Investigating the lateral mobility of outer hair cell plasma membrane constituents by fluorescence recovery after photobleaching
Organ, Louise E.
Raphael, Robert M.
Doctor of Philosophy
Mammalian hearing exhibits exquisite sensitivity and frequency selectivity attributed to the unique properties of cochlear outer hair cells (OHCs). These sensory epithelial cells are electro-mechanical transducers, capable of converting sound-induced electrical signals into mechanical forces which provide feedback via a mechanism known as the cochlear amplifier. In a process aptly termed electromotility, electro-mechanical transduction manifests as whole-cell axial length changes in OHCs that occur in response to changes in the transmembrane potential. The polytopic motor protein prestin functions as the voltage sensor and molecular motor, both in OHCs and when expressed in heterologous systems. As the molecular mechanism(s) of electromotility remain unknown, examining the structure and function of prestin is a major focus of ongoing research. Since changes in membrane composition and biophysical properties affect protein function and organization, we are particularly interested in membrane-protein interactions. Recent studies suggest that manipulations in membrane cholesterol levels reversibly shift the membrane microdomain distribution of prestin, modulate prestin oligomerization states, and alter prestin function, thus regulating electromotility through membrane-protein interactions. Measurements of protein and lipid lateral mobility provide a powerful tool to dynamically examine such interactions. We hypothesize that OHC plasma membrane cholesterol levels affect electromotility either through microdomain-mediated mechanisms that cluster or segregate prestin molecules or via alterations in the material properties of the membrane, which in turn affect the resident proteins. Using fluorescence recovery after photobleaching (FRAP), we evaluated the lateral mobility of both protein and lipid components of the OHC. Then we showed that the diffusion of both prestin in HEK cells and lipids in OHCs is altered in response to changes in membrane cholesterol concentration. Cumulatively, this work demonstrates the complexity of prestin-membrane interactions and highlights the importance of their inclusion in current models of prestin function and electromotility.