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First published online 14 November 2006
doi: 10.1242/jcs.03272

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Dynamic regulation of ERK2 nuclear translocation and mobility in living cells

¹ Institute of Neuroscience CNR, Via Moruzzi 1, 56100 Pisa, Italy
² NEST, Scuola Normale Superiore, Pisa, Italy
³ Italian Institute of Technology, Italy
⁴ Department of Physics and Meteorology, Indian Institute of Technology, Kharagpur, India
⁵ Laboratory of Neurobiology, Scuola Normale Superiore, Pisa, Italy

View larger version (25K): [in this window] [in a new window]   Fig. 1. Estimate of ERK2-GFP concentration. (A) Starved cells with an high expression of ERK2-GFP exhibited strong nuclear localization (green), independently of ERK phosphorylation (red, pERK immunofluorescence). Bar, 20 µm. (B) Relationship between the concentration of ERK2-GFP and nuclear localization in starved cells. The concentration index (CI, see Materials and Methods) measures ERK2-GFP localization: a larger CI corresponds to stronger nuclear accumulation. To extend the measures over two orders of magnitude we used variable laser power according to the best imaging conditions for each cell; data were pooled together after normalization to reference imaging conditions. The concentration of ERK2-GFP was estimated by comparing the fluorescence of the cells with artificial cells loaded with known concentrations of EGFP (supplementary material Fig. S1). In this study we selected cells with expression level less than approximately 150 nM (broken line) and that had a CI1 when starved.

View larger version (34K): [in this window] [in a new window]   Fig. 2. ERK2-GFP concentrates in the nucleus of living cells after stimulation. (A) Nuclear fluorescence increased within 4 minutes from stimulation with 10% serum. Bar, 10 µm in all panels. (B) Cumulative time course of the CI after stimulation with FGF4 (blue, n=9; ERK1-KD, red line, n=4) or 10% serum (green symbols, n=15; C-ERK2, dark-green line, n=6). In this and following figures, the vertical bars are representative of the s.e.m. at various time points. (C,D) The CI after stimulation with serum had a transient component (30% decline 40 minutes after stimulation), whereas FGF4 produced a sustained response. (E) Time course of ERK2-GFP nuclear concentration after stimulation with two different doses of serum (green, 10%, n=7; yellow, 1%, n=8) and FGF (blue, n=8). Red trace, untreated control cells (n=9).

View larger version (25K): [in this window] [in a new window]   Fig. 3. ERK2-GFP translocation in response to a transient stimulus. (A) Cells have been stimulated with FGF4 for only 4 minutes. Bar, 10 µm. (B) Comparison of the response to 4 minutes (red trace, n=15) or continuous stimulation (blue trace). The green line is an exponential best fit (=9.5 minutes, R2=0.99).

View larger version (20K): [in this window] [in a new window]   Fig. 4. Dynamic regulation of ERK2-GFP localization. (A) Treatment of starved cells with the phosphatase-inhibitor sodium-orthovanadate caused gradual nuclear translocation. Bar, 20 µm in all panels. (B) ERK activation and consequent nuclear accumulation required continuous activation of the MEK-ERK pathway. Cells were treated initially with FGF4 and with U0126 15 minutes later (20 µM). Blockage of MEK caused the rapid loss of nuclear fluorescence. (C) Comparison of the time course of ERK2-GFP translocation in response to FGF, FGF4+U0126 and orthovanadate. Traces from A and B have been normalized from 0 to 1, with 0 indicating the CI at the time of administration of the specific compound and 1 indicating the asymptotic value of the exponential fits. The CI decline caused by U0126 (open circles) has been inverted for a better comparison with the other data. The continuous lines are exponential fits to the top 80% of the data points.

View larger version (23K): [in this window] [in a new window]   Fig. 5. (A) The MEK inhibitor U0126 did not decrease nuclear ERK2-GFP after orthovanate-induced translocation. Starved cells were treated first with the phosphatase inhibitor and then with U0126 (20 µM). Bar, 10 µm in all panels. The red line represents the decline observed in U0126 in the absence of the phosphatase inhibitor (Fig. 4B). (B) FGF4 alone was not sufficient to cause maximal translocation. Cells were treated initially with FGF4 and with orthovanadate 15 minutes later, which caused a 30% increase in the CI. (C) Relationship between phosphospecific signal and ERK2-GFP localization. Transfected fibroblasts were stimulated with FGF for 15 minutes, fixed and treated for pERK immunofluorescence. (D) CI measured on a sample of 58 transfected and 57 non-transfected cells (green and red dots, respectively). A similar distribution was found in ERK1-KD cells (blue dots).

View larger version (36K): [in this window] [in a new window]   Fig. 6. (A) Fluorescence recovery after photobleaching of the nucleus in starved conditions (upper sequence) and after 30 minutes in FGF4. After the first photobleaching in starved cells, we waited at least 20 minutes to allow complete recovery of fluorescence before proceeding with stimulation and the second photobleaching. Bar, 10 µm. (B) Normalized recovery of the cell indicated by the arrowhead in A. The exponential fit of the fluorescence recovery (continuous lines) is used to compute and the IF. Stimulation caused a faster turnover of ERK2-GFP through the nuclear membrane because the of the recovery decreased from 144 seconds (red trace) to 69 seconds (blue). A similar effect occurred in the upper cell in A (starved =236 seconds; FGF4 =84 seconds). (C,D) Averaged of recovery and IF for starved cells (red symbols) or after stimulation (45 minutes: green, ERK1-KO: green open; 3 hours: dark green; starved overexpressing: magenta; **P0.0001; *P0.003; °P0.05). Time constants have been measured in cells transfected with both the N- and C-terminal fusions. (E) Scatter diagram showing recovery and CI of all paired cells (red, starved; blue, FGF4; green, serum-treated cells). Broken lines join observations relative to the same cell. Open symbols represent averages and s.e.m. of each group.

View larger version (43K): [in this window] [in a new window]   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 (P0.0001) (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.

View larger version (38K): [in this window] [in a new window]   Fig. 8. (A) High-speed FRAP measures. The cell is repeatedly imaged along the broken line at high frequency (400 Hz). Bleaching is performed along a strip covering the nucleus for 300 milliseconds. Fluorescence is corrected for background and divided by the cytoplasm fluorescence to compensate for image bleaching. (B) Recovery of fluorescence for the same cell before (red) and after stimulation with FGF4. Fluorescence has been normalized to the pre-bleach value. (C) Cumulative results. At this temporal resolution the recovery of GFP (starved cells) is clearly discernible and it almost overlaps with the recovery of ERK2-GFP measured in strongly overexpressing cells (blue, stimulation did not cause any change). Photobleaching in low-expressing cells is larger and the recovery is slower, indicating lower mobility, which is dependent on ERK activation.