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. 2004 Jun;86(6):3993–4003. doi: 10.1529/biophysj.103.038422

FIGURE 5.

FIGURE 5

Rev-CRM1 dissociation. (A) Time series of two-dimensional confocal sections of a cell cotransfected with CRM1 and Rev fused to YFP (red) and CFP (green), respectively. (B) The corresponding estimate of colocalized pixels (shown in white). (C) The corresponding two-dimensional histograms for each image in A. The red channel (CRM1) is on the x axis and the green one (Rev) on the y axis in the two-dimensional histogram. Selected colocalized areas are also shown by yellow transparent rectangular areas. Initially, CRM1 colocalized with Rev in the nucleolus. Thirty-three minutes after addition of leptomycin B, an inhibitor of the CRM1-cargo binding, most of the CRM1 has dispersed in the nucleus. The last image of the color sequence indicates the region of the nucleolus in which the amount of colocalization is computed (bold dashed line in A). The nucleus and cell limits are also shown (solid and light dashed lines, respectively, in A). (D) The graph shows the relative concentration of the bound complex Rev/CRM1 derived from the amount of colocalization of each protein (expressions in Eq. 12) as a function of time after the end of the lag period. The dissociation rate constant kd was found to be (1.25 ± 0.31) × 10−3 s−1 (fit shown by solid line). Error bars are standard errors based on all cells averaged for each time point. Average P-values for colocalization significance test are also plotted as ▴ and confirm Rev/CRM1 interaction for at least the first 20 min of dissociation (at a 95% significance level). These results match very well the two-dimensional histogram shown in C, where the linear behavior goes from a clear inclined line to a vertical line at 33 min, indicating no more colocalization. E shows the least-square fits of the import rate of Rev into the nucleolus and the diffusion of CRM1 out of the nucleolus after injection of LMB versus the dissociation rate obtained in D, which clearly indicates that simply measuring the loss of CRM1 outside the nucleolus after injection of LMB had nothing to do with measuring the dissociation of Rev/CRM1.