Figure 7.
ARHGAP17 regulates the spatiotemporal dynamics of Cdc42 activity during invadopodia turnover. (A) Representative TIRF micrographs of a SUM159 cell expressing TKS5-mTagBFP2 as a marker for invadopodia and dTomato-wGBD as a marker for Cdc42 activity and forming an invadopodia cluster after PDBu treatment (scale bar for both panels, 5 μm). The edge of the cell is marked by a dashed line. (B) Representative TIRF micrographs of a SUM159 cell forming an individual invadopodia after PDBu treatment. Cells were expressing TKS5-mTagBFP2 as a marker for invadopodia, ARHGAP17-GFP (expressed in ARHGAP17 KO cells), and dTomato-wGBD as a marker for Cdc42 activity (scale bar, 1 μm). (C) Quantification of TKS5-mTagBFP2, ARHGAP17-GFP (expressed in ARHGAP17 KO cells), and dTomato-wGBD intensity at individual invadopodia. Intensity and time have been rescaled between values of 0 and 1 for each invadopodia to allow for comparison. Lines are loess smoothing and 95% confidence from three independent experiments in which at least 10 invadopodia per condition per experiment were measured. (D) Cross-correlation analysis of TKS5-mTagBFP2, ARHGAP17-GFP (expressed in ARHGAP17 KO background), and dTomato-wGBD intensity. Higher values denote positive correlation and lag denotes the time shift effect on correlation between signals. Lines are loess smoothing and 95% confidence from three independent experiments in which at least 10 invadopodia per condition per experiment were measured. (E) Quantification of the peak dTomato-wGBD intensity at individual invadopodia after PDBu treatment, normalized to the average whole-cell intensity before treatment. Results are expressed as mean ± SEM of three independent experiments. Large circles represent biological replicates, small circles represent technical replicates. Significance was tested with a two-tailed t test: *, P < 0.05. (F) Radial intensity profiles of individual invadopodia from live cells expressing TKS5-mTagBFP2, ARHGAP17-GFP (expressed in ARHGAP17 KO cells), and dTomato-wGBD plotted over time. Time scales between invadopodia are rescaled between 0 and 1 to allow for alignment of intensity values. Results are expressed as the mean of eight representative invadopodia. (G and H) Radial profile plots taken from time 0.25 and 0.75 of F showing relative intensity and localization of the invadopodia markers while the invadopodia are assembling and disassembling, respectively. Results are expressed as mean ± SEM of eight invadopodia. (I) Radial intensity profiles of individual invadopodia from live cells expressing TKS5-mTagBFP2 and ΔC-ARHGAP17-GFP (expressed in ARHGAP17 KO cells) plotted over time. Time scales between invadopodia are rescaled between 0 and 1 to allow for alignment of intensity values. Results are expressed as the mean of 13 representative invadopodia. (J and K) Radial profile plots taken from time 0.25 and 0.75 of I showing relative intensity and localization of the invadopodia markers while the invadopodia are assembling and disassembling, respectively. Results are expressed as mean ± SEM of 13 invadopodia.

ARHGAP17 regulates the spatiotemporal dynamics of Cdc42 activity during invadopodia turnover. (A) Representative TIRF micrographs of a SUM159 cell expressing TKS5-mTagBFP2 as a marker for invadopodia and dTomato-wGBD as a marker for Cdc42 activity and forming an invadopodia cluster after PDBu treatment (scale bar for both panels, 5 μm). The edge of the cell is marked by a dashed line. (B) Representative TIRF micrographs of a SUM159 cell forming an individual invadopodia after PDBu treatment. Cells were expressing TKS5-mTagBFP2 as a marker for invadopodia, ARHGAP17-GFP (expressed in ARHGAP17 KO cells), and dTomato-wGBD as a marker for Cdc42 activity (scale bar, 1 μm). (C) Quantification of TKS5-mTagBFP2, ARHGAP17-GFP (expressed in ARHGAP17 KO cells), and dTomato-wGBD intensity at individual invadopodia. Intensity and time have been rescaled between values of 0 and 1 for each invadopodia to allow for comparison. Lines are loess smoothing and 95% confidence from three independent experiments in which at least 10 invadopodia per condition per experiment were measured. (D) Cross-correlation analysis of TKS5-mTagBFP2, ARHGAP17-GFP (expressed in ARHGAP17 KO background), and dTomato-wGBD intensity. Higher values denote positive correlation and lag denotes the time shift effect on correlation between signals. Lines are loess smoothing and 95% confidence from three independent experiments in which at least 10 invadopodia per condition per experiment were measured. (E) Quantification of the peak dTomato-wGBD intensity at individual invadopodia after PDBu treatment, normalized to the average whole-cell intensity before treatment. Results are expressed as mean ± SEM of three independent experiments. Large circles represent biological replicates, small circles represent technical replicates. Significance was tested with a two-tailed t test: *, P < 0.05. (F) Radial intensity profiles of individual invadopodia from live cells expressing TKS5-mTagBFP2, ARHGAP17-GFP (expressed in ARHGAP17 KO cells), and dTomato-wGBD plotted over time. Time scales between invadopodia are rescaled between 0 and 1 to allow for alignment of intensity values. Results are expressed as the mean of eight representative invadopodia. (G and H) Radial profile plots taken from time 0.25 and 0.75 of F showing relative intensity and localization of the invadopodia markers while the invadopodia are assembling and disassembling, respectively. Results are expressed as mean ± SEM of eight invadopodia. (I) Radial intensity profiles of individual invadopodia from live cells expressing TKS5-mTagBFP2 and ΔC-ARHGAP17-GFP (expressed in ARHGAP17 KO cells) plotted over time. Time scales between invadopodia are rescaled between 0 and 1 to allow for alignment of intensity values. Results are expressed as the mean of 13 representative invadopodia. (J and K) Radial profile plots taken from time 0.25 and 0.75 of I showing relative intensity and localization of the invadopodia markers while the invadopodia are assembling and disassembling, respectively. Results are expressed as mean ± SEM of 13 invadopodia.

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