Figure 6.
The mechanism of invadopodia formation directed by TKS4 is independent of FGD1 and CDC42.(A) Schematic representation of TKS5 and TKS4 domains and percentage of identity in the different domains (Saini and Courtneidge, 2018). Lysates of Hs578T or MDA-MB-231 human breast cancer cells expressing FGD1GFP or not (Ctrl) were immunoprecipitated with GFP antibodies. Bound proteins were analyzed with FGD1, TKS4, and TKS5 antibodies (IP). 5% of total lysate was loaded as a control (input). Equal loading was controlled using GAPDH antibody. Molecular weights are in kD. (B) MDA-MB-21 cells seeded on type I collagen (gray) were stained for TKS4 (green) and cortactin (red), and nuclei were stained with DAPI (blue). Scale bar, 10 µm; zoom-in of boxed region, 5 µm. Lower panel: Correlation of cortactin (x axis) and TKS4 (y axis) pixel normalized fluorescence intensity along the invadopodial structures. inv, number of invadopodia analyzed; pix, total number of pixels analyzed for both markers. (C) Quantification of TKS5-positive invadopodia in MDA-MB-231 cells silenced for TKS4. The y axis is the ratio of the TKS5 area to the total cell area normalized to the mean value of control siNT-treated cells (as percentage ± SEM). Mann–Whitney U test. (D and E) Hs578T cells seeded on type I collagen (gray) were stained for TKS4 (green, D) or TKS5 (green, E), cortactin (red), and DAPI (blue). Boxed regions highlight cortactin-positive invadopodia. Scale bar, 10 µm; zoom-in of boxed regions, 5 µm. The lower panels show the correlation of pixel normalized fluorescence intensity for the indicated markers along the invadopodial structures as in B. inv, number of invadopodia analyzed, pix, total number of pixels analyzed for both markers. (F) Quantification of pericellular collagenolysis by Hs578T cells treated with CDC42 or MT1-MMP siRNAs measured as mean intensity of Col1-3/4C signal per cell. Values for control cells (siNT) were set to 100%. siNT, n = 53 cells; siCDC42#07, n = 40 cells; and siMT1-MMP, n = 53 cells from three independent experiments. Kruskal–Wallis test. (G) Quantification of TKS4-positive invadopodia in Hs578T cells silenced for CDC42. siNT, n = 266 cells; siCDC42#07, n = 125 cells from three independent experiments. Statistical analysis is based on a Kruskal–Wallis test. ****, P < 0.0001; ns, not significant.

The mechanism of invadopodia formation directed by TKS4 is independent of FGD1 and CDC42. (A) Schematic representation of TKS5 and TKS4 domains and percentage of identity in the different domains (Saini and Courtneidge, 2018). Lysates of Hs578T or MDA-MB-231 human breast cancer cells expressing FGD1GFP or not (Ctrl) were immunoprecipitated with GFP antibodies. Bound proteins were analyzed with FGD1, TKS4, and TKS5 antibodies (IP). 5% of total lysate was loaded as a control (input). Equal loading was controlled using GAPDH antibody. Molecular weights are in kD. (B) MDA-MB-21 cells seeded on type I collagen (gray) were stained for TKS4 (green) and cortactin (red), and nuclei were stained with DAPI (blue). Scale bar, 10 µm; zoom-in of boxed region, 5 µm. Lower panel: Correlation of cortactin (x axis) and TKS4 (y axis) pixel normalized fluorescence intensity along the invadopodial structures. inv, number of invadopodia analyzed; pix, total number of pixels analyzed for both markers. (C) Quantification of TKS5-positive invadopodia in MDA-MB-231 cells silenced for TKS4. The y axis is the ratio of the TKS5 area to the total cell area normalized to the mean value of control siNT-treated cells (as percentage ± SEM). Mann–Whitney U test. (D and E) Hs578T cells seeded on type I collagen (gray) were stained for TKS4 (green, D) or TKS5 (green, E), cortactin (red), and DAPI (blue). Boxed regions highlight cortactin-positive invadopodia. Scale bar, 10 µm; zoom-in of boxed regions, 5 µm. The lower panels show the correlation of pixel normalized fluorescence intensity for the indicated markers along the invadopodial structures as in B. inv, number of invadopodia analyzed, pix, total number of pixels analyzed for both markers. (F) Quantification of pericellular collagenolysis by Hs578T cells treated with CDC42 or MT1-MMP siRNAs measured as mean intensity of Col1-3/4C signal per cell. Values for control cells (siNT) were set to 100%. siNT, n = 53 cells; siCDC42#07, n = 40 cells; and siMT1-MMP, n = 53 cells from three independent experiments. Kruskal–Wallis test. (G) Quantification of TKS4-positive invadopodia in Hs578T cells silenced for CDC42. siNT, n = 266 cells; siCDC42#07, n = 125 cells from three independent experiments. Statistical analysis is based on a Kruskal–Wallis test. ****, P < 0.0001; ns, not significant.

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