Vinculin facilitates cell invasion into three-dimensional collagen matrices

Abstract

The cytoskeletal protein vinculin contributes to the mechanical link of the contractile actomyosin cytoskeleton to the extracellular matrix (ECM) through integrin receptors. In addition, vinculin modulates the dynamics of cell adhesions and is associated with decreased cell motility on two-dimensional ECM substrates. The effect of vinculin on cell invasion through dense three-dimensional ECM gels is unknown. Here, we report how vinculin expression affects cell invasion into three-dimensional collagen matrices. Cell motility was investigated in vinculin knockout and vinculin expressing wild-type mouse embryonic fibroblasts. Vinculin knockout cells were 2-fold more motile on two-dimensional collagen-coated substrates compared with wild-type cells, but 3-fold less invasive in 2.4 mg/ml three-dimensional collagen matrices. Vinculin knockout cells were softer and remodeled their cytoskeleton more dynamically, which is consistent with their enhanced two-dimensional motility but does not explain their reduced three-dimensional invasiveness. Importantly, vinculin-expressing cells adhered more strongly to collagen and generated 3-fold higher traction forces compared with vinculin knockout cells. Moreover, vinculin-expressing cells were able to migrate into dense (5.8 mg/ml) three-dimensional collagen matrices that were impenetrable for vinculin knockout cells. These findings suggest that vinculin facilitates three-dimensional matrix invasion through up-regulation or enhanced transmission of traction forces that are needed to overcome the steric hindrance of ECMs.

FIGURE 1.

Vinculin dependence of cell invasion into collagen gels (three-dimensional, 3D) compared with cell migration on collagen substrates (two-dimensional, 2D). A, modulation contrast images of a representative invasive MEFvin^wt/wt cell (left panel) and a MEFvin^−/− cell (right panel) after 3 days of invasion into collagen gels (invasion depth, 50 μm). Scale bars, 20 μm. B, the percentage of invasive MEFvin^wt/wt cells is higher compared with MEFvin^−/− cells. C, the cumulative probability shows increased depth of invasion for MEFvin^wt/wt cells. D, modulation contrast images of representative invasive MEFvin^wt/wt cells after 3 days of invasion into collagen gels treated with control siRNA (left panel) or vin-siRNA (right panel). Scale bars, 20 μm. E, the percentage of invasive cells is 2.5-fold higher in control siRNA as compared with vin-siRNA treated cells. F, the invasion profile shows that control siRNA treated cells invade deeper into collagen gels than vin-siRNA-treated cells. G, phase contrast images and trajectories of MEFvin^wt/wt (left panel) and MEFvin^−/− cells (right panel) on collagen-coated glass. Scale bars, 30 μm. H and I, the motion of MEFvin^wt/wt (H) and MEFvin^−/− cells (I) was recorded over 2 h, and the starting points of the trajectories lines were transposed to the same origin. J, MSD calculated from trajectories of MEFvin^−/− cells (light gray) was significantly larger than that of MEFvin^wt/wt cells (dark gray). K and L, the apparent diffusivity D (K) and the persistence β (L) of cell motility were significantly increased in MEFvin^−/− compared with MEFvin^wt/wt cells (n > 55 cells; p < 0.05).

FIGURE 2.

Effect of MMP-inhibition and MT1-MMP expression on cell invasion. A, FACS analysis of MT1-MMP expression in MEFvin^wt/wt and MEFvin^−/− cells. The numbers in the histograms specify the differences in the mean fluorescence intensities (Δmf) between specific antibody binding (shaded gray curves) and appropriate isotype controls (white curves). MT1-MMP receptor expression was increased in MEFvin^−/− cells (A and B). B, mean values (Δmf) ± S.E. of three to five independent FACS measurements. C, the percentages (means ± S.E.) of invasive cells treated with 100 μm GM6001 was slightly decreased in MEFvin^wt/wt and slightly increased in MEFvin^−/− compared with dimethyl sulfoxide (DMSO) control treatment. D, the invasion profile of MEFvin^wt/wt cells treated with GM6001 (orange) was unchanged compared with dimethyl sulfoxide control treatment (dark gray), whereas the invasion profile of MEFvin^−/− cells treated with GM6001 (light gray) revealed more invasive behavior compared with the dimethyl sulfoxide control (red).

FIGURE 3.

Effect of vinculin expression on adhesion strength, integrin expression, focal adhesions, and spreading area. A, fluorescent images recorded at different focus depths show that FN-coated beads are tightly associated with actin stress fibers (stained with Alexafluor546 Phalloidin) in MEFvin^wt/wt (top row) and MEFvin^−/− cells (bottom row). B, percentage of beads detached from the cells versus pulling force. Adhesion strength was significantly lower in MEFvin^−/− cells (red circles) compared with MEFvin^wt/wt cells (black circles). Addition of the MLCK inhibitor ML-7 decreased adhesion strength of MEFvin^wt/wt (light gray) and MEFvin^−/− cells (orange). *, p < 0.05. C, adhesion strength was significantly lower in vin-siRNA cells (red) compared with control siRNA cells (black). *, p < 0.05. D, FACS analysis of αv, α5, and β1 integrin subunit expression on MEFvin^wt/wt cells and MEFvin^−/− cells. The numbers in the histograms specify the differences in mean the fluorescence intensities (Δmf) between specific antibody binding (shaded gray curves) and appropriate isotype controls (white curves). α5 as well as β1 integrin subunit expression was increased in MEFvin^−/− cells, whereas the αv expression was not altered. The bar graphs show the mean values (Δmf) ± S.E. of three to five independent FACS measurements. E, MEFvin^wt/wt and vin^−/− cells adhered for 24 h on collagen type I-coated glass. Actin was stained with Alexafluor546 Phalloidin (red), focal adhesions with an antibody directed against paxillin (green), and nuclei with Hoechst 33342 (blue). Scale bars are 30 μm. F, spreading area (means ± S.E.) of MEFvin^−/− cells (light gray) was significantly increased compared with MEFvin^wt/wt cells (dark gray). *, p < 0.05. G, numbers of focal adhesions/cell were slightly reduced in MEFvin^−/− cells. H and I, focal adhesion size (H) and focal adhesion length (I) showed no differences.

FIGURE 4.

Effect of vinculin expression on stiffness and fluidity of cells. Cell stiffness (A; means ± S.E.) and cell fluidity (B) of MEFvin^wt/wt (black) and MEFvin^−/− cells (red) was measured before (circles) and after (squares) inhibition of myosin light chain kinase with ML-7 and of MEFvin^wt/wt (dark gray) and MEFvin^−/− cells (orange).

FIGURE 5.

Effect of vinculin on the generation of contractile forces. A, bright field (left panels) and traction images (right panels) of MEFvin^wt/wt (top panels) and MEFvin^−/− cells (bottom panels). B, the strain energy/cell (means ± S.E.) of MEFwt cells (n = 191) was 3-fold increased compared with MEFvin^−/− cells (n = 304). The scale bar is 20 μm. *, p < 0.05.

FIGURE 6.

Effect of collagen density on the invasion of MEFs. A, three different collagen concentrations of 2.4, 3.7, and 5.8 mg/ml were tested for cell invasion. After 3 days, MEFvin^wt/wt cells were able to invade all gels, whereas MEFvin^−/− cells were able to migrate only into 2.4 mg/ml gels. B, invasion profiles of MEFvin^wt/wt and MEFvin^−/− cells (light gray) in three-dimensional ECMs with different collagen concentrations (red, 5.8; blue, 3.7; dark gray, 2.4).