LIMK inhibition blocks invadopodia formation by decreasing F-actin stability. (A) X-Y, X-Z, and Z-Y views of a fixed MDA-MB-231 cell stained for F-actin (red) plated on FITC-labeled gelatin (green). Invadopodia are actin-rich projections within dark regions, indicative of gelatin degradation. Bar, 10 µM. (B) X-Y and X-Z views of a fixed MDA-MB-231 cell stained for N-Wasp (green) and MMP9 (red) plated on Oregon Green 488–labeled gelatin matrix (gray). Invadopodia are regions of gelatin degradation that colocalize with N-Wasp and MMP9. Bar, 10 µM. (C) LIMKi or LIMK1/2 knockdown with two independent duplex pairs significantly reduced invadopodia formation and consequent area of gelatin degradation relative to vehicle control and NT controls, respectively. Microscope settings were varied to acquire images of similar intensity for comparison purposes. Insets show higher magnification. Bar, 10 µM (averages ± SEM, n = 3). (D) Cortactin localization in cells plated on gelatin was unaffected by LIMKi treatment (box equals upper and lower quartiles with line indicating median, whiskers are 5 and 95% confidence intervals, n = 90 per treatment). Bar, 10 µM. (E) FRAP analysis of cells plated on gelatin and expressing GFP-actin revealed that LIMKi significantly decreased the t1/2 of recovery relative to vehicle DMSO-treated cells. Bar, 10 µM (average ± SD; untreated n = 16; LIMKi n = 26).
