Application of fluorescence recovery after photobleaching to study prestin lateral mobility in the human embryonic kidney cell

Abstract

The transmembrane protein prestin is crucial to outer hair cell (OHC) electromotility and contributes to the sensitivity and frequency selectivity of mammalian hearing. The molecular mechanisms of electromotility remain unclear, but prestin is purported to function as both a voltage sensor and a molecular motor. Understanding the role of prestin requires characterizing its organization and behavior in the plasma membrane. Fluorescence recovery after photobleaching (FRAP) provides a powerful means to quantitatively study molecular diffusion. However, OHCs are inherently fragile ex vivo, and dynamic studies of prestin require model systems, such as human embryonic kidney (HEK) cells, expressing fluorescently labeled prestin. Utilizing this system, we provide the first direct, quantitative measurement of prestin lateral mobility. The results show remarkably different diffusion behavior for prestin-green fluorescent protein (GFP) as compared to a control protein, human somatostatin receptor 5 (SSTR5). Prestin-GFP FRAP experiments reveal immobile fractions approaching 50%, low effective diffusion coefficients, and recovery times slower than those of SSTR5. Secondary bleaching of a region reveals distinctly different diffusion parameters, which we propose reflect the transient confinement of prestin in the HEK cell. Although uncharacterized, intermolecular interactions between prestin and the membrane and/or cytoskeleton may be important for the unique properties of prestin in electromotile OHCs.

Cochlear Outer Hair Cell Motility

“…In expression systems, such as in HEK cells, the lateral diffusion of GFP-labeled prestin appears, on the basis of FRAP experiments, to be considerably lower than expected for freely diffusing proteins (254). This study suggests that there may be intermolecular interactions between prestin and a lipid membrane and/or the underlying cytoskeleton, even in these cells.…”

Immune atomic force microscopy of prestin-transfected CHO cells using quantum dots

“…This discrepancy probably relates to the diffusibility of prestin in the plasma membrane. The prestin molecules are considered to be diffusible in the lateral plasma membrane of OHCs [34][35][36][37][38][39]. In addition, in between the intramembrane molecules, including prestin, there are some spaces [13][14][15][16][17][18].…”

Membrane Cholesterol Strongly Influences Confined Diffusion of Prestin

“…In addition, depletion of membrane cholesterol enhances the mechanical response of OHCs to electrical stimuli (19). Moreover, confocal fluorescence images of prestin-transfected cells show punctate foci in the membrane (16,17,20,21). Puncta reversibly disappear upon cholesterol depletion and colocalize with known lipid raft markers (16,17).…”

“…Early indications that prestin is mobile came from indirect electrophysiological measurements (24,25). Later, direct measurements of prestin diffusion employing fluorescence recovery after photobleaching (FRAP) revealed that immobile fractions remained after dual bleaches, suggesting that prestin is transiently confined (20). However, because FRAP is an ensemble measurement, a detailed analysis of the size and character of the putative confinements was not possible, and other, more-complex modes of diffusion could not be ruled out (20).…”