Figure 6.
Figure 6. MT1-MMP traffics from a late endosomal compartment to the plasma membrane and associates with N-WASP. (A) NT and N-WASP knockdown cells invading in CIA were fixed and stained with anti-Arp2/3 and cortactin to reveal actin-rich puncta (arrowheads) in NT cells (top). (B) Immunofluorescence images of invading cells expressing mCherry-MT1-MMP (red) and staining for endogenous N-WASP (green) in CIA, with arrows pointing toward the wound edge. Bar, 10 µm. Enlarged images show details (boxed regions) of the invasive pseudopods containing MT1-MMP vesicles and N-WASP puncta. (C) Endogenous MT1-MMP vesicles colocalize with endogenous Rab7. Arrows point toward the wound edge. Bar, 20 µm. Inset images show the LE/LY vesicles containing both MT1-MMP and Rab7 (enlarged views of the boxed regions). (D) MDA-MB-231 cells expressing PA mCherry-MT1-MMP (red) and GFP-Rab7 (green) were plated in CIA and imaged by confocal microscopy. Photo-activation was achieved with a 405-nm laser aimed at a small area (region A) containing GFP-Rab7–positive vesicles (also MT1-MMP), marked by the red box. Images were then captured at 3.2 s per frame over a period of >6 min (Video 6). Single-section confocal images of activated mCherry-MT1-MMP and images of merged mCherry-MT1-MMP and GFP-Rab7 at certain time points were presented. The insets are enlarged images showing increased signals of photoactivated mCherry-MT1-MMP on the plasma membrane area near the activated vesicles. The enlarged area is indicated with the yellow dotted line at time point “2 m 29 s.” The same areas are shown in time points “0 s” and “3 m 37 s.” The integrated fluorescence intensity of activated region A (red) and a area of plasma membrane (region B) near the activated vesicles (light blue) was quantified for each frame of Video 6, and the values are plotted against elapsed time. (E) Quantification of fluorescence intensity of activated mCherry-MT1-MMP vesicles in multiple experiments indicated the exit rate of photoactivated mCherry-MT1-MMP from the Rab7-positive compartment (n = 10). Error bars indicate means ± SEM. Bars: (A) 20 µm; (B) 5 µm; (C) 1 µm; (D) 10 µm; (B–D, insets) 1 µm.

MT1-MMP traffics from a late endosomal compartment to the plasma membrane and associates with N-WASP. (A) NT and N-WASP knockdown cells invading in CIA were fixed and stained with anti-Arp2/3 and cortactin to reveal actin-rich puncta (arrowheads) in NT cells (top). (B) Immunofluorescence images of invading cells expressing mCherry-MT1-MMP (red) and staining for endogenous N-WASP (green) in CIA, with arrows pointing toward the wound edge. Bar, 10 µm. Enlarged images show details (boxed regions) of the invasive pseudopods containing MT1-MMP vesicles and N-WASP puncta. (C) Endogenous MT1-MMP vesicles colocalize with endogenous Rab7. Arrows point toward the wound edge. Bar, 20 µm. Inset images show the LE/LY vesicles containing both MT1-MMP and Rab7 (enlarged views of the boxed regions). (D) MDA-MB-231 cells expressing PA mCherry-MT1-MMP (red) and GFP-Rab7 (green) were plated in CIA and imaged by confocal microscopy. Photo-activation was achieved with a 405-nm laser aimed at a small area (region A) containing GFP-Rab7–positive vesicles (also MT1-MMP), marked by the red box. Images were then captured at 3.2 s per frame over a period of >6 min (Video 6). Single-section confocal images of activated mCherry-MT1-MMP and images of merged mCherry-MT1-MMP and GFP-Rab7 at certain time points were presented. The insets are enlarged images showing increased signals of photoactivated mCherry-MT1-MMP on the plasma membrane area near the activated vesicles. The enlarged area is indicated with the yellow dotted line at time point “2 m 29 s.” The same areas are shown in time points “0 s” and “3 m 37 s.” The integrated fluorescence intensity of activated region A (red) and a area of plasma membrane (region B) near the activated vesicles (light blue) was quantified for each frame of Video 6, and the values are plotted against elapsed time. (E) Quantification of fluorescence intensity of activated mCherry-MT1-MMP vesicles in multiple experiments indicated the exit rate of photoactivated mCherry-MT1-MMP from the Rab7-positive compartment (n = 10). Error bars indicate means ± SEM. Bars: (A) 20 µm; (B) 5 µm; (C) 1 µm; (D) 10 µm; (B–D, insets) 1 µm.

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