Munc13-4 function in exosome release is dependent on Rab11a. (A) MDA-MB-231 cells expressing GFP-Rab11, GFP-Rab11 Q70L, or GFP-Rab11 S25N were immunolabeled for myc-Munc13-4 (red) and endogenous CD63 (cyan). Bar, 5 µm. (B) Pearson’s correlation coefficient for GFP-Rab11 versus CD63 immunolocalization. Mean values ± SE (15 cells/group from three separate preparations) are shown. *, P < 0.05; **, P < 0.01. (C) MDA-MB-231 cells expressing GFP, GFP-Rab11, or GFP-Rab11 S25N were immunoblotted for the indicated proteins. Arrow indicates GFP-Rab11; lower band in upper panel corresponds to Rab11. (D) Cell medium supernatants (as in Fig. 1 B) from untreated MDA-MB-231 cells or cells treated with 1.25 µM ionomycin were filtered onto membrane and analyzed for CD63 and GM130. (E) Quantification of CD63 exosome release as percentage of total cellular shown as mean values ± SE (n = 3) with *, P < 0.05 for comparison to GFP-alone samples. (F) Model for the generation of secretion-competent MVBs as arising from the transient fusion of Rab11⁺ endosomes with MVB precursors, which requires Munc13-4, Ca²⁺, and Rab11a. MT1-MMP (green bar) is found in the recycling endosome (RE) and delivered to the MVB dependent on Munc13-4 for incorporation to ILVs and exosome release. The secretion-competent MVBs generated acquire components for Ca²⁺-dependent fusion with the plasma membrane. Bars, 5 μm.