Vinculin Regulates Directionality and Cell Polarity in 2D, 3D Matrix and 3D Microtrack Migration

Abstract

During metastasis, cells can use proteolytic activity to form tube-like "microtracks" within the extracellular matrix (ECM). Using these microtracks, cells can migrate unimpeded through the stroma. To investigate the molecular mechanisms of microtrack migration, we developed an in vitro 3D micromolded collagen platform. When in microtracks, cells tend to migrate unidirectionally. Since focal adhesions are the primary mechanism by which cells interact with the ECM, we examined the roles of several focal adhesion molecules in driving unidirectional motion. Vinculin knockdown results in the repeated reversal of migration direction compared with control cells. Tracking the position of the Golgi centroid relative to the position of the nucleus centroid reveals that vinculin knockdown disrupts cell polarity in microtracks. Vinculin also directs migration on 2D substrates and in 3D uniform collagen matrices, indicated by reduced speed, shorter net displacement and decreased directionality in vinculin-deficient cells. In addition, vinculin is necessary for Focal Adhesion Kinase (FAK) activation in 3D as vinculin knockdown results in reduced FAK activation in both 3D uniform collagen matrices and microtracks, but not on 2D substrates, and accordingly, FAK inhibition halts cell migration in 3D microtracks. Together, these data indicate that vinculin plays a key role in polarization during migration.

FIGURE 1:

Unidirectional migration of cancer cells in microtracks. (A) Ex situ confocal reflectance image of an in vivo microtrack in the stromal extracellular matrix (purple) in a murine mammary tumor model; arrowheads denote a microtrack, width 10 μm; 3× inset magnification. (B) Time-lapse phase contrast image of an MDA-MB-231 cell migrating unidirectionally through a microfabricated 3D collagen microtrack in vitro. (C) Quantification of the displacement of MDA-MB-231 cells migrating through in vitro collagen microtracks, seven cells. Scale bars, 50 μm.

FIGURE 2:

Vinculin is critical for maintaining directionality within collagen microtracks. (A) Western blotting confirmed knockdown of focal adhesion proteins vinculin, p130Cas, and zyxin. (B) Quantification of MDA-MB-231 cell displacement from the cells’ original location as a function in time after knockdown of p130Cas, zyxin, or vinculin. Note that vinculin siRNA–treated MDA-MB-231 cells migrate back and forth, reversing direction several times. (C) Quantification of the number of reversals in migration direction over a 6-h period of time in micromolded microtracks in control or p130Cas-, zyxin-, or vinculin-knockdown 231 cells; ***p < 0.001, *p < 0.05; >50 cells. (D) Time-lapse phase contrast images and displacement curves of a representative single control (blue) and vinculin (red) siRNA–treated MDA-MB-231 cell migrating through an in vitro collagen microtrack. Whereas control MDA-MB-231 cells migrate in one direction, vinculin siRNA–treated MDA-MB-231 cells reverse directions several times; arrowheads indicate reversals. Scale bars, 100 μm. (E) Quantification of MDA-MB-231 cell migration speed with and without vinculin knockdown; >100 cells. All quantitative data are pooled from three individual equivalent experiments.

FIGURE 3:

Control siRNA cells maintain cell polarity during unidirectional microtrack migration. (A) Representative time-lapse phase contrast and confocal fluorescence images (overlaid) of a control siRNA–treated MDA-MB-231 cell, which maintains cell polarity during unidirectional microtrack migration by positioning the nucleus (blue) at the leading edge, followed by the Golgi apparatus (red). Arrow indicates the direction of movement. Scale bar, 50 μm. (B) Quantification of the position of the cell centroid relative to the centroid of the nucleus and Golgi during migration in a microtrack. Whereas the nucleus is strictly positioned at the leading edge of a control siRNA–treated MDA-MB-231 cell, the nucleus and Golgi positions alternate in an MDA-MB-231 cell treated with vinculin siRNA during microtrack migration. (C) Quantification of the centroid position of the nucleus relative to the Golgi of migrating MDA-MB-231 cells in microtracks. The nucleus is observed ahead of the cell centroid and Golgi relative to the direction of cell motion in control siRNA–treated MDA-MB-231 cells throughout the period of observation and only ∼50% of the time in vinculin siRNA–treated MDA-MB-231 cells; *p < 0.05.

FIGURE 4:

3D collagen matrix migration is regulated by vinculin. (A) Rose plots show trajectories of control siRNA– and vinculin siRNA–treated MDA-MB-231 cells in 3D collagen matrices over several hours. Vinculin siRNA cells (B) show slower migration speed (μm/min), (C) travel less far over the same observation window (μm), (D) lose persistent directionality, and take more time to (E) elongate and (F) establish front–rear polarization compared with control siRNA cells. ***p < 0.001; 45 cells. All quantitative data are pooled from three individual equivalent experiments.

FIGURE 5:

2D migration is regulated by vinculin. (A) Rose plots of the trajectories of control siRNA– and siRNA vinculin–treated MDA-MB-231 cells on a 2D plastic surface over several hours. (B) Migration speed of control and siRNA vinculin–treated MDA-MB-231 cells (***p < 0.001; 45 cells). (C) Net displacement of control and siRNA vinculin–treated MDA-MB-231 cells in 2D (***p < 0.001; 45 cells). (D) Directionality of control and siRNA vinculin–treated MDA-MB-231 cells, **p < 0.01; 45 cells. (E) Time-lapse phase contrast images of control and vinculin siRNA–treated MDA-MB-231 cells during wound healing; scale bar, 100 μm. (F) Wound closure rate for control and siRNA vinculin–treated MDA-MB-231 cells; ***p < 0.001. (G) Quantification of the wound closure rate for wild-type vinculin MEFs compared with vinculin-null MEFs; ***p < 0.001. All quantitative data are pooled from three individual equivalent experiments.

FIGURE 6:

Vinculin siRNA cells generate reduced traction force. (A) Traction force color contour maps and phase contrast images of a control and vinculin siRNA–treated MDA-MB-231 cells; scale bar, 30 μm. (B) Quantification of the total force, |F|, generated (nN), (C) spread area (μm²), and (D) normalized force per cell area (nN/μm²) in control and siRNA vinculin–treated MDA-MB-231 cells. (E) Corresponding scatter plot of traction force as a function of cell area with linear regression lines for control and vinculin knockdown–treated MDA-MB-231 cells. *p < 0.05; >40 cells. All quantitative data are pooled from three individual equivalent experiments.

FIGURE 7:

Vinculin regulates FAK activity in 3D migration. (A) Western blotting of MDA-MB-231 cells in 2D and 3D environments with and without knockdown of vinculin. Vinculin knockdown reduces FAK phosphorylation of MDA-MB-231 cells in 3D matrices but not 2D substrates. (B) Quantification of pFAK activation in control and vinculin siRNA–treated MDA-MB-231 cells during 2D (p = 0.475) and 3D matrix migration; *p < 0.05. (C) Confocal fluorescence images of control and vinculin siRNA cells on 2D substrate, in 3D uniform collagen matrices, and in 3D collagen microtracks; scale bars, 25 μm. (D) Fraction of motile cells in microtracks with and without FAK inhibition using PF573228; ***p < 0.001; >60 cells. (E) Migration speed of control MDA-MB-231 cells and MDA-MB-231 cells treated with the FAK inhibitor in microtracks; ***p < 0.001; >60 cells. All quantitative data are pooled from three individual equivalent experiments.