A distinctive role for focal adhesion proteins in three-dimensional cell motility - PubMed (original) (raw)

. 2010 Jun;12(6):598-604.

doi: 10.1038/ncb2062. Epub 2010 May 16.

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A distinctive role for focal adhesion proteins in three-dimensional cell motility

Stephanie I Fraley et al. Nat Cell Biol. 2010 Jun.

Abstract

Focal adhesions are large multi-protein assemblies that form at the basal surface of cells on planar dishes, and that mediate cell signalling, force transduction and adhesion to the substratum. Although much is known about focal adhesion components in two-dimensional (2D) systems, their role in migrating cells in a more physiological three-dimensional (3D) matrix is largely unknown. Live-cell microscopy shows that for cells fully embedded in a 3D matrix, focal adhesion proteins, including vinculin, paxillin, talin, alpha-actinin, zyxin, VASP, FAK and p130Cas, do not form aggregates but are diffusely distributed throughout the cytoplasm. Despite the absence of detectable focal adhesions, focal adhesion proteins still modulate cell motility, but in a manner distinct from cells on planar substrates. Rather, focal adhesion proteins in matrix-embedded cells regulate cell speed and persistence by affecting protrusion activity and matrix deformation, two processes that have no direct role in controlling 2D cell speed. This study shows that membrane protrusions constitute a critical motility/matrix-traction module that drives cell motility in a 3D matrix.

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Figures

Fig. 1

Fig. 1. Regulated formation of FAs in 2-dimensional, 2.5-dimensional, and 3-dimensional collagen matrix microenvironments

A and B. Schematics of type I collagen microenvironments studied here, including cells on conventional flat type I collagen-coated glass substrates (“2-D”, A), cells sandwiched between a collagen-coated substrate and coated with a thick layer of collagen (“2.5-D”, A), and cells fully immersed inside a 3-D collagen matrix (“3-D”, B). C and D. Confocal fluorescence micrographs of vinculin and zyxin, two major constituents of standard FAs in 2-D, in wild type (WT) HT-1080 human fibrosarcoma cells, which were either plated on conventional substrates (2-D case, C) or partially embedded in a matrix (2.5-D case, D). Scale bar, 10 μm. As a control, we verified that we were able to visualize the microtubule network using antibodies against tubulin and the same secondary antibodies used to stain FA proteins (data not shown). E. Phase contrast and fluorescence micrographs of live wild-type cells stably expressing either EGFP-vinculin (top panels) or EGFP-zyxin (bottom panels) on flat 2-D substrates (left panels) and inside a 3-D matrix (right panels). Insets show large vinculin or zyxin-containing FAs in the 2-D case, and diffuse staining in the 3-D case. Scale bar, 10 μm.

Fig. 2

Fig. 2. Regulation of 2-D cell motility by FA proteins is not predictive of regulation of 3-D cell motility in matrix

A. Typical trajectories of individual matrix-embedded WT HT-1080 cells and HT-1080 cells RNAi-depleted of major FA proteins p130Cas, talin, FAK, and vinculin. Scale bar, 10 μm. B and C. Average random-motility speed of WT cells and multiple cells stably depleted of major FA proteins on 2-D substratum (B) and inside a 3-D collagen matrix (C). D. Lack of correlation between 2-D cell speed and 3-D cell speed. Cell speeds were normalized by the maximum mean value in each data set (here zyxin-depleted cells in 3-D and p130Cas-depleted cells in 2-D). Slope evaluated from a linear fit of the data, R squared value, and p value of correlation are indicated. E and F. Persistence time (E) and persistence distance of migration (F) of cells in matrix for WT cells and FA protein-depleted cells. G. Correlation function between the 2-D and the 3-D persistence distances normalized by maximum persistence distance mean value in each data set. H. 3-D cell speed of WT E006AA human prostate cancer cells and E006AA cells depleted of either p130Cas or zyxin. I. Lack of correlation between 3-D cell speed and 2-D cell speed of WT, p130Cas-depleted, and zyxin-depleted E006AA cells. 2-D and 3-D cell speeds were normalized by the maximum mean value in each data set. *** in panels B, C, E, F and H indicate p values <0.001 between the type of cell considered and WT cells. The speed, persistence time, and persistence distance of at least 35 cells were measured on three different days for each condition. Bar graphs show mean and SEM values of three independent experiments. Arrows point to the WT case.

Fig. 3

Fig. 3. Extent of focal adhesion protein-mediated protrusion activity predicts 3-D cell speed

A. Typical time-dependent morphological changes of WT, FAK-depleted and talin-depleted cells embedded in a 3-D matrix showing actively growing protrusions (indicated by arrows). Scale bar, 10 μm.B. Actin filament organization in WT, FAK-depleted and talin-depleted cells in a 3-D matrix. Insets show cross-sectional view. Scale bar, 10 μm. C. Averaged number of actively growing protrusions per 90 min (i.e. protrusion activity) for matrix-embedded WT cells and cells RNAi-depleted of major FA proteins. **D.**Correlation function between 3-D cell speed and cellular protrusion activity. Values are normalized by corresponding maximum mean values. Slope evaluated from a linear fit of the data, R squared value, and p value of correlation are indicated. E. Averaged lifetime of actively growing protrusions. F. Averaged growth rate of individual protrusions. G. Correlation between 3-D cell speed and growth rate of protrusions. Values were normalized by maximum mean value in each data set. H. Time-dependent angular distributions of actively growing protrusions along the matrix-embedded cell periphery after 90 min, 3h, and 12 h. The largest protrusion at time 0 was arbitrarily taken as being pointing in the 0 degree direction. I. Averaged number of actively growing protrusions per 90 min for WT E006AA cells and E006AA cells depleted of either p130Cas or zyxin. J. Correlation between 3-D cell speed and protrusion activity for the cells characterized in panel I and in Fig. 2H. *, **, and *** in panels C, E, and I indicate p values <0.05, <0.01, and <0.001, respectively, between the KD cells considered and WT cells unless indicated. The pseudopodial protrusions of at least 35 cells were characterized on three different days for each condition. Bar graphs show mean and SEM values of three independent experiments.

Fig. 4

Fig. 4. Regulation of 3-D cell-matrix interactions by FA proteins

A. Schematic of the method used for the measurements of local matrix traction mediated by embedded cells, whereby large fiduciary beads are tightly embedded in the matrix and are monitored by high-resolution 3-D multiple-particle tracking. B. Typical movements of fiduciary beads in the vicinity of a WT cell and cells depleted of talin and FAK, as indicated. Left and right micrographs respectively show the initial and final positions of the beads after 90 min. Arrows indicate the magnitude and direction of the displacements of the matrix-bound beads, which were magnified three times for ease of visualization. Scale bar, 10 μm.C. Typical x, y, and _z_displacements of an individual matrix-bound bead in the vicinity of a WT cell in the 3-D matrix. D. Maximum displacements of the fiduciary beads in the matrix (i.e. traction). E. Correlation function between 3-D cell speed and the maximum bead displacement (i.e. traction). Values were normalized by maximum mean value in each data set. Slope evaluated from a linear fit of the data, R squared value, and p value of the correlation are indicated. F. Percentage deformation (i.e. matrix remodeling) calculated as the ratio of the final distance between initial and final bead position and the total bead displacement. This percentage is 0 when the matrix deformation is purely elastic and 100 when the matrix deformation is irreversible. See more details in the Materials and Methods section. ** and *** in panels D and F indicate p values <0.01 and <0.001, respectively, between the KD cell considered and WT cells. The local matrix traction in the vicinity of at least 5 cells (~30 beads per cell) was measured on three different days for each condition. Bar graphs show mean and SEM values of three independent experiments.

Fig. 5

Fig. 5. Regulation of cell motility on compliant substrates by FA proteins

A. Wild-type and zyxin-depleted cells on soft collagen-coated bis-crosslinked polyacrylamide gels and stiff collagen-coated glass substrates. Scale bar, 10 μm. B. Averaged cell speed of wild-type and zyxin-depleted cells on collagen-coated glass_vs_. soft substrates. ** and *** indicate p value <0.01 and <0.001 respectively comparing cells on the softest substrates to the same cells on glass. The speed of at least 35 cells were measured on three different days for each condition. Bar graphs show mean and SEM values of three independent experiments.

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