DENND2B C-terminal interacts with MICAL-L2. (A) GST-Rab13 Q67L or T22N, bound to glutathione beads, were incubated with HEK-293T cell lysates expressing GFP–MICAL 1 or GFP–MICAL-L2, and the indicated proteins were detected by blotting. Starting material (SM) equals 5% of the lysate used per condition. (B) GST-DENND2B C-terminal (C-term) region or GST coupled to a scrambled version of that sequence, bound to glutathione beads, were incubated with HEK-293T cell lysates expressing GFP–MICAL 1, GFP–MICAL-L2, or GFP-Scyl1, and the indicated proteins were detected by blotting. Starting material equals 2.5% of the lysate used per condition. (C) Lysates from HEK-293T cells coexpressing Flag-DENND2B full length (FL) or Flag-DENND2BΔC with GFP–MICAL-L2 were immunoprecipitated with the anti-GFP antibody, and the indicated proteins were detected by blotting. IP, immunoprecipitation. (D) MCF10A cells were transfected with mCh-DENND2B and GFP–MICAL-L2, fixed, and processed for fluorescence for GFP and mCh. Bar, 10 µm. (E) Protrusion length of transfected MCF10A cells was quantified (mean ± SEM of three independent experiments measuring a minimum of 40 cells per conditions per experiment; one-way ANOVA with Dunnett’s post-test; ***, P < 0.001). (F) Proposed model whereby vesicles carrying GDP-bound Rab13 encounter DENND2B in a complex with MICAL-L2 on cortical actin (blue lines). DENND2B activates Rab13, and the Rab13-GTP–positive vesicles fuse with the plasma membrane. Rab13-GTP also binds to and opens MICAL-L2, which then stimulates actin assembly for the formation of membrane ruffles.