|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
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.
|
