Figure 4.

Diffusion of fluorescent proteins is dependent on their cellular scaffolds or relation with membrane environment. (a) GFP-Ga_i1 was stably expressed in a C6 glioma cell line and FRAP was used to assess the mobility of GFP-Ga_i1 after antidepressant treatment. Half-time to recovery of GFP-Ga_i1 is unaffected after chronic (3-day) escitalopram and fluoxetine treatments. Colchicine and methyl-b-cyclodextrin are presented as positive controls of cytoskeletal and membrane disruption on G protein distribution. (b) C6 glioma were transiently transfected with various fluorescent fusion proteins and FRAP was performed 24 h after transfection. Half-time of recovery was faster for peripheral membrane and cytosolic proteins relative to transmembrane proteins. (c) FRAP was performed on cells expressing GFP-Gα_s under a variety of conditions that alter Gα_s membrane association. Cytosolic GFP-Gα_s, whether ‘normal' or resulting from a mutation (C3S) that blocks palmitoylation (and subsequently, membrane attachment) shows significantly faster half-time to recovery. Furthermore, agents that remove Gα_s from membrane, either by blocking palmitoylation (2-bromopalmitate) or by activation and subsequent internalization (isoproterenol) also enhance FRAP recovery. Data were analyzed by one-way ANOVA followed by Tukey's test for post hoc multiple comparisons of means (control versus treatment, *p<0.05, **p<0.01, ***p<0.001). Error bars represent SEM.