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First published online 7 August 2007
doi: 10.1242/jcs.003061

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Modulation of lamellipodial structure and dynamics by NO-dependent phosphorylation of VASP Ser239

¹ Division of Immunology, Infection and Inflammation, University of Glasgow, Glasgow Biomedical Research Centre, 120, University Place, Glasgow, G12 8TA, UK
² Department of Urology, Gartnavel General Hospital, Great Western Road, Glasgow, G12 0YN, UK
³ Institute for Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Josef-Schneider Strasse 2, 97080 Würzburg, Germany

View larger version (69K): [in this window] [in a new window]   Fig. 1. Distribution of VASP within human PTECs. (a) Cells stained for VASP (green), actin (red) or merged with images of nuclei stained with DAPI (blue). The arrowhead in the upper panel indicates the edge of a lamellipodium. (b,c) Human PTECs were transfected with WT GFP-VASP (b) or GFP-S239A VASP (c), then fixed 16 hours after transfection and imaged for GFP (green) and nuclei stained with DAPI (blue). (d) Western blot of cell extracts from human PTECs either untransfected control (C) or transfected with WT GFP-VASP (WT) or S239A GFP-VASP (S239A). Molecular mass markers (kDa) are to the left of the panel. Arrowheads indicate bands of endogenous VASP (50 kDa) and GFP-VASP fusion proteins (75 kDa). The usual doublet band of VASP is not seen on this gel as it was optimized to show both endogenous and transfected protein. (e) Human PTECs transfected with a GFP control vector alone. (f) Untransfected human PTECs visualised using phase-contrast microscopy (upper panel) or GFP fluorescence (lower panel). Bars, 10 µm.

View larger version (68K): [in this window] [in a new window]   Fig. 2. Movement of human PTECs transfected with GFP-VASP and GFP-S239AVASP. (a) Sequence of frames taken from Movie 1 (see supplementary material), showing one cell imaged at the indicated intervals (in minutes). The final frame (t=65 minutes) shows the outline of the original cell at its starting position at t=0 minutes. The white line indicates the axis used to generate the kymograph in panel c. (b) Still frames from another cell transfected with WT GFP-VASP and imaged at the indicated times (in minutes) after the start of the experiment. The white line indicates the axis used to generate the kymograph in panel d. (c) Kymograph of the cell shown in panels a, taken along the axis indicated by the white line. (d) Kymograph of the cell shown in panels b, taken along the axis indicated by the white line. (e) Sequence of frames from Movie 2 (see supplementary material), showing one cell imaged at the indicated intervals (in minutes). The final frame (t=64.5 minutes) shown the outline of the original cell at its starting position at t=0 minutes. The white line indicates the axis used to generate the kymograph in panel g. (f) Still frames from another cell transfected with S239A GFP-VASP and imaged at the indicated times (in minutes) after the start of the experiment. The white line indicates the axis used to generate the kymograph in panel h. (g) Kymograph of the cell in shown panels e, taken along the axis indicated by the white line. (h) Kymograph of the cell shown in panels f, taken along the axis indicated by the white line. Bars, 10 µm. (i) Box and whisker plot of cell migration and lamellipodial projection speeds for human PTECs transfected with WT GFP-VASP and S239A GFP-VASP (WT and S239A, respectively). Horizontal line is the median value, the box encloses the 25th and 75th percentiles. The bars indicate the range of the values. The differences in cell migration and lamellipodial projection speeds between WT and S239A were not significant (n.s.) as determined by Mann-Whitney test; n=7-12 independent observations.

View larger version (58K): [in this window] [in a new window]   Fig. 3. Effect of NO on VASP and lamellipodial dynamics. (a) Western blot showing Ser239-phosphorylation status of VASP at various times (in minutes) after the addition of NO donor. VASP proteins are indicted by square brackets at the right of the gel. VASP was immunoprecipitated from human PTECs and analysed for the presence of VASP phosphorylated at Ser239 (P-Ser239-VASP) (upper panel). The blot was then stripped and reprobed for total VASP (lower panel). Immunoprecipitates from untreated positive-control (C) and NO-treated (+) platelets are shown in the first three lanes from the left. Control immunoprecipitates with control immunoglobulin at 30 minutes after addition of NO donor showed no VASP (data not shown). The experiment was repeated three times with similar results. The fraction of VASP phosphorylated at Ser157 following addition of NO donor was calculated as described in Materials and Methods and is plotted in the diagram on the right. Values are the means of duplicates; error bars are ± s.e.m. (b,c) Sequence of frames taken from time-lapse Movie 3 (see supplementary material), showing one human PTECl transfected with (b) WT GFP-VASP or (c) S239A GFP-VASP and treated with NO donor. Frames were taken at the indicated times. NO donor was added at 2250 seconds for panels b, and at 1080 seconds for panels c. Bars is 10 µm. The white line in the first panel of b indicates the axis used to generate the kymograph in d. (d) Kymograph of the cell shown in panels b, taken along the axis indicated by the white line. (e) Effects of NO on the localization of VASP in lamellipodia. The area of lamellipodia occupied by WT GFP-VASP was calculated just prior to (control) and after the addition of NO donor at the times indicated, and are expressed as a percentage of the control value prior to NO addition. Values are the means ± s.e.m.; n= 12. The effect of NO was significant as determined by ANOVA, P<0.0001. The diagram in the inset shows an exponential decay curve fitted to the data; dotted lines are 95% confidence limits. The calculated half-life of reduction of VASP localization within lamellipodia after NO addition was 12.6 ± 0.45 minutes (± s.e.m.). (f) Data from experiment as described for panel e, but from cells transfected with S239A GFP-VASP. No significant effect after the addition of NO donor was observed (ANOVA, P=0.2461). Values are the mean ± s.e.m.; n= 13. (g) GFP-VASP distribution in lamellipodia following induction of iNOS with cytokines for 16 hours. Box indicates the 25th to 75th percentiles of the percentage of lamellipodial area occupied by VASP, the line the median value and the bars the range. Hatched bars show data of WT GFP-VASP (n=11), white bars of S239A GFP-VASP (n=6) after the treatment as indicated. Difference between the median values of WT and S239A GFP-VASP following cytokine stimulation is significant (*P=0.0022, Mann-Whitney test). (h) Percentage change in mean speed of cells following addition of NO donor. Bars show mean speed of cell centroids (calculated as described in the Materials and Methods) for 20 minutes, following addition of NO donor to WT GFP-VASP (n=8) and S239A GFP-VASP (n=8); bars are ± 1 s.e.m. *P=0.0033 significantly different from no change (one sample t-test); N.S. not significant difference from no change.

View larger version (43K): [in this window] [in a new window]   Fig. 4. Influence of VASP phosphorylation on cell area after the addition of NO donor. (a) Sequence of frames taken from Movie 1 (see supplementary material), showing distribution of WT GFP-VASP (green) in transfected human PTEC before (t=0 minutes) and after (t=30 minutes) addition of NO donor. The outline of the cells is shown in the phase-contrast micrographs. Merged images are on the right. Bars, 10 µm. Arrowheads mark edge of cell. (b) Mean cell area normalized to that just prior to addition of NO donor. Black bars, cells transfected with WT GFP-VASP; white bars cells transfected with S239A GFP-VASP. Areas were measured 30 minutes before (Control) and after (After NO donor) NO donor addition. Values are the mean ± s.e.m.; n=12 or more. Reduction in area in cells transfected with WT GFP-VASP following NO donor addition was significantly reduced compared with control value (P<0.01, two-tailed t-test).

View larger version (49K): [in this window] [in a new window]   Fig. 5. Effects of NO on lamellipodial VASP and cell shape in human embryonic fibroblasts (HEK cells). (a,b) Montage from time-lapse sequence of HEK cells transfected with WT GFP-VASP and S239A GFP-VASP respectively at the indicated times. NO donor was added at 25 minutes in panel a and 24 minutes in panel b. Last panel shows outline of cell from first panel. (c) Effects of NO on the localization of VASP in lamellipodia. The area of lamellipodia occupied by WT GFP-VASP was calculated just prior to addition of NO donor (control) and at the indicated times after NO addition, expressed as a percentage of the control value prior to NO addition. Values are the mean ± s.e.m.; n=7. The effect of NO was significant as determined by ANOVA, P<0.001. The inset shows an exponential decay curve fitted to the data; dotted lines are 95% confidence limits. The calculated half-life of reduction of VASP localization within lamellipodia after NO addition was 18.6 minutes. (d) As panel c, but from cells transfected with S239A GFP-VASP. No significant effect after NO donor addition was observed (ANOVA, P=0.32). Values are the mean ± s.e.m.; n=6. (e) Mean cell area normalized to that just prior to NO donor addition. Black bars, cells transfected with WT GFP-VASP, white bars cells transfected with S239A GFP-VASP. Areas were measured 30 minutes before (Control) and after (After NO donor) NO donor addition. Values are the mean ± s.e.m.; n=6. Decrease of area in cells transfected with WT GFP-VASP following NO donor addition was significantly reduced compared with control value (***P<0.001, two-tailed t-test).

View larger version (36K): [in this window] [in a new window]   Fig. 6. Effects of the phosphomimetic S239D mutation on VASP distribution. (a) Distribution of WT GFP-VASP and S239D GFP-VASP fusions (green) in human PTECs with corresponding phase-contrast views. Edges of lamellipodia are shown by arrowheads. Bar is 10 µm. (b) Quantification of VASP in lamellipodia. Percentage of lamellipodia containing VASP measured as described in the Materials and Methods for WT GFP-VASP (WT; n=30) and S239D GFP-VASP (SDT; n=61). *P<0.0001 (Fisher's exact test), significant difference from WT GFP-VASP.

View larger version (59K): [in this window] [in a new window]   Fig. 7. Dependence of NO effects on PKG I and PKG II and guanylate cyclase. (a,c) Distribution of WT GFP-VASP in human PTEC co-transfected with siRNA targeting (a) PKG I or (c) PKG II (supplementary material Movies 5 or 6, respectively). Montage of cell images taken immediately before (left panels) and after (right panels) addition of NO donor, showing distribution of WT GFP-VASP. Time points at which images were taken are shown in each panel. White lines in each image show the axises used to generate the kymographs shown in b and d. Bars, 10 µm. (b and d) Kymographs of the cell in panels a and c, respectively, using the axis as shown. Arrows indicate the time at which the NO donor was added. (e,f) Localization of WT GFP-VASP within lamellipodia of cells co-transfected with siRNA targeting (e) PKG I or (f) PKG II, measured as a percentage of the value immediately before addition of NO donor (control=100%). Values are the mean ± s.e.m. at the indicated times after addition of NO donor; n=8 (PKG I) and n=10 (PKG II) separate determinations. The change in percentage of WT GFP-VASP within lamellipodia in response to NO donor addition was significant for cells transfected with siRNA targeting PKG I (P<0.0001, ANOVA) but not for siRNA targeting PKG II (P=0.8841, ANOVA). (g) Effect of the siRNA on PKGI or PKG II protein levels in cells transfected with control siRNA (C) or siRNAs specifically targeting the indicated PKG isoforms (+). Loading control (actin) is shown in the lower panel. (h) Change in cell area following addition of NO donor to cells transfected with siRNA targeting PKG I (black bars) or PKG II (white bars). Values were determined as described for Fig. 4b. Results are given as the mean ± s.e.m.; n=13 (PKG I) and n=11 (PKG II) individual determinations. The difference between the area after addition of NO donor in PKG I transfected cells was significantly reduced by NO addition (***P<0.01, two-tailed t-test). (i) Localization of WT GFP-VASP within lamellipodia of cells treated with ODQ, measured as a percentage of the value immediately before addition of NO donor (control=100%). Addition of ODQ for 30 minutes produced a small and non-significant decline in WT GFP-VASP within lamellipodia. Values are the mean ± s.e.m. at the indicated times after addition of NO donor of six separate determinations. The change in percentage of WT GFP-VASP within lamellipodia in response to NO donor was not significant for cells treated with ODQ (P>0.05, ANOVA). (j) Change in cell area following addition of NO donor to cells treated with ODQ. Values were determined as described for Fig. 4b. Results are mean ± s.e.m.; n=6. The difference between the area after NO addition in ODQ treated cells was not significant (P>0.05, two-tailed t-test).

View larger version (105K): [in this window] [in a new window]   Fig. 8. Effects of NO on F-actin and focal adhesions in PTECs. (a) Untreated cells stained for VASP (green), actin (red) and nuclei (blue). Arrowheads indicate focal adhesions, arrows indicate actin stress fibres. (b) Changes in F-actin/VASP in cells following addition of NO donor and the effects on lamellipodia. After 30-minute treatment with NO donor, cells were fixed and stained for VASP (green), actin (red) and nuclei (blue). Cellular actin was not seen in cell processes. Occasionally, cells retained a cortical actin cytoskeleton (arrows). (c) Changes in F-actin/VASP in cells following addition of NO donor and the effects on focal adhesions. Cells treated and stained as described for panel b. This panel shows specifically cells with focal adhesions (arrowheads) or actin stress fibres (arrow) that show no effect following treatment with NO donor. Bars, 10 µm.

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