The actin binding site in the talin rod domain is required for talin localization to invadopodia and matrix degradation. (A) Representative images of MDA-MB-231 cells plated on 405-labeled gelatin, showing that WT GFP-talin, the GFP–talin rod domain, and the talin rod-LI/A mutant localize to the cortactin-rich invadopodium core. Inset shows magnified image of invadopodia in the box. Bars: (main panel) 10 µm; (inset) 1 µm. (B) Quantification of talin enrichment at invadopodia relative to cytosolic levels in talin siRNA-treated cells expressing GFP-tagged talin constructs. n > 33 invadopodia; three independent experiments. *, P = 0.032; **, P = 0.015; ***, P < 0.0019. Cartoon depicting the domain structure of talin is shown, including the C-terminal integrin binding site 2 (IBS2) and actin binding site (ABS). (C) Quantification of the number of mature invadopodia per cell in cells treated with either control or talin siRNA and transfected with GFP or GFP-tagged talin constructs. n > 41 cells; three independent experiments. *, P < 0.048; **, P = 0.0093. Error bars represent the SEM.