Experimental method for determining Rac dissociation rate from the membrane. (A) Representative GFP-wtRac–expressing cells, either 30 min (left) or 12 h (right) after replating on fibronectin. The red lines delimit the photobleached areas. (B) Cell in A at the indicated times during the photobleaching protocol. (C) Normalized fluorescence in the unbleached region (protrusive or quiescent) of individual cells expressing GFP-wtRac and the unbleached and bleached region of cells expressing GFP-PBR/C189SRac, plotted as a function of time. Lines are the best-fit curves to Equation (4). (D) Average best-fit curves to Equation (4) for protrusive (n = 11) and quiescent (n = 5) unbleached regions of cells expressing GFP-wtRac. Values are means ± SD. (E and F) Apparent k_off and r values for GFP-wtRac in protrusive (n = 11) and quiescent (n = 5) areas of cells. Values are means ± SD (∗p <; 0.05 by Student's t test). (G) Fluorescence intensity as a function of time (relative to time 0) in the absence of photobleaching. Values represent the mean of three independent experiments ± SD. (H) Apparent k_off in protrusive areas of cells expressing low (n = 8), medium (n = 11), and high (n = 10) levels of GFP-wtRac. Levels of expression were monitored by the total fluorescence intensities across the cell and normalized to the lowest value. k_off values are the means ± SD.

Effect of Rac regulators on membrane targeting of GFP-Rac. (A) Postnuclear lysates from NIH3T3 cells expressing the indicated constructs were separated into membrane (M) and cytosolic (C) fractions. Equal percentages of cytosolic and membrane material were resolved by SDS-PAGE and immunoblotted for GFP (Rac), and integrin β1 and RhoGDI as membrane and cytosol markers, respectively. (B) Percentage of GFP-Rac in the membrane fraction; values are means ± SD (n = 3).

Role of RhoGDI in Rac–membrane dissociation. (A and B) k_off values (A) and r values (B) of GFP-wtRac (•) or GFP-G12VRac (○) with increasing RFP-RhoGDI as determined by total RFP intensity. ▴ and ▵, the mean k_off and r values ± SD of GFP-wtRac and GFP-G12VRac alone, respectively, as in Figure 4, C and D. (C) Cells were electroporated with pSUPER RNAi constructs for RhoGDI or control sequences. Total lysates were analyzed by immunoblotting for RhoGDI and RhoGDI2. The antibody to RhoGDI2 cross-reacts with RhoGDI, but RhoGDI2 runs at a lower position on the gel (arrow). Equal loading was verified by immunoblotting with anti-actin. Data are representative of at least three independent experiments. (D) Average best-fit curves to Equation (4) from cells expressing GFP-wtRac in the presence (n = 8) or absence (n = 11) of RhoGDI RNAi. Values are means ± SD. (E and F) k_off (E) and r (F) values in protrusive areas of cells expressing GFP-wtRac alone (n = 11) or GFP-wtRac in the presence of RhoGDI RNAi (n = 8). Values are means ± SD (∗p <; 0.05 by Student's t test).

Estimation of Rac membrane diffusion rate constant. (A) Fluorescence intensity in the bleached and unbleached regions of individual cells expressing GFP-G12VRac. The width of the unbleached area was varied as indicated in the inset. Values are relative to t = 0. (B) Apparent k_off values for GFP-G12VRac as a function of the width of the unbleached area. (C) Postnuclear lysates of NIH3T3 cells expressing GFP or Fyn-GFP were fractionated into membrane (Memb.) and cytosolic (Cyt.) fractions. Equal percentages of cytosolic, membrane, and total material were resolved by SDS-PAGE and immunoblotted for GFP, integrin β1, and RhoGDI. (D) Normalized fluorescence intensity in the unbleached region (8-μm width) of a representative cell expressing Fyn-GFP. Line is the best fit to a single exponential function. The average membrane diffusion rate constant (k_diff) was 0.017 s⁻¹ (n = 13). (E) Computer simulations of the effect of membrane diffusion on fluorescence decay in the unbleached region. Cell geometry and the masked region (in red): cell diameter is 52 μm, mask width is 8.5 μm, and mask arc length is 42 μm (left). Fluorescence distribution at 22.5 s into bleaching simulated on the same domain (middle). A color scale with the equivalent numerical values is displayed (right). The simulated bleaching rate is 1 s⁻¹, and the protein diffusion coefficient is 0.25 μm²/s. (F) Normalized decay of protein concentration (fluorescence) averaged over the unbleached region. The simulation parameters are as in E. The best fit to a single exponential function Ae^−k_difft yields A = 0.95 and k_diff = 0.016 s⁻¹. (G) Dependence of the simulated decay rate constant due to membrane diffusion (k_diff) on the width of the unbleached region. k_diff values were derived as in F. The arc length was held constant at 42 μm. The estimated relative numerical error in the presented data is 5%.

Role of GAPs and GEFs on Rac–membrane dissociation. (A) Fluorescence decay in the unbleached region of representative individual cells expressing GFP-wtRac, alone or coexpressed with active Tiam1, or GFP-G12VRac. Lines are the best-fit curves to Eq (4). The width of the unbleached region was 8 μm. (B) Best-fit curves to Eq (4) averaged from multiple cells as in A. Values are means ± SD (n = 11–13). (C–D) Average k_off (after correcting for membrane diffusion) and r values in protrusive areas of cells expressing GFP-wtRac (n = 11), GFP-wtRac and active Tiam1 (n = 13) or GFP-G12VRac (n = 12). Error bars: SD (∗p <; 0.05, ∗∗p <; 0.01 by Student's t test). (E) k_off for GFP-wtRac as a function of RFP-Q61LRac (filled circles) determined from total RFP intensity (arbitrary units). Open circle represents the mean k_off ± SD of GFP-wtRac alone as in C. (F) Cytosolic fractions of cells transfected with GFP-wtRac, GFP-G12VRac, GFP-Q61LRac or empty vector were subjected to immunoprecipitation with anti-GFP and immune complexes were resolved by SDS-PAGE and blotted for GFP and RhoGDI. Equal loading was verified by immunoblotting the cytosolic fractions (Total) for RhoGDI. Data are representative of three independent experiments.