Figure 2.
Figure 2. Regulation of MT1-MMP–positive endosome dynamics by WASH and exocyst complex. (A and B) MDA-MB-231 cells treated with the indicated siRNAs were plated on cross-linked gelatin and stained for endogenous MT1-MMP. Bars, 5 µm. Cells were scored according to the distribution of MT1-MMP–positive endosomes, i.e., normal distribution (#1, blue boxes in B), clustered endosomes (#2, green box), and aggregated endosomes (#3, red box). Values ploted in B are mean ± SEM of normalized endosome distribution from three independent experiments scoring ∼100 cells for each siRNA. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with siNT-treated cells (B). (C) MDA-MB-231 cells expressing MT1-MMP-mCh were treated as in A and time series were acquired by spinning-disk microscopy (see Video 2). Still images from the time series are shown. Arrows point to tubular extensions emanating from MT1-MMP–positive endosomes in cells depleted of WASH. Bars, 5 µm. (D) MDA-MB-231 cells treated with the indicated siRNA were processed for immunofluorescence microscopy with antibodies against cortactin (red) and WASH (green). Images were acquired by 3D deconvolution microscopy of the middle plane of the cells. A region of interest (dashed line) was drawn manually around WASH-cortactin structures for quantification (shown in E). Bars, 5 µm. (E) Quantification of the integrated intensity of perinuclear endosomal cortactin and WASH in cells treated as in D and normalized to the integrated intensity in control cells in two independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01 compared with control. (F) MDA-MB-231 cells treated with the indicated siRNAs were processed for immunofluorescence microscopy with cortactin and WASH antibodies and analyzed by high-resolution (∼100-nm spatial resolution) 3D SIM. Bar, 1 µm. (G) Quantification of the projected surface of WASH and cortactin puncta in cells as described in F, normalized to mean projected surface of the puncta in control cells set to 100 (three independent experiments; n = 25 puncta for each condition; *, P < 0.05; ***, P < 0.001 compared with control).

Regulation of MT1-MMP–positive endosome dynamics by WASH and exocyst complex. (A and B) MDA-MB-231 cells treated with the indicated siRNAs were plated on cross-linked gelatin and stained for endogenous MT1-MMP. Bars, 5 µm. Cells were scored according to the distribution of MT1-MMP–positive endosomes, i.e., normal distribution (#1, blue boxes in B), clustered endosomes (#2, green box), and aggregated endosomes (#3, red box). Values ploted in B are mean ± SEM of normalized endosome distribution from three independent experiments scoring ∼100 cells for each siRNA. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with siNT-treated cells (B). (C) MDA-MB-231 cells expressing MT1-MMP-mCh were treated as in A and time series were acquired by spinning-disk microscopy (see Video 2). Still images from the time series are shown. Arrows point to tubular extensions emanating from MT1-MMP–positive endosomes in cells depleted of WASH. Bars, 5 µm. (D) MDA-MB-231 cells treated with the indicated siRNA were processed for immunofluorescence microscopy with antibodies against cortactin (red) and WASH (green). Images were acquired by 3D deconvolution microscopy of the middle plane of the cells. A region of interest (dashed line) was drawn manually around WASH-cortactin structures for quantification (shown in E). Bars, 5 µm. (E) Quantification of the integrated intensity of perinuclear endosomal cortactin and WASH in cells treated as in D and normalized to the integrated intensity in control cells in two independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01 compared with control. (F) MDA-MB-231 cells treated with the indicated siRNAs were processed for immunofluorescence microscopy with cortactin and WASH antibodies and analyzed by high-resolution (∼100-nm spatial resolution) 3D SIM. Bar, 1 µm. (G) Quantification of the projected surface of WASH and cortactin puncta in cells as described in F, normalized to mean projected surface of the puncta in control cells set to 100 (three independent experiments; n = 25 puncta for each condition; *, P < 0.05; ***, P < 0.001 compared with control).

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