EGFP-SV punctae colocalize with cortactin and exogenous Tks5 and Cdc42 but not with phospho-FAK or vinculin. COS7 cells expressing EGFP-SV (A, D, G, J, M, and P) were stained with antibodies against endogenous cortactin (B), phosphotyrosine (pTyr; K), vinculin (N), or phosphorylated FAK (pFAK; Q) or with antibodies against tags on exogenously expressed Tks5 (E) or Cdc42 (H). EGFP-SV is green in the merged images (C, F, I, L, O, and R); other proteins are shown in magenta; overlap is white. Insets, fourfold enlargements of areas within boxes. Arrows denote focal adhesions. Bar, 10 μm.

Expression of EGFP-SV leads to redistribution of cortactin. (A) COS7 cells expressing EGFP alone (a; green in c) or EGFP-SV (d; green in f) for 48 h were immunostained for endogenous cortactin (b and e; red in c and f). Cells were scored as containing (arrowheads) or lacking (arrows) peripheral cortactin. Bar, 50 μm. (B) Percentages of cells with continuous peripheral cortactin staining 48 h after mock transfection (Mock) or expression of EGFP alone (EGFP) or EGFP-SV (EGFP-SV). Means ± SD; ***p < 0.0001. (C) Two-color FACS analyses showing no change in the levels of total cellular cortactin caused by expression of EGFP or EGFP-SV in COS7 cells. Control, secondary antibody alone.

EGFP-SV redistributes lamellipodin/RAPH1/PREL1, but not filamin A, within 24 h. (A and D) COS7 cells expressing EGFP alone (a) or EGFP-SV (c) for 24 h were immunostained for endogenous lamellipodin (b and d in A) or filamin A (b and d in D). Cells were scored as containing (arrowheads) or lacking (arrows) peripherally localized protein. Bars, 50 μm. Percentages of cells with peripheral (B) lamellipodin or (E) filamin staining 24 h after mock transfection (Mock) or expression of EGFP alone (EGFP) or EGFP-SV (EGFP-SV). Means ± SD; *p < 0.05; n = 2 (B); n = 3 (E). Overlap (arrows) or lack of overlap (arrowheads) of EGFP-SV punctae with endogenous lamellipodin (C) and filamin (F). Bars, 20 μm.

Cortactin binds SV in vitro and contributes to the formation of EGFP-SV–induced punctae in vivo. (A) His-cortactin(ΔSH3) (input, lane 1) was mixed with glutathione-Sepharose beads and the indicated GST fusion proteins (lanes 2 and 4–8) or with glutathione-Sepharose beads alone (lane 3) for 1 h. Beads were washed, pelleted, and heated in SDS sample buffer and interacting proteins were subjected to SDS-polyacrylamide gel electrophoresis and stained with Coomassie blue (a) or transferred to nitrocellulose and immunoblotted for the His tag (b) (n = 3). Arrowhead denotes His-cortactin(ΔSH3). Input lane (a) was digitally moved to match immunoblot (b). (B) Cortactin protein levels in COS7 cells 4 d after transfection with control siRNA (CON, lane 1) or cortactin siRNA (CT1, CT2, lanes 2, 3). β-actin was the loading control. Percent of cortactin remaining is shown under cortactin bands (n = 1). (C) Cortactin knockdown cells were transfected with plasmids encoding EGFP or EGFP-SV at day 3 of siRNA treatment, and F-actin punctae were visualized with phalloidin after an additional 24 h (n = 3). Statistically significant differences were determined by unpaired two-tailed Student's t tests and with the Student-Newman-Keuls multiple comparison test (*p < 0.05, ***p < 0.001).

Endogenous supervillin localizes to the cores of Src-induced podosomes. COS7 cells were transfected with Y527F Src, immunostained for SV, and stained for F-actin with phalloidin (A) or with antibody against endogenous cortactin (B), α-actinin (C), pFAK (D), or vinculin (E) and visualized after deconvolution of images acquired with 0.2-μm Z-steps. F-actin and cortactin localize to podosome cores, α-actinin to cores + rings, and vinculin and pFAK to podosome rings (Linder and Aepfelbacher, 2003). Individual podosomes in the XY view (arrowheads) have been enlarged to the same scale as the XZ and YZ views. SV, green; other proteins magenta; and overlaps in white. Bars, 10 micrometers in main image, 1 μm in inset.

Supervillin localizes at and within MDA-MB-231 cell invadopodia. (A) MDA-MB-231 cells were plated on unlabeled gelatin for 3 h, fixed, and stained with antibodies (SV and cortactin) and/or phalloidin (F-actin) as noted, and then they were imaged by wide-field microscopy. Merges show supervillin (red), cortactin (green), and F-actin (blue). Bars, 10 μm (top row) and 1 μm (bottom row). (B) MDA-MB-231/WT Src cells were transfected with mRFP-SV and EGFP-MT1-MMP. Cells were plated on unlabeled gelatin-coated coverslips for 6 h, fixed, and stained with phalloidin (F-actin) and imaged by wide-field microscopy. Merges show mRFP-SV (red), EGFP-MT1-MMP (green), and F-actin (blue). Arrows indicate colocalization; arrowheads denote proximate localization. (C) MDA-MB-231 cells were plated on TRITC-labeled gelatin for 6 h, fixed, and stained with anti-SV antibodies, and then they were imaged by confocal microscopy as a Z-series with 0.2-μm steps. Merges show supervillin (green) and TRITC-gelatin (red). An XY view of such a cell is shown (top row). Areas inside the boxes were enlarged fourfold (second row). Eightfold enlargements of XZ and YZ views corresponding to a, b, c, and d are shown below XY views. Bars, 10 μm (top row), 5 μm (second row), and 1 μm (bottom row).

Overexpression of EGFP-SV increases the number of matrix holes formed per cell. MDA-MB-231/WT Src cells were transfected with either EGFP alone (A) or EGFP-SV (B) or they were mock transfected (C). Cells were plated on TRITC-labeled gelatin for 6 h before visualization by green (a) and red (b) fluorescence and phase contrast (d); merged green and red images also are shown (c). Bar, 20 μm. (D) The numbers of holes in the red gelatin matrix (representative of invadopodia) per cell were counted. EGFP-SV cells had a significant (***p < 0.001; n = 8) increase in the number of associated holes per cell. (E) The percentage of cells with some matrix degradation was not significantly different among the three conditions.

Underexpression of endogenous SV decreases the numbers of matrix holes per cell, especially under the center of the cell. MDA-MB-231/WT Src cells were transfected with either control siRNA (Con) or siRNAs targeting SV (SV1 and SV2), the related protein, gelsolin (Gel), or both SV and gelsolin (SV1/Gel). These cells were analyzed for protein levels by immunoblotting (A); the percentages of cells associated with at least one area of degraded matrix (hole), relative to control-treated cells (B); the mean numbers of holes per cell, relative to control-treated cells (C); and the percentages of cells with holes underneath the cell center (D). The percentages in A denote the mean levels (n = 4) of SV and gelsolin, relative to control-treated cells, after normalization to the β-actin loading control. (B–D) Means ± SD; n = 6. Asterisks in C and D denote significant differences (***p < 0.001, **p < 0.01, and *p < 0.05) from controls in unpaired t tests. (E) Representative micrographs of cells treated with control siRNA (a–c), SV1 siRNA (d–f), or both SV1 and gelsolin (SV1/Gel) siRNAs (g–i) and stained for F-actin (a, d, and g) or sites of matrix degradation (b, e, and h). For merged images (c, f, and i), holes in the matrix were visualized as red spots by pseudocoloring the images in b, e, and h after inverting and thresholding their luminosities. Note the sites of matrix degradation at the cell periphery (arrows) and underneath the cell center (arrowheads). Overlaps are yellow or orange. Bar, 20 μm.

Overexpression of EGFP-SV in COS7 cells increases the numbers of cytoplasmic F-actin punctae. (A) COS7 cells were transfected for 48 h with vectors encoding EGFP (a–c), EGFP-SV (d–f), or GFP plus untagged supervillin (g–i). Cells were fixed and stained for F-actin using Texas Red phalloidin (b, e, and h). Bar, 20 μm. Insets enlarged fourfold. Overlap in white. (B and C) F-actin punctae were counted in untransfected cells after a mock treatment with transfection agent alone, or in cells expressing EGFP, EGFP-SV, or GFP as a marker for untagged SV. Distributions of individual counts and their means are shown. Both EGFP-SV and untagged SV differed from controls, p ≤ 0.001, n = 25–28/group. (D) The relative amounts of EGFP (closed symbols) or EGFP-SV (open symbols) in individual cells were estimated from total luminosities of wide-field microscopic images. The plot shows the number of F-actin punctae per cell versus luminosity. EGFP-SV expression levels were 12.5 ± 0.7 (mean ± SD; n = 3) times that of endogenous SV. At these expression levels, no significant trend was detected for either data set.

Most EGFP-SV punctae are dynamic and short lived; a few are longer lived and relatively stable. (A) Time-lapse sequence of EGFP-SV punctae in COS7 cells on glass coverslips from Supplemental Video 1. Images were taken every 12 s for 60 min. Bar, 10 μm. Examples of short-lived (arrowheads) and long-lived (arrows) punctae are indicated. (B) Distribution of individual punctae lifetimes; n = 99. (C) Sample trajectories of EGFP-SV punctae with associated lifetimes.

Invasion indices are decreased in cells depleted of both SV and gelsolin. MDA-MB-231/WT Src cells were treated with either control siRNA (Control) or with siRNAs targeting either SV (SV1 and SV2), gelsolin (Gel), or both proteins (SV1/Gel) and assayed for invasion through Matrigel-coated filters. Cells depleted in both SV and gelsolin were significantly less invasive than controls, based on unpaired t tests (*p < 0.05; n = 3).