Rac3 FRET biosensor reveals two spatially distinct pools of Rac3 activity at invadopodia. (A) Biosensor design: inactive (left) and active (right) conformations. Black Xs indicate H83D/H86D GTPase binding–deficient mutations. Cyan cylinders indicate monomeric Cerulean fluorescent protein, and yellow cylinders indicate circularly permuted (cp229) monomeric Venus fluorescent protein. (B) Representative normalized emission spectra of biosensor mutants in HEK293 cells excited at 433 nm and normalized to donor emission at 474 nm. G12V (active, black line), T17N (inactive, blue line), and G12V coexpressed with 2× excess GDI (red line) are shown. (C) Representative image of an invadopodium showing the two spatially distinct pools of Rac3 activity in MDA–MB-231 cells. (C, top) Ringlike region of Rac3 activity around the invadopod. (C, bottom) Rac3 activity at the invadopod core (denoted by cortactin). Dashed lines indicate core and ringlike invadopod regions. (D) Percentage of invadopodia with high core or ring Rac3 activation. n ≥ 100 invadopodia from ≥10 cells; three independent experiments. (E, left) Linescan measurement along invadopodia of maximum-intensity projections of Rac3 activity and cortactin fluorescence. n = 7 invadopodia from ≥5 different cells imaged on ≥3 independent days. (E, right) Maximum-intensity projection of FRET/donor ratio for Rac3 at an invadopod. Projection is 21 frames (1 min/frame). (F, left) Autocorrelation function of the fluctuation of Rac3 activity in the ringlike region around the invadopodia. The autocorrelation function did not inflect after the zero crossing (no periodicity). n = 9 invadopodia ring measurements from ≥6 different cells; three or more independent experiments. (F, right) Autocorrelation function of the fluctuation of Rac3 activity within the invadopodia core. Period of oscillation: 12 min ± 3 min (±SD). n = 6 invadopodia core measurements from ≥5 different cells; three or more independent experiments. All error bars are SEM. CI, confidence interval.