WASH- and cortactin-positive MT1-MMP–containing endosomes are recruited in the periphery of linear invadopodia in association with collagen fibers. (A) MDA-MB-231 cells on a layer of collagen I fibers stained with anti-Col1-^3/4C antibodies recognizing MMP-cleaved collagen I (red) and for F-actin (green) revealing collagenolytic linear invadopodia. Inset is a higher magnification of the boxed region. Bars, 5 µm. (B and C) Cells were plated on a layer of Alexa Fluor 647–conjugated type I collagen (blue) for 30 min and stained for N-WASP (green) and F-actin (red; B) or WASH (green) and cortactin (red; C). Insets are higher magnification of boxed regions. Bars: (main and B [inset]) 5 µm; (C, inset) 1 µm. (D) MDA-MB-231 cells expressing GFP-WASH (green) and DsRed-cortactin (red) were plated on a layer of Alexa Fluor 647–conjugated type I collagen (blue) for 30 min and imaged by confocal spinning disk microscopy. A still image from a representative time-lapse series is shown (t = 25 min). Arrowheads point to WASH- and cortactin-positive puncta (associated with late endosomes) apposed to a cortactin-positive linear invadopodium aligned on collagen fibers. Insets show a higher magnification of WASH and cortactin endosomal puncta. The corresponding time-lapse series is shown in Video 4. Bars: (main) 5 µm; (inset) 1 µm. (E) Higher magnification of the boxed region in D at representative time points. A linear cortactin-positive invadopodium aligned with collagen fibers forms on the inner face of the plasma membrane (13–14 min; red arrowheads); at 60 min, this structure disassembled while collagen I fibers were remodeled. Time is in minutes. Bar, 5 µm. (F–H) Cells on Alexa Fluor 647–conjugated type I collagen (blue) stained for MT1-MMP (red) and cortactin (green). G and H are higher magnification of the boxed region in F. Arrows point to cortactin-positive puncta associated with MT1-MMP–containing endosomes in the vicinity of cortactin- and MT1-MMP–positive linear invadopodia (arrowheads). Bars, 5 µm.