(A) Intravital imaging of tumors generated by injection of MTLn3 EGFP-Mena cells into rats. Scale bar, 20 μm. Green = EGFP-Mena; purple = collagen second harmonic.
(B) Intravital imaging of tumors generated by injection of MTLn3 EGFP-Mena cells into rats. Scale bar, 20 μm. Green = EGFP-Mena; purple = collagen second harmonic. Protrusion containing Mena is outlined in white.
(C) Table showing the quantification of motile cells in orthotopic breast tumors derived from injection of MTLn3 cells into SCID mice. *p < 0.002, **p < 0.04, unpaired t test with Welch’s correction for unequal variance.
(D) Average weight of primary tumors in xenograft SCID mice derived from injection of each cell type. Error bars indicate SEM.
(E) Spontaneous lung metastases quantified using multiphoton microscopy. Insert depicts picture of lung metastasis from an SCID mouse with a tumor derived from injection of MTLn3-EGFP-Mena^INV, taken using multiphoton microscopy. Green = MTLn3-EGFP-Mena^INV; purple = collagen second harmonic. Scale bar, 50 μm. Error bars indicate SEM. **p < 0.001; ***p < 0.00001.
(F) Spontaneous lung metastases quantified using H&E stained lung sections. Insert depicts H&E staining of lung section from MTLn3-EGFP-Mena^INV-derived xenograft. Dark purple = metastasis. Scale bar, 20 μm. Error bars indicate SEM. ***p < 0.001.

(A) 3D Collagen invasion assay using MTLn3 cells expressing Mena or Mena^INV in the absence (white bars) and presence (gray bars) of macrophages. Error bars indicate SEM. *p < 0.005 compared to parental MTLn3 cells; ***p < 0.005 compared to MTLn3 Mena cells.
(B) 3D Collagen invasion assay using MTLn3 cells expressing Mena^INV in the presence of DMSO or 6 μM AG1478. Error bars indicate SEM.
(C) 3D reconstruction of MTLn3 EGFP-Mena cells transiently transfected with RFP-cortactin invading collagen I. Arrows indicate 3D protrusions (collagen I gel on bottom). Scale bar, 10 μm. Higher magnifications of the protrusion are shown on the bottom right; scale bar, 5 μm.
(D) Schematic diagram of the invadopodium assay. Images in (E) and (G) represent the plane of the matrix in this diagram.
(E) Immunofluorescence of MTLn3 cells using anti-Mena or anti-cortactin antibodies. Arrowheads indicate invadopodia. Scale bar, 10 μm.
(F) Matrix degradation by MTLn3 cells expressing EGFP, EGFP-Mena, or EGFP-Mena^INV. Bars represent the mean of two independent experiments and over 40 cells analyzed. Error bars indicate SEM. *p < 0.00005.
(G) Images of Texas Red-fibronectin matrix indicating degradation by invadopodia visible as black holes (arrowheads). Scale bar, 10 μm.
(H) Analysis of invadopodium lifetime of MTLn3 GFP-Actin cells transiently transfected with mCherry-Mena or mCherry-Mena^INV. Non-transfected cells on the same dish served as control. Error bars indicate SEM. *p < 0.005.
(I) Histogram showing the distribution of invadopodium lifetimes.

(A) EGF dose-response curve of EGFP- and EGFP-Mena^INV-expressing MTLn3 cells. The area of the cells was determined 3 min after stimulation with the indicated EGF concentrations and normalized to the cell area before EGF treatment. Results represent duplicates; over 30 cells analyzed. Error bars indicate SEM.
(B) In vivo invasion assay: EGF dose-response curve of invasive cells collected from EGFP and EGFP-Mena^INV tumors. Data normalized to average number of cells in needles containing 0.0 nM EGF for each type of xenograft. Error bars indicate SEM.
(C) Lamellipod protrusion after 0.5 nM EGF stimulation. Results represent triplicates; over 45 cells analyzed. Error bars indicate SEM. Magnification of the boxed area from the left graph is shown on the right.
(D) Lamellipod protrusion after 5 nM EGF stimulation. Results represent triplicates; over 40 cells analyzed. Error bars indicate SEM.

(A) Immunofluorescence of MTLn3 cells using anti-Mena antibody and Alexa594-phalloidin. Time in seconds after 5 nM EGF stimulation. Scale bar, 10 μm.
(B) Immunofluorescence of MTLn3 EGFP-Mena cells stained for F-actin 1 min after EGF stimulation. Scale bar, 5 μm. Lower panel: frames with 10 s intervals of MTLn3 EGFP-Mena cells. Time in seconds after 5 nM EGF stimulation. Scale bar, 5 μm.
(C) Kinetics of EGFP-Mena leading-edge recruitment. Time in seconds after 5 nM EGF stimulation. The mean fluorescence intensity of 10 different areas of the leading edge is plotted and normalized to the fluorescent intensity before EGF stimulation. Error bars represent SEM.
(D) Immunofluorescence of MTLn3 EGFP-Mena cells using anti-p34Arc antibody and Alexa350-phalloidin. Time in seconds after 5 nM EGF stimulation. Scale bar, 10 μm.
(E) Immunofluorescence of MTLn3 EGFP-Mena cells using anti-p34Arc antibody and Alexa350-phalloidin 1 min after 5 nM EGF stimulation. Scale bar, 5 μm.
(F) Representative line scans of the leading edge of MTLn3 EGFP-Mena cells stained for Arp2/3 (red) and F-actin (blue) at indicated time points after 5 nM EGF stimulation. Arrowheads indicate the leading edge of the cell.

(A) Immunofluorescence of MTLn3 EGFP-Mena cells using anti-p34Arc antibody and Alexa350 phalloidin at indicated time points after 5 nM EGF stimulation in the presence of DMSO or 50 nM CD. Scale bar, 5 μm.
(B) Representative line scans of the leading edge of MTLn3 EGFP-Mena cells stained for Arp2/3 (red) and F-actin (blue) at indicated time points after 5 nM EGF stimulation in the presence of DMSO or 50 nM CD. Arrowheads indicate the leading edge of the cell.
(C) Schematic of signaling cascades leading to protrusion and motility.

(A) Barbed end incorporation after 0.5 nM EGF stimulation in MTLn3 EGFP, EGFP-Mena, or EGFP-Mena^INV cells using Biotin-labeled actin. Scale bar, 10 μm.
(B) Relative number of barbed-end incorporation at the leading edge 1 and 3 min after 0.5 nM EGF stimulation. Results represent duplicates; over 30 cells analyzed. Error bars indicate SEM.
(C) Relative number of barbed end incorporation at the leading edge 20, 40, and 60 s after 0.5 nM EGF stimulation. Results represent triplicates; over 40 cells analyzed. Error bars indicate SEM.
(D) Barbed end incorporation after 5 nM EGF stimulation in MTLn3 EGFP-FP₄-Mito or EGFP-AP₄-Mito cells using Biotin-labeled actin. Scale bar, 10 μm.
(E) Relative number of barbed-end incorporation at the leading edge after 5 nM EGF stimulation in MTLn3-EGFP-AP4 or EGfP-FP4-Mito cells. Results represent triplicates; over 40 cells analyzed. Error bars indicate SEM.
(F) Relative number of barbed-end incorporation at the leading edge after 5 nM EGF stimulation. Results represent duplicates; over 35 cells analyzed. Error bars indicate SEM.

In unstimulated cells, actin filaments are capped. EGF stimulation leads to PLCγ-mediated release and activation of cofilin from PIP₂. Cofilin severs capped actin filaments and Mena^INV binds newly generated free barbed ends. Actin filament elongation and the resulting membrane protrusion are supported by the recruitment of polymerization-competent monomers through profilin binding and the anticapping activity of Mena. Subsequently, the Arp2/3 nucleates new filaments that branch off the growing filaments.