Figure 6.

MT1-MMP recruits Bet1 to endocytic compartments, where they are in proximity to each other. (A) Depletion of MT1-MMP decreases the endosomal localization of Bet1. MDA-MB-231 cells stably expressing Bet1-GFP were transfected with MT1-MMP siRNA, fixed, and immunostained, and then Bet1-GFP and CD63 were visualized by confocal microscopy. (B) Quantitation of colocalization of Bet1-GFP with CD63 by using the Manders’ overlap coefficient. (C) Bet1-GFP reaching the plasma membrane is reduced through MT-MMP depletion. MDA-MB-231 cells stably expressing Bet1-GFP were cultured in the medium containing GFP antibodies for the indicated periods of time, fixed, and stained, and then Bet1-GFP and the endocytosed GFP antibodies were visualized by confocal microscopy. (D) Quantitation of the level of the endocytosed GFP antibodies. (E) MT1-MMP is in proximity to Bet1. PLA was performed with antibodies to MT1-MMP and GFP in parental MDA-MB-231 cells and Bet1-GFP stable cells. (F) Quantitation of the ratio of the number of PLA dots. (G) Representative pictures of the cells with PLA signals on Bet1-GFP–positive endosomes. (H) Interaction between MT1-MMP and Bet1 in endomembrane compartments is detected by BiFC. Bet1 and MT1-MMP fused with the N- and C-terminal fragments of split Venus, Vn-Bet1 and MT1-MMP-Vc, respectively, were transiently transfected to MDA-MB-231 cells, and the cells were spread on a fibronectin-coated coverslip, fixed, and counterstained with antibodies to CD63 or GFP, and the BiFC signals, and the counterstained proteins were visualized by confocal microscopy. Of note is that the BiFC signal was observed only when Vn-Bet1 and MT1-MMP-Vc were coexpressed, i.e., not when each of them was expressed together with a Vc or Vn fragment. Scale bar: 10 μm. **, P < 0.01; vs. mock in B; vs. mock at each time point in D; vs. GFP + MT1-MMP in F.