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Fig. 7. Reduced mobility of ERK2-GFP in the nucleus. (A) Imaging before photobleaching of a small area of the nucleus (dotted circle in the magnified image in B and at the indicated time during recovery. (B) The false-colour sequence shows the difference with the last frame of the recovery, and therefore the bleached spot disappears as time passes by. Bar, 10 µm. (C) Difference between the pre-bleach image and the last image of the recovery sequence (36 seconds). The signal in the nucleus indicates that the pre-bleach fluorescence is still recovering; this requires slower equilibration through the nuclear envelope. (D) Time course of the normalized fluorescence recovery in the nuclei of starved (red) and stimulated (green) cells. GFP recovery is shown in light blue. Estimate of the asymptotic value reached by the recovery showed the presence of a small but significant IF (P
0.0001) (percentage of total normalized fluorescence: 3.3±0.6 starved, 3.2±0.7 FGF). (E) ERK activation caused a considerable decrease in the recovery speed. The data in D have been normalized to allow a better comparison of the time course, and have been fitted with an approximate solution of the diffusion equation (solid lines). (F) Computed effective diffusion coefficient for the starved and stimulated cells.
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