Retromer mediates cell surface recycling of MT1-MMP but not MT2-MMP. (A–C) MT2-MMP is overexpressed and invadopodia associated in MDA-MB-231 cells. (A) Indicated cells were lysed and immunoblotted with anti–MT2-MMP and anti-actin antibody. (B) Control and MT2-MMP–depleted cells were lysed and immunoblotted to determine KD levels. Subsequently, cells were seeded on Matrigel-coated cell inserts and allowed to invade for 20 h. The number of invasive cells was quantified and plotted (N = 3, scale bars = 50 µm). Values in the graph represent means ± SEM. Two-tailed Student’s t test, *** P < 0.001. (C) MDA-MB-231 cells transiently expressing mCherry–MT2-MMP and GFP-Tks5 were stained with phalloidin and DAPI and analyzed with a confocal microscope. The number of MT2-MMP endosomes positive for Tks5 was counted and plotted (N = 3, n = 45; scale bar = 10 µm, inset = 4 µm). The insets show the MT2-MMP vesicles positive for actin and Tks5. (D) Confocal images of MDA-MB-231 cells that were stably expressing GFP–MT1-MMP or transfected with mCherry–MT2-MMP and immunostained to label Vps26 and Vps35 (N = 3, n = 40; scale bars = 10 µm, inset = 4 µm). The insets show Vps35 and Vps26 localizing on the MT1-MMP or MT2-MMP endosomes. (E) MDA-MB-231 cells depleted of Vps26A and Vps35 were transfected with pHluorin–MT1-MMP or pHluorin–MT2-MMP and analyzed by TIRF microscopy. The number of vesicles and their total area was quantified and plotted (N = 3, n = 40; scale bars = 10 µm). Values are means ± SEM. One-way ANOVA, ** P < 0.01, *** P < 0.001, **** P < 0.0001. (F) mCherry–MT1-MMP and GFP-Vps29 were cotransfected in MDA-MB-231 cells and subjected to live-cell confocal imaging (Scale bars = 5 µm, inset = 4 µm). The insets show the multiple events over time where retromer is associated with the MT1-MMP endosome. (G) Surface levels of MT1-MMP were measured in control and Vps26A-depleted cells. Biotin labeling was done at 4°C for 45 min, followed by quenching and lysing the cells (refer to Materials and methods). The lysate was allowed to bind with Neutravidin beads followed by immunoblotting. Quantification of immunoblots was done to measure intensity of MT1-MMP (refer to Materials and methods; N = 3). Additional blots in Fig. S5. Values in the graph are means ± SEM. Two-tailed Student’s t test, * P < 0.05. WCL, whole cell lysate. (H) The KD efficiency of VAMP7 was measured by qPCR (N = 3). Two-tailed Student’s t test. MDA-MB-231 cells treated with indicated siRNA were surface labeled with MT1-MMP antibody at 4°C for 1 h, shifted to 37°C, fixed at 15 min, and immunostained to label with EEA1. Cells positive for MT1-MMP and EEA1 were counted and the percentage of cells that endocytosed MT1-MMP antibody was calculated. The insets represent the MT1-MMP–positive EEA1 endosome. The arrowheads in the enlarged insets show the internalized MT1-MMP antibody inside the vesicles positive for EEA1. (N = 4, n = 300; scale bars = 10 µm, inset = 4 µm). The graphs represent three independent experiments; values are means ± SEM. One-way ANOVA, * P < 0.05, ** P < 0.01. N, number of experimental repeats.
Figure 2.

Retromer mediates cell surface recycling of MT1-MMP but not MT2-MMP. (A–C) MT2-MMP is overexpressed and invadopodia associated in MDA-MB-231 cells. (A) Indicated cells were lysed and immunoblotted with anti–MT2-MMP and anti-actin antibody. (B) Control and MT2-MMP–depleted cells were lysed and immunoblotted to determine KD levels. Subsequently, cells were seeded on Matrigel-coated cell inserts and allowed to invade for 20 h. The number of invasive cells was quantified and plotted (N = 3, scale bars = 50 µm). Values in the graph represent means ± SEM. Two-tailed Student’s t test, *** P < 0.001. (C) MDA-MB-231 cells transiently expressing mCherry–MT2-MMP and GFP-Tks5 were stained with phalloidin and DAPI and analyzed with a confocal microscope. The number of MT2-MMP endosomes positive for Tks5 was counted and plotted (N = 3, n = 45; scale bar = 10 µm, inset = 4 µm). The insets show the MT2-MMP vesicles positive for actin and Tks5. (D) Confocal images of MDA-MB-231 cells that were stably expressing GFP–MT1-MMP or transfected with mCherry–MT2-MMP and immunostained to label Vps26 and Vps35 (N = 3, n = 40; scale bars = 10 µm, inset = 4 µm). The insets show Vps35 and Vps26 localizing on the MT1-MMP or MT2-MMP endosomes. (E) MDA-MB-231 cells depleted of Vps26A and Vps35 were transfected with pHluorin–MT1-MMP or pHluorin–MT2-MMP and analyzed by TIRF microscopy. The number of vesicles and their total area was quantified and plotted (N = 3, n = 40; scale bars = 10 µm). Values are means ± SEM. One-way ANOVA, ** P < 0.01, *** P < 0.001, **** P < 0.0001. (F) mCherry–MT1-MMP and GFP-Vps29 were cotransfected in MDA-MB-231 cells and subjected to live-cell confocal imaging (Scale bars = 5 µm, inset = 4 µm). The insets show the multiple events over time where retromer is associated with the MT1-MMP endosome. (G) Surface levels of MT1-MMP were measured in control and Vps26A-depleted cells. Biotin labeling was done at 4°C for 45 min, followed by quenching and lysing the cells (refer to Materials and methods). The lysate was allowed to bind with Neutravidin beads followed by immunoblotting. Quantification of immunoblots was done to measure intensity of MT1-MMP (refer to Materials and methods; N = 3). Additional blots in Fig. S5. Values in the graph are means ± SEM. Two-tailed Student’s t test, * P < 0.05. WCL, whole cell lysate. (H) The KD efficiency of VAMP7 was measured by qPCR (N = 3). Two-tailed Student’s t test. MDA-MB-231 cells treated with indicated siRNA were surface labeled with MT1-MMP antibody at 4°C for 1 h, shifted to 37°C, fixed at 15 min, and immunostained to label with EEA1. Cells positive for MT1-MMP and EEA1 were counted and the percentage of cells that endocytosed MT1-MMP antibody was calculated. The insets represent the MT1-MMP–positive EEA1 endosome. The arrowheads in the enlarged insets show the internalized MT1-MMP antibody inside the vesicles positive for EEA1. (N = 4, n = 300; scale bars = 10 µm, inset = 4 µm). The graphs represent three independent experiments; values are means ± SEM. One-way ANOVA, * P < 0.05, ** P < 0.01. N, number of experimental repeats.

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