FIGURE 8.

Lipid rafts disruption with mβCD alters distribution and trafficking of GPR37, PS-TX14(A)^TAMRA, and the complex between GPR37^tGFP and PS-TX14(A)^TAMRA at the plasma membrane. A, comparison of FCCS curves for GPR37^tGFP and CTxB from untreated, mβCD-treated, or cholesterol oxidase-treated cells shows a significant cross-correlation (i.e. complex formation) between GPR37^tGFP and CTxB in control cells with 85% of the complexes diffusing at τ_D1 = 0.5 ms and 15% at τ_D2 = 0.2 s. This cross-correlation was largely disrupted by mβCD and cholesterol oxidase. B, FRAP measurements for GPR37^tGFP were performed on live cells (n = 23 cells for each treatment). Measurements were made on the same cell before and after treatment. Either mβCD or cholesterol oxidase increased GPR37^tGFP fluorescence recovery compared with untreated cells, and the average amplitude of the FRAP curves at t = 350 s was significantly higher after mβCD or cholesterol oxidase treatment than before (**, p < 0.01; ***, p < 0.001, Student's t test). C, comparison of FCCS curves for GPR37^tGFP and PS-TX14(A)^TAMRA averaged from three separate untreated, mβCD-treated, or cholesterol oxidase-treated cells shows that cholesterol depletion shifts the curve toward shorter diffusion times. In untreated cells, 75 ± 15% of the complexes diffused at τ_D1 = 1.2 ± 0.6 ms and 25 ± 15% at τ_D2 = 330 ± 200 ms. In mβCD-treated cells, 80 ± 12% of the complexes diffused at τ_D1 = 550 ± 250 μs and 20 ± 12% at τ_D = 29 ± 15 ms. In cholesterol oxidase-treated cells, 89 ± 5% of the complexes diffused at τ_D1 = 170 ± 40 μs and 11 ± 5% at τ_D2 = 15 ± 9 ms.