Fig. 4

Ultrastructural organization of collagenolytic invadopodia. a Platinum replica electron microscopy (PREM) survey view of the cytoplasmic surface of the adherent plasma membrane in unroofed MDA-MB-231 cells plated for 60 min on a thin layer of collagen I (image is inverted). Extracellular space is pseudo-colored in purple. Inset is a zoom-in view of the boxed region to show the continuity of the collagen fibers outside (yellow arrowhead) and underneath the ventral plasma membrane (white arrowheads). Scale bars, 10 µm; 1 µm (inset). b PREM image showing increased electron density (pseudo-colored in yellow) corresponding to curvilinear collagen fibers underneath the ventral plasma membrane. Scale bar, 5 µm. c Zoomed-in region corresponding to the boxed region in panel b. Arrowheads indicate bow-shaped proteinaceous material in association with the membrane above a collagen fiber. Scale bar, 1 µm. d High magnification PREM view of a bow-shaped collagen fiber (arrowheads) and associated branched actin network (pseudo-colored in yellow) along the concave side of the fiber, i.e. an invadopodium. Clathrin-coated pits (CCP), some pinching the underlying collagen fibers, are visible. Inset is a zoomed-in view of the boxed region showing the differential width of ~ 10 nm-wide actin filaments and ~ 15 nm wide intermediate filament. Scale bars, 200 nm; 100 nm (inset). e, f Anti-GFP immunogold PREM of MDA-MB-231 cells expressing Arp2/3 complex subunit ArpC5B^GFP. Immunogold beads are pseudo-colored in yellow. A zoomed-out PREM image corresponding to panel e is shown in Supplementary Figure 1c. Collagen fibers, arrowheads. Scale bars, 200 nm. g, h Anti-GFP immunogold PREM analysis of MDA-MB-231 cells expressing Tks5^GFP. Panel h is a zoomed-in view of the boxed region in g. A zoomed-out PREM image corresponding to panel e is shown in Supplementary Figure 1f. Collagen fibers, arrowheads. Scale bars, 200 nm