Figure 6. RCP’s C2 domain controls... | Rockefeller University Press

Figure 6.

$Figure 6. RCP’s C2 domain controls recruitment to pseudopod tips. (A and B) A2780 cells were transfected with an siRNA targeting RCP (si-RCP) in combination with either GFP, siRNA-resistant GFP-RCPWT (GFP-RCPWT(resc)), or siRNA-resistant GFP-RCP200–649 (GFP-RCP200–649(resc)). In A, 2.5 µM cRGDfV was included during the recycling period. Values are means ± SEM of ≥12 replicates. ***, P < 0.0005 GFP versus GFP-RCPWT; Mann–Whitney test. ##, P < 0.0012 GFP versus GFP-RCP200–649. In B, cells were plated onto the CDM, and videos were collected in the absence (−) or presence of 2.5 µM cRGDfV as for Fig. 3. Pseudopod length was determined as for Fig. 3. Data are represented as box and whiskers plots (black lines show medians; whiskers: 5–95 percentile); n = 3 independent experiments. ***, P < 0.0001; Mann–Whitney test. (C–F) A2780 cells (C and D) or vector and p53273H-expressing H1299 cells (E and F) were transfected with GFP-RCP200–649. Cells were plated onto the CDM, and videos were collected in the absence (−) or presence of 2.5 µM cRGDfV as for Fig. 4 (Videos 9 and 10). Single-section confocal image stills corresponding to individual frames from these videos are presented. The yellow arrows indicate the direction of migration, and the portion of the cell within the squares is presented in the bottom images. Bars, 10 µm. (D and F) Photobleaching was performed as described in the Material and methods section. Recovery curves were generated using the FV10-ASW software and the immobile fraction (percentage) was extracted from these. In D and F, the recovery curves and immobile fraction of GFP-RCP200–649, which was photoactivated in either vesicular structures (vesic.) or diffusely cytoplasmic regions (cyto.), were analyzed either in combination (all) or separately (vesicular structures and cytoplasmic regions)—see Fig. S5 B for examples of stills from these videos. The number of cells represented by each analysis is depicted in parentheses. Values are means ± SEM.$

RCP’s C2 domain controls recruitment to pseudopod tips. (A and B) A2780 cells were transfected with an siRNA targeting RCP (si-RCP) in combination with either GFP, siRNA-resistant GFP-RCP^WT (GFP-RCP^WT(resc)), or siRNA-resistant GFP-RCP^200–649 (GFP-RCP^200–649(resc)). In A, 2.5 µM cRGDfV was included during the recycling period. Values are means ± SEM of ≥12 replicates. ***, P < 0.0005 GFP versus GFP-RCP^WT; Mann–Whitney test. ^##, P < 0.0012 GFP versus GFP-RCP^200–649. In B, cells were plated onto the CDM, and videos were collected in the absence (−) or presence of 2.5 µM cRGDfV as for Fig. 3. Pseudopod length was determined as for Fig. 3. Data are represented as box and whiskers plots (black lines show medians; whiskers: 5–95 percentile); n = 3 independent experiments. ***, P < 0.0001; Mann–Whitney test. (C–F) A2780 cells (C and D) or vector and p53^273H-expressing H1299 cells (E and F) were transfected with GFP-RCP^200–649. Cells were plated onto the CDM, and videos were collected in the absence (−) or presence of 2.5 µM cRGDfV as for Fig. 4 (Videos 9 and 10). Single-section confocal image stills corresponding to individual frames from these videos are presented. The yellow arrows indicate the direction of migration, and the portion of the cell within the squares is presented in the bottom images. Bars, 10 µm. (D and F) Photobleaching was performed as described in the Material and methods section. Recovery curves were generated using the FV10-ASW software and the immobile fraction (percentage) was extracted from these. In D and F, the recovery curves and immobile fraction of GFP-RCP^200–649, which was photoactivated in either vesicular structures (vesic.) or diffusely cytoplasmic regions (cyto.), were analyzed either in combination (all) or separately (vesicular structures and cytoplasmic regions)—see Fig. S5 B for examples of stills from these videos. The number of cells represented by each analysis is depicted in parentheses. Values are means ± SEM.