Fig 1.
MCF7 cells at the time of release from circle (A), square (B) and cross (C) shaped constraints are shown.
A representation of the regions chosen as the vertices (red), sides (blue) and intersections (green) in the three shapes are indicated in the boxes. Scale bar = 500 μm.
Fig 2.
The edges of the MCF7 clusters in circle (A), square (B) and cross (C) are shown at the start of the experiment (red), at 6 hours (green), 30 hours (blue) and 48 hours after removal of constraint (magenta).
Scale bar = 500 μm.
Fig 3.
Velocity and vorticity fields in MCF7 cell monolayers in the circle (A), square (B), cross vertex (C) and intersections (D) at 30 hours summed every hour.
The solid black line is the cluster border at the start of migration. Scale bar = 250 μm.
Fig 4.
Cluster speeds show temporal differences in the different regions of the circle (A), square (B) and cross (C) shapes respectively.
Speeds from the sides increase (I), plateau (II) and finally decrease (III) over time. These results show clear differences in cluster edge speeds which depend on spatial location in the geometry. Significant differences (p<0.05) in speeds are indicated in each figure.
Fig 5.
Migrations of MCF7 cells located on the advancing edges of the circle (A), square (B) and cross shapes (C) were obtained using individual cell tracking and are illustrated on a windrose plot to show the cell migration trajectories from the cluster edge.
We see differences in motility that depend on the cell spatial positions which are labelled corresponding to sides, vertices and intersections.
Fig 6.
Velocity fields and vorticity maps for MDCK cells are shown at 2 hours (A, D), 8 hours (B, E) and 16 hours (C, F) after removal of constraint.
The cluster border is also shown at 0 hours for reference. Cells in the vertices show minimal movement as compared to those in the sides and intersections (D, E, F). We see the development of vortices, in both clockwise (blue) and counter clockwise (yellow-red) directions at intersections by 16 hours. Scale bar = 250 μm.
Fig 7.
A: We delineate three phases in the MDCK clusters based on the temporal speed variations of initially constrained cells in the cross shape in regions corresponding to sides, vertices and intersections (p<0.05). We see an increase in the speeds of cells from the cluster sides and vertices up to 8 hours (Phase I), a plateau region observed until 12 hours (Phase II), followed by a decrease in the cluster speeds by 16 hours (Phase III) B. Cell trajectories depend on their spatial location in the cross shape.
Fig 8.
Fluorescence images show actin (red) and the nucleus (blue) for MCF7 clusters at 0 hours (A), 24 hours (B), and 48 hours (C) after release from constraints.
There is no clear cable-like structure bounding the cluster. Scale bar = 100 μm.
Fig 9.
MDCK cells were stained for actin (red) and nucleus (blue) and are shown at 0 (A) and 2 hours (B) after removal of constraint. The acto-myosin cable at cluster borders is visible. Higher magnification (C) image clearly shows the presence of the actin cable.
Fig 10.
Clusters were stained to explore the presence of E-cadherin in A. MCF7 clusters and B. MDCK cells constrained in the cross shape. The cell nucleus was labelled using DAPI.
Fig 11.
A. To assess emergence of leader cells in the MDCK clusters, speeds corresponding to the edges were quantified starting at the vertex marked using an asterisk in the figure. B. Raw data from MDCK cell clusters in cross shapes from two opposite edges at 2 hours after removal of constraint show variations in their speeds with location. C. Data were smoothened and the peaks were identified. D. Data were averaged from four cross arms to quantify average distances between peaks.
Fig 12.
Velocity and vorticity plots are shown for cell clusters treated with 50 μM blebbistatin to inhibit myosin2 and alter cytoskeletal tension.
A. MCF7 at 30 hours and B. MDCK at 16 hours. Scale bar = 200 μm.
Fig 13.
Effects of cytochalasin-D are shown for A. MCF7 cells and B. MDCK cells in cross shape at the start of the migration experiment. The edges of the cell ensembles are shown in red for MCF7 at 48 hours and MDCK at 16 hours which show minimal migrations over time.
Fig 14.
Fluorescence images of A. MCF7 cells at 48 hours and B. MDCK cells at 16 hours in the cross shape. There are few stress fibers seen in both cell types at the end of the experiments.