Figure 4. Tubular connection between MT1-MMP–containing late endosomes and the invadopodial plasma membrane. (A) MDA-MB-231 cells expressing MT1-MMPmCh were plated on cross-linked gelatin. One optical section was taken in the ventral plane of the cells by confocal spinning disk microscopy (top). A second optical section was taken after a 1-min pulse of FITC-dextran added to the medium (bottom). Arrowheads point to MT1-MMP–positive endosomes filled with FITC-dextran during the pulse. The double-headed arrow points to an endosome that lost its MT1-MMP content and filled up with FITC-dextran. Bar, 5 µm. (B) MDA-MB-231 cells expressing DsRed-cortactin treated as in A and imaged immediately after the FITC-dextran pulse. Arrowheads point to cortactin-positive puncta adjacent to dextran-labeled endosomes. Bars, 5 µm. (C) MDA-MB-231 cells expressing MT1-MMPmCh (not depicted) were plated on cross-linked fluorescent gelatin, pulsed with FITC-dextran, and analyzed by confocal spinning disk microscopy 1–2 min after the pulse. Arrowheads point to focal degradation of the matrix adjacent to a FITC-labeled endosome. Dashed line represents the contour of the FITC-dextran–containing endosome projected on the gelatin image. (D and E) MDA-MB-231 cells expressing Rab7-GFP (D) or VAMP7-GFP (E) were plated on cross-linked gelatin for 5 h. An optical section was taken immediately after a 1-min pulse of A647-dextran added to the medium. Insets are higher magnification views of the boxed regions that point to Rab7- (D) or VAMP7 (E)-positive endosomes filled with A647-dextran during the pulse. Bars: (main) 5 µm; (insets) 1 µm). (F) Live-cell imaging of MDA-MB-231 cells analyzed by TIRFM immediately after the FITC-dextran pulse. The indicated times in the image gallery are seconds after the pulse. Arrowhead points to a tubular extension emanating from the FITC-dextran–labeled endosome during regurgitation of the fluid-phase tracer. Bar, 2 µm. (G) MDA-MB-231 cells expressing MT1-MMPmCh analyzed by TIRFM. The arrowhead points to an MT1-MMP–positive tubule. Bar, 1 µm.
Figure 4.

Tubular connection between MT1-MMP–containing late endosomes and the invadopodial plasma membrane. (A) MDA-MB-231 cells expressing MT1-MMPmCh were plated on cross-linked gelatin. One optical section was taken in the ventral plane of the cells by confocal spinning disk microscopy (top). A second optical section was taken after a 1-min pulse of FITC-dextran added to the medium (bottom). Arrowheads point to MT1-MMP–positive endosomes filled with FITC-dextran during the pulse. The double-headed arrow points to an endosome that lost its MT1-MMP content and filled up with FITC-dextran. Bar, 5 µm. (B) MDA-MB-231 cells expressing DsRed-cortactin treated as in A and imaged immediately after the FITC-dextran pulse. Arrowheads point to cortactin-positive puncta adjacent to dextran-labeled endosomes. Bars, 5 µm. (C) MDA-MB-231 cells expressing MT1-MMPmCh (not depicted) were plated on cross-linked fluorescent gelatin, pulsed with FITC-dextran, and analyzed by confocal spinning disk microscopy 1–2 min after the pulse. Arrowheads point to focal degradation of the matrix adjacent to a FITC-labeled endosome. Dashed line represents the contour of the FITC-dextran–containing endosome projected on the gelatin image. (D and E) MDA-MB-231 cells expressing Rab7-GFP (D) or VAMP7-GFP (E) were plated on cross-linked gelatin for 5 h. An optical section was taken immediately after a 1-min pulse of A647-dextran added to the medium. Insets are higher magnification views of the boxed regions that point to Rab7- (D) or VAMP7 (E)-positive endosomes filled with A647-dextran during the pulse. Bars: (main) 5 µm; (insets) 1 µm). (F) Live-cell imaging of MDA-MB-231 cells analyzed by TIRFM immediately after the FITC-dextran pulse. The indicated times in the image gallery are seconds after the pulse. Arrowhead points to a tubular extension emanating from the FITC-dextran–labeled endosome during regurgitation of the fluid-phase tracer. Bar, 2 µm. (G) MDA-MB-231 cells expressing MT1-MMPmCh analyzed by TIRFM. The arrowhead points to an MT1-MMP–positive tubule. Bar, 1 µm.

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