Tractions and Stress Fibers Control Cell Shape and Rearrangements in Collective Cell Migration

Key to collective cell migration is the ability of cells to rearrange their position with respect to their neighbors. Recent theory and experiments demonstrate that cellular rearrangements are facilitated by cell shape, with cells having more elongated shapes and greater perimeters more easily sliding past their neighbors within the cell layer. Though it is thought that cell perimeter is controlled primarily by cortical tension and adhesion at each cell’s periphery, experimental testing of this hypothesis has produced conflicting results. Here we study collective migration in an epithelial monolayer by measuring forces, cell perimeters, and motion, and find all three to decrease with either increased cell density or inhibition of cell contraction. In contrast to previous understanding, the data suggest that cell shape and rearrangements are controlled not by cortical tension or adhesion at the cell periphery but rather by the stress fibers that produce tractions at the cell-substrate interface. This finding is confirmed by an experiment showing that increasing tractions reverses the effect of density on cell shape and rearrangements. Our study therefore reduces the focus on the cell periphery by establishing cell-substrate traction as a major physical factor controlling shape and motion in collective cell migration.

Popular Summary

For a group of cells to collectively flow or change shape, as would occur in tissue development or wound healing, each cell must be able to rearrange its local position with respect to its neighbors. However, current knowledge of tissue development and wound healing is limited by the lack of understanding of how these collective rearrangements result from the forces produced by each individual cell. To address this problem, we design experiments to investigate the relationship between forces produced by the cell and the collective migration in single layers of canine kidney cells, a cell line common to mammalian biomedical research.

Our experiments use fluorescent imaging and laser ablation to assess forces at the periphery of each cell. We also quantify the traction applied by the cell to its underlying surface by placing the cells on a gel laced with fluorescent beads, whose displacement reveals how strongly the cells tugged on the substrate. We alter the forces by controlling cell density and by using chemicals known to decrease or increase forces produced by each cell. We find that cell shape and migration depend primarily on the traction at the cell-substrate interface.

Our research gives a new interpretation of recent theoretical models, which have demonstrated that cell rearrangements are related to average cell shape. Most models have suggested that cell shape is controlled primarily by the forces at the periphery of each cell. This finding redirects focus towards the cell-substrate interface, thereby establishing traction as a dominant physical factor controlling shape and motion in collective cell migration.

Figure 1

Cell density decreases cell rearrangements and perimeter $q$. (a),(c) Phase contrast images of low-density ($1200\mathrm{cells}/{\mathrm{mm}}^2$) (a) and high-density ($4200\mathrm{cells}/{\mathrm{mm}}^2$) (c) cell islands. (b),(d) Cell trajectories for the highlighted regions over 8 h in islands of low (b) and high density (d). Trajectories for the entire islands are shown in Fig. 7. (e) MSD for low- and high-density islands plotted on logarithmic axes. In this and all subsequent figures, each line of the MSD corresponds to a different cell island; dots represent the individual data points that make up the curve. (f) The MSD is fit to MSD $\sim {\mathrm{\Delta }t}^{\alpha }$, with exponent $\alpha$ characterizing cell rearrangements. High-density islands have a smaller value of $\alpha$ than low-density islands ($p<0.001$). In this and all subsequent figures, each dot of $\alpha$ corresponds to a different cell island. (g) Low-density islands have a larger exponent ${\alpha }_{\mathrm{CR}}$ than high-density islands ($p<0.001$). (h) Average perimeter $⟨q⟩$ is greater for cells at low density compared to high density ($p<0.001$). In this and all subsequent figures, each dot of $⟨q⟩$ corresponds to an average over 100–150 cells in a field of view, with at least two fields of view from at least three biologically different samples.

Figure 2

Cell density has no effect on cortical tension or E-cadherin, but it decreases tractions. (a),(b) Confocal images of PMLC at low ($1200\mathrm{cells}/{\mathrm{mm}}^2$) and high density ($4200\mathrm{cells}/{\mathrm{mm}}^2$). (c),(d) Confocal images of actin and E-cadherin for cells at low (c) and high density (d). (e) Drawing showing analysis of fluorescent intensities at cell peripheries. Segmented cell boundaries (blue) are dilated by $1\mu \mathrm{m}$ (gray); pixels within the dilated region are used for quantifying peripheral fluorescent intensities. (f) Fluorescent intensity of peripheral PMLC is unaffected by cell density ($p=0.4$). For all data quantifying fluorescent images in this manuscript, each dot corresponds to an average over 100–150 cells in a field of view, with at least two fields of view from at least three biologically different samples. (g) Fluorescent intensity of peripheral E-cadherin is unaffected by cell density ($p=0.7$). (h) Change in distance between the vertices of an ablated edge $d(t)-d(0)$ in cells at low and high densities. Each line corresponds to an ablated cell edge. (i) Cells at low density show higher initial recoil velocity compared to cells at high density ($p<0.01$). For all data quantifying recoil velocity in this manuscript, each dot corresponds to a different ablated edge. (j) Average rms traction decreases with increase in cell density ($p<0.001$). (k) Average traction imbalance computed over different circular regions of radius $R$. In this and all subsequent figures, quantifying traction or traction imbalance, each dot corresponds to a different cell island. (l) Average traction imbalance for $R=12\mu \mathrm{m}$ is statistically larger in islands of low density than high density ($p<0.001$).

Figure 3

Effect of blebbistatin and cytochalasin D on cortical tension and cell perimeter. (a) Cortical actin imaged for control cells (top) or cells treated with $20\mu \mathrm{M}$ of blebbistatin (bottom). (b) Cell curvature index (defined as total edge length divided by end-to-end distance) increases after treating with $20\mu \mathrm{M}$ of blebbistatin ($p<0.01$). (c) PMLC imaged for control cells (top) or cells treated with $0.05\mu \mathrm{M}$ of cytochalasin D (bottom). (d) Peripheral PMLC fluorescent intensity is decreased by cytochalasin D ($p<0.05$). (e),(f) Change in distance between the vertices of an ablated edge $d(t)-d(0)$ for the control and cells treated with $20\mu \mathrm{M}$ of blebbistatin (e) or $0.05\mu \mathrm{M}$ of cytochalasin D (f). (g),(h) Initial recoil velocity decreases after treating with $20\mu \mathrm{M}$ of blebbistatin [$p<0.01$ (g)] or $0.05\mu \mathrm{M}$ of cytochalasin D [$p<0.001$ (h)]. (i),(j) Average perimeter $⟨q⟩$ measured after treating with blebbistatin (i) or cytochalasin D (j) at different concentrations. Both $20\mu \mathrm{M}$ of blebbistatin ($p<0.01$) and $0.05\mu \mathrm{M}$ of cytochalasin D ($p<0.001$) decrease $⟨q⟩$. For all data in this figure, the cell density is approximately $1200\mathrm{cells}/{\mathrm{mm}}^2$.

Figure 4

Blebbistatin and cytochalasin D show dose-dependent reduction in tractions. (a),(b) Average rms traction for different cell islands measured over time before ($t<0$) and after ($t>0$) treating with blebbistatin (a) or cytochalasin D (b). For each island, the average rms traction is normalized by its average value before the treatment. (c),(d) Normalized rms tractions are averaged for $t=60–120\min$. All groups are statistically different from each other ($p<0.001$). (e),(f) Average traction imbalance over different circular regions of radius $R$ for control islands and islands treated with blebbistatin (e) and cytochalasin D (f). (g),(h) The magnitude of average traction imbalance for $R=12\mu \mathrm{m}$ is reduced by both blebbistatin (g) and cytochalasin D (h) ($p<0.001$ for both treatments compared to the control). For all data in this figure, the cell density is approximately $1200\mathrm{cells}/{\mathrm{mm}}^2$.

Figure 5

Effect of density on stress fiber organization. (a),(b) Confocal images of actin stress fibers in low- ($1200\mathrm{cells}/{\mathrm{mm}}^2$) (a) and high-density ($4200\mathrm{cells}/{\mathrm{mm}}^2$) (b) monolayers. (c) Order parameter $S$ quantifying average stress fiber alignment in different monolayers. $S$ is lower at high density compared to low density ($p<0.001$). (d),(e) Confocal images of actin stress fibers of control cells (d) and cells treated with $40\mu \mathrm{M}$ of SMIFH2 (e) (the density for both panels is approximately $1200\mathrm{cells}/{\mathrm{mm}}^2$). (f) The average dimensionless perimeter $⟨q⟩$ decreases with SMIFH2 treatment ($p<0.001$) for both treatments compared to the control.

Figure 6

Increasing cell-substrate tractions reverses the effect of density on the cell perimeter and cell rearrangements. (a) Average perimeter $⟨q⟩$ for cells in islands of low density ($1200\mathrm{cells}/{\mathrm{mm}}^2$), high density ($2600\mathrm{cells}/{\mathrm{mm}}^2$), or high density with the Rho activator CN03 ($2\mu \mathrm{g}/\mathrm{mL}$). The low-density and CN03 groups are statistically different from the high-density groups ($p<0.005$). (b) MSD for low-density, high-density, and CN03-treated high-density islands. (c) Exponent $\alpha$ of the MSD for low-density and CN03-treated high-density islands is different from the untreated high-density islands ($p<0.005$). (d) Exponent $\alpha$ of CRMSD for low-density and CN03-treated high-density islands is different from the untreated high-density islands ($p<0.01$). (e) Average rms traction for high-density and CN03-treated high-density islands measured over time before ($t<0$) and after ($t>0$) treatment with vehicle control or CN03. Shading shows time points for which tractions produced by the CN03-treated cells are statistically different from the high-density control ($p<0.001$). (f) Average traction imbalance for high-density and CN03-treated high-density islands measured over time before ($t<0$) and after ($t>0$) treatment with vehicle control or CN03 for a circular region of radius $R=12\mu \mathrm{m}$. Shading shows time points for which CN03-treated cells are statistically different from the high-density control ($p<0.001$). (g) Average cell perimeter becomes statistically larger than the control at 230 min after the CN03 treatment ($p<0.005$).

Figure 7

Effect of density on collective motion. Cell trajectories computed over a time span of 8 h in low- ($1200\mathrm{cells}/{\mathrm{mm}}^2$) (a) and high-density ($4200\mathrm{cells}/{\mathrm{mm}}^2$) (b) islands. These are the same cell islands as in Figs. 1 and 1. (c) Mean-square displacement computed from trajectories of selected cells (${\mathrm{MSD}}_i$) from single islands of low and high density. (d) Exponent ${\alpha }_i$ of the ${\mathrm{MSD}}_i$ curves in panel (c) ($p<0.001$). Each dot in panel (d) represents an individual cell. (e) Exponent ${\alpha }_{\mathrm{CR}}$ of the CRMSD curves for different cell pack sizes $N$ for cell islands of low and high densities. (f) CRMSD of low- and high-density islands for $N=20$.

Figure 8

Correlation between the aspect ratio and dimensionless perimeter. (a) Scatter plot between the aspect ratio and dimensionless perimeter for cells at low ($1200\mathrm{cells}/{\mathrm{mm}}^2$) and high density ($4200\mathrm{cells}/{\mathrm{mm}}^2$). Each dot corresponds to a different cell. Aspect ratio is computed from the cell vertices using the regionprops command of matlab 2015a. (b) Mean aspect ratio is smaller at high density compared to low density ($p<0.001$). Each dot corresponds to an average of 100–150 cells in a different field of view.

Figure 9

Recoil in vertices that connect a cell edge after laser ablation. Change in distance $d(t)-d(0)$ between vertices after laser ablation of cells in a monolayer of low density ($1200\mathrm{cells}/{\mathrm{mm}}^2$). Initial recoil velocity is computed by fitting a line to data points in the gray region corresponding to $t=0–11\mathrm{s}$. Each line corresponds to an ablated cell edge.

Figure 10

(a),(b) Color maps of traction magnitude in low-density ($1200\mathrm{cells}/{\mathrm{mm}}^2$) (a), high-density ($4200\mathrm{cells}/{\mathrm{mm}}^2$) (b), blebbistatin-treated ($1200\mathrm{cells}/{\mathrm{mm}}^2$) (c), and cytochalasin D–treated ($1200\mathrm{cells}/{\mathrm{mm}}^2$) (d) islands.

Figure 11

Effect of blebbistatin and cytochalasin D on E-cadherin and stress fibers. (a)–(c) Confocal images of cortical actin, E-cadherin, and actin stress fibers for the control cell monolayers (a) and cell monolayers treated with $20\mu \mathrm{M}$ of blebbistatin (b) or $0.05\mu \mathrm{M}$ of cytochalasin D (c). (d),(e) Peripheral E-cadherin intensities are unaffected by $20\mu \mathrm{M}$ of blebbistatin [(d) $p=0.45$] or $0.05\mu \mathrm{M}$ of cytochalasin D [(e) $p=0.23$]. The cell density in all panels is $1200\mathrm{cells}/{\mathrm{mm}}^2$.

Figure 12

Cell rearrangements decrease with inhibition of actomyosin contractility. (a),(c) MSD of control cell islands and islands treated with $20\mu \mathrm{M}$ of blebbistatin (a) or $0.05\mu \mathrm{M}$ of cytochalasin D (c). (b),(d) Exponent $\alpha$ of MSD decreases after treating with $20\mu \mathrm{M}$ of blebbistatin [(b) $p<0.05$] or $0.05\mu \mathrm{M}$ of cytochalasin D [(d) $p<0.001$]. (e),(g) CRMSD of control cell islands and islands treated with $20\mu \mathrm{M}$ of blebbistatin (e) or $0.05\mu \mathrm{M}$ of cytochalasin D (g). (f),(h) Exponent ${\alpha }_{\mathrm{CR}}$ of CRMSD decreases after treating with $20\mu \mathrm{M}$ of blebbistatin [(f) $p<0.001$] or $0.05\mu \mathrm{M}$ of cytochalasin D [(h) $p<0.001$]. (i),(k) ${\mathrm{MSD}}_i$ for selected cell trajectories in the control islands and islands treated with $20\mu \mathrm{M}$ of blebbistatin (i) or $0.05\mu \mathrm{M}$ of cytochalasin D (k). (j),(l) Exponent ${\alpha }_i$ of ${\mathrm{MSD}}_i$ of cell trajectories in the control islands and islands treated with $20\mu \mathrm{M}$ of blebbistatin [(j) $p<0.001$] or control islands and islands treated with $0.05\mu \mathrm{M}$ of cytochalasin D [(l) $p<0.001$]. Dots in panels (j) and (l) represent individual cells. The cell density in all panels is $1200\mathrm{cells}/{\mathrm{mm}}^2$.

Figure 13

Ratio of total PMLC intensity in the bulk and peripheral region. Peripheral PMLC is quantified as described in Sec. 4. Bulk PMLC is quantified in the same region as the stress fibers, namely, at the base of the cell layer and away from the cell peripheries. The cell density is $1200\mathrm{cells}/{\mathrm{mm}}^2$.

Figure 14

Stress fiber inhibition. (a) Average rms traction for each island is measured over time before ($t<0$) and after ($t>0$) treating with the formin inhibitor SMIFH2. For each island, the rms traction is normalized by its average value before the treatment. (b) Normalized rms traction for $t=60–180\min$. Relative to control, both concentrations reduce the rms traction ($p<0.001$). (c) Order parameter $S$ decreases after stress fiber inhibition ($p<0.001$). The cell density is $1200\mathrm{cells}/{\mathrm{mm}}^2$. (d) Change in distance between the vertices of an ablated edge $d(t)-d(0)$ for the control and cells treated with $40\mu \mathrm{M}$ of SMIFH2. (e) Initial recoil velocity after laser ablation is decreased by SMIFH2 ($p<0.01$). (f) Peripheral PMLC fluorescent intensity is increased by SMIFH2 ($p<0.05$). (g) Peripheral E-cadherin intensity is decreased by SMIFH2 ($p<0.01$).

Figure 15

Rho activator CN03 increases stress fibers and tractions. (a)–(c) Confocal images of actin stress fibers for the control cells at high density (a) and cells at high density at $t=180\min$ (b) or 320 min (c) after treating with CN03 ($2\mu \mathrm{g}/\mathrm{mL}$). (d) Order parameter for high density is smaller than for high density treated with CN03 ($p<0.001$ for both groups compared to the high-density control). (e),(f) Color maps of traction magnitude of high density (e) and high density treated with CN03 at $t=320\min$ (f). Cell density for all data in this figure is $2600\mathrm{cells}/{\mathrm{mm}}^2$.

Figure 16

Effect of CN03 ($2\mu \mathrm{g}/\mathrm{mL}$) on line tension. (a) Fluorescent intensity of peripheral PMLC ($p=0.12$). (b),(c) Change in distance $d(t)-d(0)$ after laser ablation (b) and initial recoil velocity [$p=0.86$ (c)]. (d) Fluorescent intensity of peripheral E-cadherin ($p=0.15$). Cell density for all data in this figure is $2600\mathrm{cells}/{\mathrm{mm}}^2$.

Figure 17

Changes in traction precede changes in dimensionless cell perimeter. (a) Average rms traction for the high-density island ($2600\mathrm{cells}/{\mathrm{mm}}^2$) and high-density islands treated with CN03 ($2\mu \mathrm{g}/\mathrm{mL}$) measured over time before ($t<0$) and after ($t>0$) treatment with vehicle control or CN03. (b),(c) Phase contrast images of control cells (b) and CN03-treated cells (c) at different times during the treatment.

Figure 18

Rho activator CN03 increases cell rearrangements. (a) CRMSD for islands of low density, high density, and high density treated with CN03 ($2\mu \mathrm{g}/\mathrm{mL}$). (b) ${\mathrm{MSD}}_i$ for selected cell trajectories in islands of low density ($1200\mathrm{cells}/{\mathrm{mm}}^2$), high density ($2600\mathrm{cells}/{\mathrm{mm}}^2$), and high density treated with CN03 islands ($2\mu \mathrm{g}/\mathrm{mL}$). (c) Exponent ${\alpha }_i$ of ${\mathrm{MSD}}_i$ of cell trajectories in islands of low density, high density, and high density treated with CN03 ($2\mu \mathrm{g}/\mathrm{mL}$). Each dot represents an individual cell.