Vinculin is required for cell polarization, migration, and extracellular matrix remodeling in 3D collagen

Abstract

Vinculin is filamentous (F)-actin-binding protein enriched in integrin-based adhesions to the extracellular matrix (ECM). Whereas studies in 2-dimensional (2D) tissue culture models have suggested that vinculin negatively regulates cell migration by promoting cytoskeleton-ECM coupling to strengthen and stabilize adhesions, its role in regulating cell migration in more physiologic, 3-dimensional (3D) environments is unclear. To address the role of vinculin in 3D cell migration, we analyzed the morphodynamics, migration, and ECM remodeling of primary murine embryonic fibroblasts (MEFs) with cre/loxP-mediated vinculin gene disruption in 3D collagen I cultures. We found that vinculin promoted 3D cell migration by increasing directional persistence. Vinculin was necessary for persistent cell protrusion, cell elongation, and stable cell orientation in 3D collagen, but was dispensable for lamellipodia formation, suggesting that vinculin-mediated cell adhesion to the ECM is needed to convert actin-based cell protrusion into persistent cell shape change and migration. Consistent with this finding, vinculin was necessary for efficient traction force generation in 3D collagen without affecting myosin II activity and promoted 3D collagen fiber alignment and macroscopical gel contraction. Our results suggest that vinculin promotes directionally persistent cell migration and tension-dependent ECM remodeling in complex 3D environments by increasing cell-ECM adhesion and traction force generation.

Keywords: 3D cell migration; cell morphodynamics; integrin; traction force generation.

Figure 1.

Vinculin is necessary for the persistent migration and normal polarization of primary MEFs in 3D collagen. A) Tracks of 17 control and 16 Vcl-KO MEFs, migrating in 2.4 mg/ml collagen I gels, monitored for 12 h at a 10 min frame rate. Tracks were recorded in the x/y dimensions. B) Quantification of distance from the origin over time. n = 17 (control) and 16 (Vcl-KO) MEFs; error bars, sem. C) Box-and-whisker plot of cell migration velocity. n = 17 (control) and 16 (Vcl-KO) MEFs. Numbers indicate means; P = 0.062, Student’s t test. D) Plots of turning angle distribution. n = 17 (control) and n = 16 (Vcl-KO) MEFs; data as in (C). *P < 0.05, Student’s t test. E) Plot of persistence ratio (distance to the origin:total distance) of control and Vcl-KO MEFs after 12 h migration. n = 17 (control) and 16 (Vcl-KO) MEFs; data as in (C). *P < 0.05, Student’s t test. F) Maximum projections of confocal stacks of live control and Vcl-KO MEFs expressing tdTomato-farnesyl. Scale bar, 10 μm. G) Plot of aspect ratio of live control and Vcl-KO MEFs in 3D collagen. n = 27 (control) and 33 (Vcl-KO) MEFs; data as in (C). *P < 0.005, Student’s t test. H) Cumulative probability distribution of the reorientation angle of the cell main axis of tdTomato-farnesyl labeled control and Vcl-KO MEFs at 5 min frame rate (Fig. 3A; Supplemental Movie 3). n = 336 frames from 12 control MEFs and 280 frames from 10 Vcl-KO MEFs, Kolmogorov-Smirnov test.

Figure 2.

Vinculin is dispensable for lamellipodia formation and leading-edge protrusion of primary MEFs in 3D collagen. A) Projections of Bessel beam image stacks of live control and Vcl-KO MEFs expressing tdTomato–F-tractin, to label actin-based cell protrusions (insets). Note the presence of sheetlike, lamellipodial protrusions of similar size and morphology in control (arrow) and Vcl-KO MEFs (arrowhead). Scale bar, 10 μm. B) Kymographs placed along the direction of protrusion through lamellipodia of live control and Vcl-KO MEFs in 3D collagen I ECM, showing lamellipodium dynamics. Cell exterior: top, cell interior: bottom. SDC confocal time lapses, 5 s frame rate. D, distance; T, time. C) Kymograph-based quantification of lamellipodium protrusion velocity of control and Vcl-KO MEFs in 3D collagen I. n = 35 (control) and 26 (Vcl-KO) kymographs from 12 (control) and 9 (Vcl-KO) MEFs. Numbers indicate means; *P < 0.005, Student’s t test.

Figure 3.

Vinculin promotes persistent protrusion and stable orientation of primary MEFs migrating in 3D collagen. A) Projections of confocal image stacks of live tdTomato-farnesyl–expressing control and Vcl-KO MEFs in 3D collagen; 5 min frame rate. Persistent protrusion and stable main axis orientation (red rods) were observed in control MEFs, but repeated protrusion–retraction and frequent change in main axis orientation in Vcl-KO MEFs. Scale bar, 10 μm. B) Magnification of insets from (A) showing overlay of cell segments at t = 0 min (black outline) and at the indicated time points (gray outlines). Dashed lines: 10 μm distance from the leading edge (D_LE; minimum bounding circle at D_LE = 0 μm is not shown) for both time points in each panel (black: t = 0 min; gray: t = 5, 10, 15, 20, and 25 min). Red areas: overlap of peripheral cell area at D_LE = 10 μm limits between t = 0 min and the respective time points. There was a persistent decrease in overlap in control MEFs, but a repeated decrease–increase of overlap in Vcl-KO MEFs. C) Decay of peripheral cell area (protrusion) overlap at increasing time intervals for D_LE = 2, 5, 10, and 15 μm in control and Vcl-KO MEFs. Dots: original data from 12 control and 10 Vcl-KO MEFs with multiple overlap measurements; lines: stretched exponential fits. Decay of protrusion overlap was slower in Vcl-KO vs. control MEFs for all D_LE. D) Quantification of time constant (τ), representing protrusion turnover, for D_LE = 2, 5, 10, and 15 μm, derived from stretched exponential fits. n = 12 control and 10 Vcl-KO MEFs; error bars, sem; *P < 0.05, Student’s t test.

Figure 4.

Vinculin promotes protrusion of primary MEFs in 3D collagen independent of myosin II contractility. A) Western blot analysis of vinculin, phosphor-Ser¹⁹ MLC-2, total MLC-2, and β-actin (loading control) in control and Vcl-KO MEFs. B) Quantification of Western blot analyses as shown in (A); *P > 0.05, Student’s t test, n = 4. C) Maximum projections of confocal image stacks from control and Vcl-KO MEFs treated with 5 or 50 μM blebbistatin, or with DMSO (0). Large ellipses outside of cells (arrow) denote smallest outer-bounding ellipses for quantification of whole-cell aspect ratio and protrusion range. Small ellipses inside the cell body (arrowhead) denote largest inner-bounding ellipses for quantification of cell body aspect ratio. With increasing blebbistatin concentrations, the aspect ratio of the inner ellipse area decreased in control MEFs and increased for the outer ellipse area in control and Vcl-KO MEFs. Scale bar, 20 μm. D) Quantification of the inner ellipse aspect ratio, representing the cell body aspect ratio. *P < 0.05, Student’s t test, n = 20 cells per condition. E) Quantification of outer ellipse aspect ratio, representing the whole-cell aspect ratio. *P < 0.05, Student’s t test; n = 20 cells per condition. F) Quantification of outer ellipse area, representing protrusion range. *P < 0.05, Student’s t test, n = 20 cells per condition.

Figure 5.

Vinculin promotes traction force generation of primary MEFs in 3D collagen. A) Traction force microscopy of control and Vcl-KO MEFs in 3D collagen, using confocal reflection (collagen) and bright-field (cell) imaging. The x/y/z axes are labeled for orientation. Left to right: bright-field projections of respective cells. Scale bar, 20 μm. 3D collagen displacement fields overlaid with negative minimum projections of the cells. 3D traction force reconstructions overlaid with negative minimum projections of the cells. Z-projections of reconstructed 3D forces, overlaid with negative minimum projections of the cells, showing localization of the highest forces at the cell poles (arrows). B) Box-and-whisker plot of total traction force per cell generated by control and Vcl-KO MEFs in 3D collagen. n = 18 (control) and 15 (Vcl-KO) MEFs; numbers indicate means; *P < 0.05, Mann-Whitney U test. C) Scatterplot of the fraction of cellular forces directed along a main force axis vs. total traction force for individual control and Vcl-KO MEFs. n = 18 (control) and 15 (Vcl-KO) MEFs.

Figure 6.

Vinculin promotes assembly of soluble FN into fibers. A) Projections of confocal image stacks of control and Vcl-KO MEFs after FN immunofluorescence staining (green) and DRAQ5 labeling of nuclei (purple), showing FN assembly over 15 h, showing fibrous FN morphology in control MEFs (arrow) and homogenous FN morphology in Vcl-KO MEFs (arrowhead). Scale bar, 10 μm. B) Box-and-whisker plot of total assembled FN per cell for control and Vcl-KO MEFs (15 h). n = 15 (control) and 12 (Vcl-KO) MEFs; numbers indicate means; P = 0.93, Student’s t test. C) FN-immunofluorescence intensity histograms across confocal stacks of control and Vcl-KO MEFs. There was a progressive decrease in the number of high-intensity pixels in Vcl-KO vs. control MEFs. n = 15 (control) and 12 (Vcl-KO) MEFs; *P < 0.05 for gray values >95, Student’s t test.

Figure 7.

Vinculin promotes macroscopic contraction and fiber alignment of 3D collagen. A) 3D collagen contraction by control and Vcl-KO MEFs after 24 h of culture. Collagen plugs containing Vcl-KO MEFs were increased compared with controls. Scale bar, 1 mm. B) Box-and-whisker plot of 3D collagen contraction by control and Vcl-KO MEFs after 24 h. n = 12 (control) and 12 (Vcl-KO) MEFs. Numbers indicate means; *P < 0.0001, Mann-Whitney U test. C) LCPolScope micrographs of collagen gels containing control or Vcl-KO MEFs, overlaid with rod maps showing spatial distribution of preferred collagen fiber orientation (rod orientation, insets) and degree of fiber alignment (rod length/image brightness, insets). Rod length was reduced and homogenous rod orientation lessened in gels containing Vcl-KO vs. control MEFs. Scale bar, 10 μm. D) Scatterplot of retardance (rod length/image brightness) of collagen containing control or Vcl-KO MEFs 4, 24, and 48 h after gelation. n = 22 (4 h), 16 (24 h), and 9 (48 h) control MEFs and 11 (4 h), 11 (24 h), and 13 (48 h) Vcl-KO MEFs. E) Quantification of slow axis angle distribution (rod orientation) of collagen containing control or Vcl-KO MEFs 4, 24, and 48 h after gelation. Number of cells at each time point is as in (D).