Cortactin, Coronin 1b, and Rab27a coordinately control invadopodial actin dynamics. (A) Representative confocal immunofluorescence images showing localization of coronin 1b and actin to invadopodia. ECM degradation evident as dark spots in FITC-FN images. Fluorescence levels of the images were enhanced equally across all images for visualization only, not for quantitation. Bar, 15 µm. Boxes outline example invadopodia and are shown in B. (B and C) Quantitation of the absolute intensity of coronin 1b at invadopodia was measured using 4 × 4–pixel boxes. Representative blue 4 × 4–pixel boxes are shown within the coronin 1b zooms and are further zoomed 4× and shown on the left (B). Quantitations shown in C. n ≥ 5 invadopodia per cell from ≥35 cells per condition, n ≥ 3 independent experiments Scatterplot with median is plotted. Analyzed by Mann–Whitney U test. (D and E) Quantification of functional invadopodia per cell and ECM degradation area percentage. Box and whisker plots with median shown as a line, box indicates 25–75th percentile and whiskers indicate 5–95th percentile. n ≥ 3 independent experiments. ***, P < 0.001, determined by Mann–Whitney U test. (F) Actin turnover in invadopodia after 10 µM latrunculin A (LatA) treatment was assessed by live TIRF imaging. See also Video 10. Actin fluorescence intensity at invadopodia after LatA treatment is plotted. Five or more invadopodia per cell were quantitated for ≥10 cells for each condition; n ≥ 3 independent experiments.