JIP4 recruitment on MT1-MMP–positive endosomes depends on WASH. (A) JIP4 and cortactin staining of MDA-MB-231 cells expressing MT1-MMPmCh treated with the indicated siRNAs. Bars: 5 µm; (insets) 1.25 µm. (B) JIP4 and cortactin association with MT1-MMPmCh–positive endosomes as in Fig. 3 H. ***, P < 0.001 (as compared with siNT-treated cells in which association was set to 100). (C) Inverted still images from time-lapse sequences of MDA-MB-231 cells expressing KIF5B-YFP and MT1-MMPmCh treated with the indicated siRNAs. Bar, 10 µm. Galleries show images with 30-s intervals corresponding to the boxed regions. Arrows point to tubulated endosomes. Bar, 5 µm. (D) Quantification of tubulation from time-lapse sequences as in C. All values are mean ± SEM. n, number of cell regions scored for each cell population. ***, P < 0.001 (as compared with siNT-treated cells). (E) FITC-gelatin degradation by MDA-MB-231 cells overexpressing MT1-MMPmCh and KIF5B-YFP treated with indicated siRNAs. n, number of cells scored for each cell population. **, P < 0.01 (compared with siNT-treated cells). (F) Scheme depicting ARF6 and JIP3/JIP4-mediated regulation of a kinesin-1 and dynactin–dynein tug of war leading to MT1-MMP endosome tubulation and surface delivery of MT1-MMP (see Discussion). (G) Model of trans-interaction between plasma membrane GTP-ARF6 and endosomal JIP4. ARF6 myristoylated amphipatic N-terminal helix is indicated as a green cylinder lying against the plasma membrane. Only the second coiled coil domain of JIP4 is shown. Binding of GTP-ARF6 to JIP4 dimer is compatible with the dynactin–dynein interaction with JIP4, whereas it prevents kinesin-1 interaction (based on structural and biochemical data; Isabet et al., 2009; Montagnac et al., 2009).