Discrepancy between fluorescence correlation spectroscopy and fluorescence recovery after photobleaching diffusion measurements of G-protein-coupled receptors

Abstract

Fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) are the two most direct methods to measure the diffusion of molecules in intact living cells. Ideally, these methods should produce similar results for an identical system. We have used these methods to monitor the diffusion of two G-protein-coupled receptors and their associated proteins in the plasma membranes of cells that do not or do contain invaginated protein domains called caveolae. FRAP studies show that caveolae domains increase the immobile fraction of receptors without significantly changing their mobility. On the other hand, FCS studies show an unexpected increase the mobility of caveolae-associated proteins. Our data suggest that the geometry of caveolae domains gives rise to a confined diffusion of its attached proteins, resulting in an apparent increase in mobility.

Keywords: Caveolae; Fluorescence correlation spectroscopy; Fluorescence recovery after photobleaching; G-protein-coupled receptors; Protein diffusion.

Fig. 1

(A) Example of the distribution of Cav1–eGFP in the Z direction expressed in an FRTwt cell. (B) Corresponding image of the cell. (C) Expanded view of a region of the image in black and white and binary depiction. (D) Cartoon depicting caveolae in an FCS-based illuminated measurement.

Fig. 2

FRAP studies of Cav1–eGFP (A), membrane marker–eYFP (Clontech) (B), and G_αq–eGFP (C) diffusing in the basolateral membrane of FRTwt and FRTcav+ cells. The open and closed circles are for data taken in FRTwt and FRTcav+ cells, respectively, where n = 10 to 13 (see Table 1). The data shown are average values and standard errors.

Fig. 3

(A and B) FRAP studies of B₂R–GFP (A) and μOR–GFP (B) diffusing in the basolateral membrane of FRTwt cells (●) and FRTcav+ cells (○), showing compiled data (top) and sample images (bottom), where n = 12 and 13 (see Table 1) and the arrow points to the bleached spot. (C) Comparison of mobile fractions in FRTwt and FRTcav+ cells obtained from FRAP curves in Fig. 5A below. Data are shown with standard errors. Because the data have a normal distribution as determined by a Shapiro–Wilk test, a Student t test was performed between FRTwt and FRTcav+ for each membrane protein. An asterisk indicates statistical difference in the mobile fractions of membrane proteins between FRTwt and FRTcav (P < 0.001).

Fig. 4

FRAP studies of B₂R–GFP (A) and μOR–GFP (B) diffusing in the basolateral membrane of FRTwt and FRTcav+ cells where the size of the bleach spot was varied. Although the calculated diffusion coefficient decreased approximately 3-fold with bleach size, accompanied by a decrease in mobile fraction, no significant differences between mobility or mobile fraction for either receptor in FRTwt and FWTcav+ were found.

Fig. 5

Representative FCS curves of B₂R–GFP in FRTwt and FRTcav cells showing the fit and residuals for each curve.

Fig. 6

Distribution of diffusion coefficients extracted from FCS data showing the broadening toward slower coefficients for membrane marker (E), similar coefficients for μOR (B) and G_αi (D), and faster values for B₂R–GFP (A) and G_αq (C) in the presence of caveolae. Although the curves are fit to Gaussian distributions, no model is intended.

Fig. 7

Cartoon describing our proposed model of B₂R diffusion in the presence of caveolae. (A) B₂R (orange rectangle), with its attached G_αq (green triangle), is transiently confined to diffuse on the periphery of caveolae (small blue circle) due to interactions between Cav1 and G_αq. (B) Cartoon depicts the illumination diameter in an FCS measurement focused on a plasma membrane region rich in caveolae (blue dots). (C) Side view of a caveolae domain in which B₂R (orange rectangle) with its attached G_αq (green triangle) diffuses on the membrane until it encounters a caveolae invagination. The G protein interacts with the caveolin proteins while the receptor remains bound. It is possible for G_αq to diffuse on the surface of the caveolae domain, becoming detached from the receptor, which does not enter the domain. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)