future, this technological advancement will allow studying the function of individual nanoscale surface structures (such as individual membrane proteins or protein complexes) in a living cell. The technique that we have developed is not yet commercially available and, therefore, the proposed supplement would contribute to the development of the research infrastructure in the US.