Investigating the role of the outer hair cell plasma membrane in electromotility and prestin function using quantitative optical microscopy techniques
Greeson, Jennifer N.
Doctor of Philosophy
Almost 25 years ago, the unique electromotile activity of mammalian outer hair cells (OHCs) was discovered. This cylindrical neuro-epithelial cell is capable of transducing changes in transmembrane potential into whole-cell axial deformations. The resulting contractions and elongations feedback into the sound-induced vibrations of the basilar membrane, enhancing hearing sensitivity and frequency detection capabilities. Although the complete mechanism of electromotility is not presently understood, the discovery and localization of the putative motor protein, prestin, to the OHC plasma membrane promotes the importance of the membrane in both OHC and cochlear behavior. A thorough understanding of the constituents of the OHC membrane, both lipids and proteins, as well as their interactions, is then crucial to current and future analyses of OHC function. In this work, we describe the use of quantitative optical microscopy to investigate protein-protein and membrane-protein interactions. For the former, we employ fluorescence resonance energy transfer (FRET) microscopy to assess prestin-prestin interactions in a heterologous expression system. For the latter, we have developed a specific implementation of fluorescence polarization microscopy (FPM) to measure the orientation of the fluorescent lipid analog di-8-ANEPPS in OHCs. Using both of these techniques, we can investigate the effects of treatments known to affect prestin function and non-linear capacitance in a membrane-dependent manner. The results undeniably highlight the importance of the plasma membrane in regulating prestin and OHC electromotile function.