Figure 3. Association of WASH endosomal puncta with invadopodia. (A) MDA-MB-231 cells plated on cross-linked FITC-labeled gelatin (blue) were stained with antibodies against MT1-MMP (red) and WASH (green) and imaged by 3D deconvolution microscopy. Image is a single section in the ventral plane of the cell in contact with gelatin. Insets corresponding to the boxed region show split channels with dashed line representing the contour of the MT1-MMP–containing endosome projected on the FITC-gelatin image. Arrowhead points to matrix degradation restricted to WASH puncta adjacent to MT1-MMP–positive endosome. Bars: (main) 5 µm; (inset) 1 µm. (B) Orthogonal section through the dashed line in A. From left to right: merged image, MT1-MMP (M), WASH (W), and FITC-gelatin (G) signals. (C) Fluorescence intensity profile through the dashed line in the boxed region in A (x-axis, in pixels; y-axis, in arbitrary units). (D and E) MDA-MB-231 cells expressing MT1-MMPmCh and GFP-WASH (D) or GFP-Exo84 (E) plated on cross-linked gelatin and analyzed by live-cell dual color TIRFM. Time interval between each image is 1 s. Bars, 1 µm. (F) BT-549 cells plated on cross-linked gelatin were fixed and immunostained for MT1-MMP and WASH and analyzed by TIRFM. Insets are higher magnification views of the boxed regions. (G and H) Cells treated as in A, stained with antibodies against MT1-MMP (red) and p34-Arc subunit of Arp2/3 complex (G, green) or FAM21 (H, green). Arrowheads point to the accumulation of the markers near degradative invadopodia adjacent to MT1-MMP–positive endosomes. Bars: (main) 5 µm; (inset) 1 µm. (I) MDA-MB-231 cells expressing GFP-cortactin and MT1-MMP-mCh plated on cross-linked gelatin and analyzed by dual color TIRFM. A still image from a representative time-lapse series is shown. (J) A series of time-lapse TIRFM images corresponding to the boxed region in panel I. Numbers in the series of time-lapse images represent the time in minutes. Two events of docking of MT1-MMP–containing endosomes (labeled #1 and #2) are boxed in pink. (K) Cells as in A were stained for cortactin and WASH or N-WASP. The graph shows the percentage of WASH- (pink bars) or N-WASP–positive (blue bars) degradative invadopodia positive for cortactin (not depicted) as a function of the surface of matrix degradation (in pixels). Values are means ± SEM scoring 360 invadopodia from 15 WASH-labeled cells and 135 invadopodia from five N-WASP–labeled cells.
Figure 3.

Association of WASH endosomal puncta with invadopodia. (A) MDA-MB-231 cells plated on cross-linked FITC-labeled gelatin (blue) were stained with antibodies against MT1-MMP (red) and WASH (green) and imaged by 3D deconvolution microscopy. Image is a single section in the ventral plane of the cell in contact with gelatin. Insets corresponding to the boxed region show split channels with dashed line representing the contour of the MT1-MMP–containing endosome projected on the FITC-gelatin image. Arrowhead points to matrix degradation restricted to WASH puncta adjacent to MT1-MMP–positive endosome. Bars: (main) 5 µm; (inset) 1 µm. (B) Orthogonal section through the dashed line in A. From left to right: merged image, MT1-MMP (M), WASH (W), and FITC-gelatin (G) signals. (C) Fluorescence intensity profile through the dashed line in the boxed region in A (x-axis, in pixels; y-axis, in arbitrary units). (D and E) MDA-MB-231 cells expressing MT1-MMPmCh and GFP-WASH (D) or GFP-Exo84 (E) plated on cross-linked gelatin and analyzed by live-cell dual color TIRFM. Time interval between each image is 1 s. Bars, 1 µm. (F) BT-549 cells plated on cross-linked gelatin were fixed and immunostained for MT1-MMP and WASH and analyzed by TIRFM. Insets are higher magnification views of the boxed regions. (G and H) Cells treated as in A, stained with antibodies against MT1-MMP (red) and p34-Arc subunit of Arp2/3 complex (G, green) or FAM21 (H, green). Arrowheads point to the accumulation of the markers near degradative invadopodia adjacent to MT1-MMP–positive endosomes. Bars: (main) 5 µm; (inset) 1 µm. (I) MDA-MB-231 cells expressing GFP-cortactin and MT1-MMP-mCh plated on cross-linked gelatin and analyzed by dual color TIRFM. A still image from a representative time-lapse series is shown. (J) A series of time-lapse TIRFM images corresponding to the boxed region in panel I. Numbers in the series of time-lapse images represent the time in minutes. Two events of docking of MT1-MMP–containing endosomes (labeled #1 and #2) are boxed in pink. (K) Cells as in A were stained for cortactin and WASH or N-WASP. The graph shows the percentage of WASH- (pink bars) or N-WASP–positive (blue bars) degradative invadopodia positive for cortactin (not depicted) as a function of the surface of matrix degradation (in pixels). Values are means ± SEM scoring 360 invadopodia from 15 WASH-labeled cells and 135 invadopodia from five N-WASP–labeled cells.

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