Invadopodia display a dynamic oscillatory behavior in 2D. (A) Representative images of MDA-MB-231 cells expressing WT cortactin-RFP and cofilin-GFP. The cells were imaged for 4 h and the fluctuation in the relative fluorescence intensity of cofilin and cortactin was analyzed and plotted. The insets show the boxed region. (B) Representative traces of WT cortactin and cofilin pixel intensity representative of 1 invadopodia from 12 analyzed. (C and D) The fluctuations in WT cortactin and cofilin fluorescence intensity were analyzed using autocorrelation (see Material and methods). The autocorrelation for WT cortactin is shown in C and the corresponding cofilin-GFP autocorrelation is shown in D. Data are based on the analysis of 12 invadopodia in three independent experiments. (E–G) The experiment was repeated in cells expressing 3YF cortactin-RFP and cofilin-GFP. (E) Representative traces of cofilin and 3YF cortactin pixel intensity (1/10). Autocorrelation analysis of the fluorescence intensity of 3YF cortactin is shown in F and the corresponding cofilin-GFP signal is shown in G. Red traces represent the standard error of the mean. (H) Representative images of cells expressing WT cortactin and the SNARF-5F pH indicator. Inner white circles represent the invadopodial core and the outer circles represent the invadopodium periphery. (I) Quantification of the coefficient of pH variation. n = 18 invadopodia in three experiments; ***, P < 0.001. Error bars indicate SEM.