Fig. 5
From: MT1-MMP directs force-producing proteolytic contacts that drive tumor cell invasion
MT1-MMP is required for invadopodia assembly independently of its catalytic activity. a MDA-MB-231 cells expressing MT1-MMPpHLuorin were cultured on type I collagen (gray), stained for GFP (green), Tks5 (red) and DAPI (blue). Right panels are zoom-in of the boxed region with separated channels. Arrowheads point to surface MT1-MMPpHLuorin signal localized to a Tks5-positive invadopodium in association with a collagen fiber. Normalized fluorescence intensity profiles along the white line are shown in the inset. EL, perinuclear endolysosomes positive for MT1-MMPpHLuorin. Scale bar, 10 µm; 5 µm in zoom-in. b Cells were treated with siRNA against MT1-MMP 3′ and 5′ UTR sequences (siMT1UTR) and plated on collagen (gray) and stained for F-actin (green), Tks5 (red) and DAPI (blue). Bottom panels are zoom-in of the boxed region with separated channels. Scale bar, 10 µm; 5 µm in zoom-in. c Cells treated with siMT1UTR siRNA as in panel b were transfected with MT1-MMPpHLuorin rescue construct and stained for GFP to detect MT1-MMPpHLuorin (green), Tks5 (red) and DAPI (blue). Bottom panels are zoom-in of the boxed region with separated channels. Arrowheads point to MT1-MMPpHLuorin in Tks5-positive invadopodia. Scale bar, 10 µm; 5 µm in zoom-in. The cell contour is shown with a dashed line (panels a–c). d Quantification of Tks5-positive invadopodia in MDA-MB-231 cells knocked down for MT1-MMP or mock-treated and rescued with the indicated MT1-MMP constructs (see Supplementary Figure 5). The Y-axis is the ratio of the Tks5 area to the total cell area normalized to the mean value of corresponding Mock-treated cells (as percentage). n: number of cells analyzed; (n): number of independent experiments. Mann–Whitney tests. e Quantification of Tks5-positive invadopodia in MDA-MB-231 mock- (EtOH) or GM6001 (GM)-treated cells plated on type I collagen (see images in Supplementary Figure 5d,e) as in d. n number of cells analyzed from three independent experiments. Mann–Whitney test. f Rupture index (i.e. rupture events/cell/hr) calculated in mock- and GM-treated cells. Data are presented as the mean from three independent experiments; Mann–Whitney test. n: cell number; (n): invadopodia number. Error bars SEM, ** P < 0.01, **** P < 0.0001; ns not significant
