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First published online April 28, 2005
doi: 10.1242/10.1242/jcs.02300

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Single-molecule diffusion measurements of H-Ras at the plasma membrane of live cells reveal microdomain localization upon activation

¹ Department of Biophysics, Leiden Institute of Physics, Leiden University, Niels Bohrweg 2, 2333 CA Leiden, The Netherlands
² Department of Molecular Cell Biology, Institute of Biology, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands

View larger version (36K): [in a new window]   Fig. 1. (A) Images of the localization of eYFP-H-Ras in transiently transfected HEK293 cells. Confocal images of HEK293 cells transiently expressing eYFP-H-Ras(N17), eYFP-H-Ras(V12) and eYFP-H-Ras(wt). Images were taken 48 hours after transfection. Fusions between eYFP and the wild-type H-Ras as well as both mutants of H-Ras showed clear plasma membrane localization, indicating that membrane targeting was not impaired by the eYFP fusion to H-Ras. Scale bar: 10 µm. (B) Insulin-induced GTP loading of eYFP-H-Ras(wt). Active Ras was specifically pulled-down in an RBD assay, separated on a PAA-gel, blotted and detected by anti-Ras antibody (see Materials and Methods). 3T3-A14 cells were transiently transfected with Ras membrane anchor (10 C-terminal amino acids) fused to eYFP (mt), eYFP-H-Ras(wt), eYFP-H-Ras(V12), or eYFP-H-Ras(N17). Results are shown for unstimulated (–) and 5-minute insulin-stimulated (+) conditions. A clear increase of GTP loading was observed for wild-type eYFP-H-Ras (50 kDa) after stimulation, as well as for endogenous Ras (21 kDa). (C) Activation of extracellular regulated kinases (ERKs) in the presence of eYFP-H-Ras(wt). Results are shown for unstimulated (–) and 5-minute insulin-stimulated (+) conditions. To show that the fusion of eYFP to H-Ras(wt) did impair downstream signaling, the phosphorylation of the MAPK-kinases ERK1 (44 kDa) and ERK2 (42 kDa) was tested. Activation of Ras by insulin, preparation of the cell lysates, SDS-PAGE and blotting were performed as described in Materials and Methods. The left part of the immunoblot (anti-ERK) shows total ERK1 and ERK2 in cell lysates of 3T3-A14 cells transiently transfected with eYFP-H-Ras(wt). The phosphorylation of ERK1 and ERK2 upon insulin addition is shown on the right (anti-ERK-P). The phosphorylation of the amino acids Thr202 and Tyr204 of ERK1 and ERK2 was specifically detected using a phospho-p44/42-MAPK antibody (Cell Signaling Technology, Beverly, MA, USA).

View larger version (35K): [in a new window]   Fig. 2. Tracking individual eYFP-H-Ras(wt) molecules in the plasma membrane of live cells. (A) Image showing three single-molecule signals of eYFP-H-Ras(wt) molecules at the dorsal plasma membrane of a 3T3-A14 cell. The image was taken using 514 nm illumination for 3 mseconds at 2 kW/cm2. Scale bar: 1 µm. (B) Example of a single-step photobleaching event of an individual eYFP-H-Ras(wt) molecule. (C) Probability density of the single molecule fluorescence intensity of eYFP-H-Ras(wt). Analysis of the signals of 3028 individual eYFP-H-Ras(wt) molecules at the dorsal plasma membrane of 3T3-A14 cells (solid line) in unstimulated conditions. The probability density of the fluorescence intensity is nearly Gaussian-shaped with a maximum of 204 counts/3 milliseconds. Statistics of the background signal (dashed line) is shown for comparison. The backgound signal had a maximum at 14 counts/3 milliseconds and a width of =10 counts/3 milliseconds. These values translate into a signal to background noise ratio of 20. (D) Trajectory of an eYFP-H-Ras(wt) molecule diffusing in the dorsal plasma membrane of a 3T3-A14 cell. The time between subsequent steps was 10 milliseconds.

View larger version (31K): [in a new window]   Fig. 3. Square displacement analysis of the different eYFP-H-Ras constructs. Cumulative probability, P(r2, tlag), plotted versus the square displacements, r2. (A) Cumulative probability, P(r2, 50 mseconds) distributions of the inactive mutant eYFP-H-Ras(N17) (red dots) and the active mutant eYFP-H-Ras(V12) (green squares). These distributions were fitted to the one-component model (Eqn 1) or two-component model (Eqn 2). (B,C) Show the distributions and the results of the fits to the one-component (dashed line) and two-component (solid line) model. (D) Cumulative probability, P(r2, 60 mseconds) distributions of wild-type eYFP-H-Ras(wt) before (red triangles) and after 5 minutes insulin stimulation (green triangles). (E,F) The distributions and the results of the fits to the one-component (dashed line) and two-component (solid line) model. B,C,E and F clearly show that the two-component model describes the cumulative probability distributions significantly better than the one-component model. For all cumulative probability distributions of H-Ras that were obtained and analyzed in this study, the two-component model yielded significantly better fit results than the one-component model.

View larger version (21K): [in a new window]   Fig. 4. Diffusion characteristics of eYFP-H-Ras(N17) and eYFP-H-Ras(V12). Fitting of the square displacement distributions (Fig. 3) to Eqn 2 (Materials and methods) yielded a fraction of fast-diffusing molecules, , and corresponding characteristic mean square displacements for the fast (r12) and slow (r22) diffusing population of molecules for each t. A and D, B and E, C and F show plots of , r12 and r22, respectively, versus tlag for both the N17 and V12 mutants. (A,D) The solid lines show the weighted mean of the fast fraction of molecules over all timelags. (B,E) Mean square displacement data of the fast-diffusing fractions were fitted according to a free diffusion model (r12=4D1tlag, solid line). (C,F) Mean square displacement data of the slow-diffusing fractions were fitted according to a free (C) and confined (F, Eqn 3) diffusion model (solid lines). The dotted lines in C and F represent the offset due to the limited positional accuracy (see Materials and Methods), although the same offset is present in B and 4E, the dotted lines were omitted here for clarity.

View larger version (26K): [in a new window]   Fig. 5. Diffusion characteristics of eYFP-H-Ras(wt) before stimulation and 5, 10 and 15 minutes after stimulation with insulin. (A,D,G,J) Fast-diffusing fraction of molecules, , versus timelag. The solid lines show the weighted mean of the fast fraction of molecules over all timelags. (B,E,H,K) Mean square displacement of the fast-diffusing fraction, r12, plotted versus timelag. The data were fitted according to a free diffusion model (r12=4D1tlag, solid line). (C,F,I,L) Mean square displacement of the slow-diffusing fraction, r22, plotted versus timelag. The data in C was fitted to a free diffusion model (solid line); the data in F,I,L were fitted according to a confined diffusion model (Eqn 3, solid line). The dotted lines in the plots of the bottom row represent the offset due to the limited positional accuracy (see Materials and Methods); although the same offset is present in the plots of the middle row, the dotted lines were omitted here for clarity.

View larger version (52K): [in a new window]   Fig. 6. (A) Schematic showing the differences in diffusion and domain localization between the inactive eYFP-H-Ras(N17) and active eYFP-H-Ras(V12) mutants. For both mutants the fast-diffusing fraction was freely diffusing (blue), but its size and diffusion coefficient were significantly smaller for the active H-Ras mutant than the inactive mutant. Also, the slow-diffusing fraction of the inactive mutant was freely diffusing (green), whereas it was confined in 200 nm domains for the active mutant (red). (B) Model depicting the effect of H-Ras(wt) activation on its diffusion characteristics. Before stimulation (left part) slow (25%, green) and fast (75%, blue) diffusing populations of H-Ras(wt) molecules were present, that were both freely diffusing. Activation of H-Ras(wt) by addition of insulin for 5 minutes resulted in the confinement of the slow-diffusing fraction in domains (red) with a diameter of 200 nm.

View larger version (14K): [in a new window]   Fig. 7. Mean square displacement of the fast fraction of the eYFP-H-Ras(V12) (A) and 15-minute stimulated eYFP-H-Ras(wt) (B) constructs. The dashed lines resulted from fits to the free diffusion model (r12=4D1tlag), the resulting diffusion coefficients (D1) are given in Table 1. The solid lines represent fits to the anomalous diffusion model: r12(tlag). The resulting anomalous diffusion exponents, , were 0.89±0.08 for eYFP-H-Ras(V12) (A) and 0.81±0.12 for 15 minute-activated eYFP-H-Ras(wt) (B), respectively.