Invadopodia are dynamic structures in 3D. (A) Representative images of MDA-MB-231 cells expressing WT cortactin-RFP incubated with Geltrex and BSA-Green 488 for 48 h. The cells were imaged for 8 h and the images represent snapshots of a time-lapse movie. The degradation marker channel was processed to highlight the degradation areas. (B) The cartoon shown represents the dynamics of invadopodia protrusion observed in these cells. Multiple cycles of invadopodia elongation and retraction are observed during cell invasion. Results are based on the analysis of 15 cells in three experiments. (C) Representative kymograph of a WT cortactin cell (left), a 3YF cortactin cell (middle), and a NHE1KD cell (right) migrating in 3D. The plots underneath represent the variations in pixel intensity of the protrusions over time. (D) Quantification of the WT cortactin protrusion autocorrelation. Cells expressing RFP-tagged WT cortactin were mixed with 10 mg/ml Geltrex and incubated for 24 h. Cells were imaged by between 24 and 48 h after plating. (E) Quantification of the 3YF cortactin protrusion autocorrelation. Cells expressing RFP-tagged 3YF cortactin were mixed with Geltrex and incubated for 24 h. Cells were imaged between 24 and 48 h after plating. n = 9 cells (WT) and 10 cells (3YF) in three independent experiments. Red traces represent the standard error of the mean.