Localization of IQGAP1 and Sec8 at F-actin–rich invadopodia in MDA-MB-231 cells plated on cross-linked fluorescent gelatin. (a) MDA-MB-231 cells were plated on fluorescent FITC-gelatin, and, after 5 h, cells were fixed and stained for immunofluorescence microscopy with anti-IQGAP1 antibodies and fluorescent phalloidin. F-actin and endogenous IQGAP1 colocalize at invadopodia corresponding to proteolytic holes in the fluorescent gelatin matrix (right). (b) Phalloidin and anti-V5 staining of MDA-MB-231 cells transiently expressing V5-tagged Sec8 after 5 h on FITC-gelatin showing colocalization of F-actin and overexpressed Sec8 at invadopodia. (c) MDA-MB-231 cells transiently transfected with V5-tagged Sec8 together with HA–MT1-MMP and Y527F c-Src were plated for 4 h on AlexaFluor350-gelatin and stained for immunofluorescence microscopy with the indicated antibodies. Higher magnification views of the boxed areas are shown underneath each image. Bars, 10 μm.