Moesin localizes to invadopodia in a talin-dependent manner to promote invadopodium maturation. (A) Moesin localizes to invadopodia. Representative TIRF images of moesin and Tks5 staining in cells plated on 405-labeled gelatin. Bars: (main panel) 10 µm; (inset) 1 µm. (B) Western blot analysis of MDA-MB-231 cells transfected with control or moesin siRNA (SMARTpool) for 72 h. Blots were stained for moesin and GAPDH (loading control). (C) Quantification of the number of mature invadopodia per cell in control and moesin- and ezrin-depleted cells. n > 56 cells; three independent experiments. **, P = 0.017. (D) Coimmunoprecipitation of moesin with talin. Cells were starved for 16 h and stimulated with 2.5 nM EGF. Endogenous talin was immunoprecipitated from MDA-MB-231 cell lysates using the 8d4 talin antibody, and the resulting immunoprecipitates were subjected to Western blotting with anti-moesin, anti-ezrin, and anti-talin antibodies. The amount of talin immunoprecipitated in each of the two experiments is shown as a control. IgG served as a negative control for the immunoprecipitation; three independent experiments. (E) Quantification of moesin enrichment at invadopodia in control and talin siRNA-treated cells and then transfected with a series of GFP-tagged talin constructs. n > 46 invadopodia; three independent experiments. ***, P < 0.0023. Error bars represent the SEM. (F) In vitro direct binding pulldown assay with His-tagged talin and GST-tagged moesin (latter fused to agarose beads). Western blots are stained for talin (TD77, R11-DD) or anti-His. Three independent experiments. (G) Schematic of the talin–moesin binding interaction. The moesin N terminus binds to talin domains R11-DD (with higher affinity for R12 and R13-DD than R11). The talin R12 domain, but not R13-DD, also binds to the moesin C terminus. Solid gray lines indicate binding sites for β1 integrin, actin, and moesin; dashed line over R11 indicates lower affinity binding.