Figure 6.
Figure 6. Cofilin and cortactin phosphorylation are required for invadopodia protrusion. (A) Representative images of MDA-MB-231 cells expressing WT cortactin-RFP incubated in Geltrex + DQ Collagen IV (1:10) or Geltrex + BSA-Green 488 degradation marker. Images are representative of >50 cells analyzed in three independent experiments. (B) Representative images of WT cortactin cells embedded in the 3D matrix. The cells were imaged by confocal microscopy between 48 and 72 h after plating. The image is a 3D reconstruction of a representative field. Arrows and insets represent cells with invadopodia. The bottom right panel shows a representative TEM image of an invadopodium in a WT cortactin cell. (C) Representative images of 3YF cortactin cells embedded in the 3D matrix. The cells expressing 3YF cortactin-GFP were incubated as described (see Materials and methods). Arrows and bottom left insets represent high-magnification images of cells with invadopodia. The bottom right panel shows a representative TEM image of an invadopodium in a 3YF cortactin cell. Images are representative of 50 cells analyzed in three experiments. (D) Quantification of invadopodia length in 3D. Dominant protrusions enriched in cortactin were measured and the mean length analyzed. Approximately 20 cells per group were analyzed in four independent experiments. Where indicated, the cells were treated with specific SiRNA 24 h before seeding. The GM6001 inhibitor was used to show that the invadopodia protrusion is proteolysis dependent. (E) Quantification of cell protrusion through a 1-µm Transwell without Matrigel coating. Where indicated, cells were treated with NHE1 siRNA 72 h before the experiment and rescued with the described NHE1 constructs 24 h before the experiment (>8 fields/group, n = 3; **, P < 0.001). (F) Quantification of cell invasion through Matrigel-coated 1-µm Transwell. The number of protrusive structures crossing to the bottom of the membrane (>12 µm) was counted and normalized to the cell area on top of the membrane (>10 fields per group; **, P < 0.01).

Cofilin and cortactin phosphorylation are required for invadopodia protrusion. (A) Representative images of MDA-MB-231 cells expressing WT cortactin-RFP incubated in Geltrex + DQ Collagen IV (1:10) or Geltrex + BSA-Green 488 degradation marker. Images are representative of >50 cells analyzed in three independent experiments. (B) Representative images of WT cortactin cells embedded in the 3D matrix. The cells were imaged by confocal microscopy between 48 and 72 h after plating. The image is a 3D reconstruction of a representative field. Arrows and insets represent cells with invadopodia. The bottom right panel shows a representative TEM image of an invadopodium in a WT cortactin cell. (C) Representative images of 3YF cortactin cells embedded in the 3D matrix. The cells expressing 3YF cortactin-GFP were incubated as described (see Materials and methods). Arrows and bottom left insets represent high-magnification images of cells with invadopodia. The bottom right panel shows a representative TEM image of an invadopodium in a 3YF cortactin cell. Images are representative of 50 cells analyzed in three experiments. (D) Quantification of invadopodia length in 3D. Dominant protrusions enriched in cortactin were measured and the mean length analyzed. Approximately 20 cells per group were analyzed in four independent experiments. Where indicated, the cells were treated with specific SiRNA 24 h before seeding. The GM6001 inhibitor was used to show that the invadopodia protrusion is proteolysis dependent. (E) Quantification of cell protrusion through a 1-µm Transwell without Matrigel coating. Where indicated, cells were treated with NHE1 siRNA 72 h before the experiment and rescued with the described NHE1 constructs 24 h before the experiment (>8 fields/group, n = 3; **, P < 0.001). (F) Quantification of cell invasion through Matrigel-coated 1-µm Transwell. The number of protrusive structures crossing to the bottom of the membrane (>12 µm) was counted and normalized to the cell area on top of the membrane (>10 fields per group; **, P < 0.01).

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