Characterization of Prestin Oligomerization and Diffusion At the Single Molecule Level

Abstract

Prestin is the putative motor protein that drives outer hair cell electromotility. Several groups have shown evidence of prestin-prestin interactions and prestin oligomerization using biochemical techniques and optical imaging. Furthermore, studies have demonstrated that alterations of cholesterol in the cell membrane, which modify prestin function, affect membrane fluidity and prestin mobility. However, the role prestin-prestin interactions or oligomerization play in electromotility and the molecular motifs that mediate these interactions are unknown. It is also unknown whether the effects of cholesterol modifications on prestin function are due to changes in prestin mobility or changes in prestin interactions. Ensemble optical measurements of prestin self-association cannot provide the molecular level details of prestin-prestin interactions, nor can they distinguish the oligomeric states the interactions produce. Likewise fluorescence recovery after photobleaching cannot elucidate non-Brownian modes of diffusion which could indicate interactions with the cytoskeleton. We have thus developed our ability to detect individual prestin-citrine molecules using single molecule fluorescence (SMF) microscopy. We have applied SMF to measure the distribution of intensities emitted by individual prestin clusters in the HEK cell membrane. The distribution displays peaks spaced at multiples of the unitary intensity which one would expect for a distribution of non-interacting fluorescent emitters. We have resolved the stoichiometries up to tetramers, however the data makes clear that higher populations also exist. We have also explored the effect of membrane cholesterol depletion on the oligomerization of prestin using SMF microscopy to asses whether this treatment dissociates prestin oligomers or simply removes a bulk population of intact oligomers from microdomains. We will further utilize total internal reflection fluorescence microscopy and site-directed labeling to measure prestin diffusion at the single molecule level and explore the effects of cholesterol depletion and cytoskeletal inhibitors.