Figure 1.
Figure 1. Talin localizes to invadopodia and is required for their stabilization and maturation. (A) Talin localizes to the invadopodium core in MDA-MB-231 cells plated on Alexa Fluor 405–labeled gelatin for 4 h. Representative confocal images of talin and Tks5 staining. Red arrowheads denote mature invadopodia with colocalization of talin and Tks5 in the invadopodium core. Inset shows magnified image of invadopodia in the box. Bars: (main panel) 10 µm; (inset) 1 µm. (B) Western blot analysis of MDA-MB-231 cells transfected with control or talin1 siRNA (SMARTpool) for 96 h. Blots were stained for talin and GAPDH (loading control). (C–E) Steady-state invadopodial matrix degradation assay. (C) Representative images of MDA-MB-231 cells stained for cortactin and Tks5. White arrowheads denote mature invadopodia; blue arrowheads denote invadopodium precursors. Inset shows magnified image of invadopodia in the box. Bars: (main panel) 10 µm; (inset) 1 µm. (D) Quantification of the number of mature invadopodia per cell. Mature invadopodia are defined as cortactin-/Tks5-rich punctate structures colocalized with a matrix degradation hole. n > 52 cells; three independent experiments. **, P < 0.0076. (E) Quantification of invadopodial matrix degradation area per cell. n > 52 cells; three independent experiments. **, P = 0.0017. (F) EGF stimulation assay. Quantification of the number of invadopodium precursors in MDA-MB-231 cells stimulated with 2.5 nM EGF for 0 (unstimulated) or 3 min. Precursors are defined as cortactin-/F-actin–rich punctate structures that do not colocalize with a matrix degradation hole. n > 58 cells; three independent experiments. **, P < 0.018. (G) Quantification of invadopodium precursor, mature invadopodium, and mean (pooled) invadopodium lifetimes in MDA-MB-231 cells treated with control or talin siRNA and transfected with TagRFP-cortactin and GFP-Tks5 plated on 405-labeled gelatin. Cells were imaged for 3 h using time lapse microscopy (see Videos 1 and 2). n > 46 precursors; n > 28 mature invadopodia; three independent experiments. ***, P < 0.00013. Error bars represent the SEM.

Talin localizes to invadopodia and is required for their stabilization and maturation. (A) Talin localizes to the invadopodium core in MDA-MB-231 cells plated on Alexa Fluor 405–labeled gelatin for 4 h. Representative confocal images of talin and Tks5 staining. Red arrowheads denote mature invadopodia with colocalization of talin and Tks5 in the invadopodium core. Inset shows magnified image of invadopodia in the box. Bars: (main panel) 10 µm; (inset) 1 µm. (B) Western blot analysis of MDA-MB-231 cells transfected with control or talin1 siRNA (SMARTpool) for 96 h. Blots were stained for talin and GAPDH (loading control). (C–E) Steady-state invadopodial matrix degradation assay. (C) Representative images of MDA-MB-231 cells stained for cortactin and Tks5. White arrowheads denote mature invadopodia; blue arrowheads denote invadopodium precursors. Inset shows magnified image of invadopodia in the box. Bars: (main panel) 10 µm; (inset) 1 µm. (D) Quantification of the number of mature invadopodia per cell. Mature invadopodia are defined as cortactin-/Tks5-rich punctate structures colocalized with a matrix degradation hole. n > 52 cells; three independent experiments. **, P < 0.0076. (E) Quantification of invadopodial matrix degradation area per cell. n > 52 cells; three independent experiments. **, P = 0.0017. (F) EGF stimulation assay. Quantification of the number of invadopodium precursors in MDA-MB-231 cells stimulated with 2.5 nM EGF for 0 (unstimulated) or 3 min. Precursors are defined as cortactin-/F-actin–rich punctate structures that do not colocalize with a matrix degradation hole. n > 58 cells; three independent experiments. **, P < 0.018. (G) Quantification of invadopodium precursor, mature invadopodium, and mean (pooled) invadopodium lifetimes in MDA-MB-231 cells treated with control or talin siRNA and transfected with TagRFP-cortactin and GFP-Tks5 plated on 405-labeled gelatin. Cells were imaged for 3 h using time lapse microscopy (see Videos 1 and 2). n > 46 precursors; n > 28 mature invadopodia; three independent experiments. ***, P < 0.00013. Error bars represent the SEM.

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