Rab13 activation occurs at the leading edge of migrating cells. (A) Schematic of a Rab13 biosensor. (B) Mean emission ratio FRET/mCer3 of the optimized Q67L and T22N Rab13 biosensors in adherent MEFs using whole cell imaging (mean ± SD measuring 14 samples per condition pooled from two independent experiments; Student’s t test; ***, P < 0.001). (C) Ratiometric image (FRET/mCer3) of the optimized Q67L and T22N Rab13 biosensors (BS) in MEFs using whole cell imaging. (D) Fluorometric analysis of HEK-293T cells in suspension coexpressing Rab13 biosensor and RabGDI with empty vector, Flag-DENND2B full length (FL) or DENN domain, and Flag–connecdenn 1 (CD1) DENN domain (mean ± SD measuring six samples per conditions pooled from two independent experiments; one-way ANOVA with Dunnett’s post-test; ***, P < 0.001). (E) Ratiometric image (FRET/mCer3) of Rab13 wild-type biosensor in MCF10A cells. (F) Ratiometric image (FRET/mCer3) of Rab13 biosensor wild type in a randomly migrating MEF. The images correspond to Video 6. Time in minutes and seconds is indicated. Bars, 10 µm.