A steering model of endothelial sheet migration recapitulates monolayer integrity and directed collective migration

Abstract

Cells in endothelial cell monolayers maintain a tight barrier between blood and tissue, but it is not well understood how endothelial cells move within monolayers, pass each other, migrate when stimulated with growth factor, and also retain monolayer integrity. Here, we develop a quantitative steering model based on functional classes of genes identified previously in a small interfering RNA (siRNA) screen to explain how cells locally coordinate their movement to maintain monolayer integrity and collectively migrate in response to growth factor. In the model, cells autonomously migrate within the monolayer and turn in response to mechanical cues resulting from adhesive, drag, repulsive, and directed steering interactions with neighboring cells. We show that lateral-drag steering explains the local coordination of cell movement and the maintenance of monolayer integrity by allowing closure of small lesions. We further demonstrate that directional steering of cells at monolayer boundaries, combined with adhesive steering of cells behind, can explain growth factor-triggered collective migration into open space. Together, this model provides a mechanistic explanation for the observed genetic modularity and a conceptual framework for how cells can dynamically maintain sheet integrity and undergo collective directed migration.

FIG. 1.

Experimental and conceptual bases for the endothelial steering model. (A) Classes of genes regulating collective endothelial migration (for further details, see reference 22). (Right) Scratch assay for endothelial sheet migration. Shown are images before and after addition of growth factor displaying growth factor-triggered migration. Bar, 150 μm. (B) (Left) Individual tracks of hundreds of cells in a HUVEC monolayer imaged every 20 min for 15 h. The tracks are colored according to the direction of migration to highlight coordinated movement of neighboring cells. Bar, 150 μm. (Right) Time lapse fluorescence images of HUVEC expressing GFP-VE-cadherin showing vertex-switching events as cells pass each other in a sheet. Bar, 25 μm. (C) (Left) Immunofluorescence image showing cell nuclei stained with Hoescht (green) and adherens junctions stained with anti-VE-cadherin antibody (red). (Middle) Predicted cell boundaries (red lines) derived from Voronoi tessellation of nuclear centroids (green circles). Cell nuclei highlighted with white asterisks represent the neighbors of the yellow cell. (Right) Overlay of measured and predicted cell boundaries. Bar, 25 μm. (D) Schematic representation of the “turtle” characteristic of autonomously migrating cells, where cell motility is generated at the basolateral surface through lamellipodial extensions while cell contacts above remodel to maintain tight barriers. (E) Schematics of the four steering mechanisms that turn autonomously migrating cells. The blue arrows are the current directions of migration. Predicted relative contributions from steering terms are represented by arrows.

FIG. 2.

Steering model predicting collective endothelial migration behaviors. (A) Computational cell monolayers respond to a large open space over 10 simulated hours in the absence (left) and presence (right) of growth factor. Enlargements of the regions outlined by yellow dashes are shown below. The colors of the nuclei indicate the dominant steering type: green, adhesive; red, directed; magenta, repulsive; and black, lateral drag. The arrows illustrate the direction and magnitude of steering terms. (B) Schematic representation of two possible migration outcomes for monolayers starting with triangular projections: finger extension versus corner retention. (C) Images of simulated monolayers responding to a cell-free area with pioneer cells at artificially generated triangular corner positions. The snapshots represent cells immediately after the introduction of cell-free space and after a simulated 12-h period, showing corner retention. (D) HUVEC subjected to a cross scratch that generated triangular projections before and after a 12-h incubation period, showing corner retention. The cells were assayed as in Fig. 1A. Bars, 150 μm.

FIG. 3.

Adhesive steering mediates follower behavior during directed sheet migration. (A) Directed migration as a function of distance from the cell-free area. Directed migration is defined as the fraction of time a cell spends moving toward the cell-free area (see the text). (B) Sheet migration rates for monolayers simulated with indicated relative changes to steering terms. The sheet migration rate was calculated as the average progression of the sheet boundary per hour. (C) Images from migrating monolayers run for 10 simulated hours for control (left), no adhesion (middle), and no drag term (right), suggesting that adhesion is important for sheet cohesion. (D) HUVEC monolayer transfected with control siRNA (siControl) or siRNA targeting alpha-catenin (siCTNNA1), a regulator of cell-cell junctions, fixed 15 h after the introduction of cell-free space and stained with fluorescein-phalloidin, showing loss of cohesion. Zoom, enlargements of the areas outlined by yellow dashes. All measurements represent the average of three replicates with error bars indicating standard errors. Bars, 150 μm.

FIG. 4.

Drag mediates coordinated migration and small-lesion closure even in the absence of directed motility. (A) Cell tracks from a computational model run for 16 simulated hours colored according to the direction of migration. (B) Quantification of coordinated migration in simulated tracks with indicated relative steering changes (for details, see the text). (C) Images of cell monolayers derived from the computational model representing 0, 4, 12, and 16 simulated hours after the introduction of a small lesion. Steering terms were included (+) or eliminated (−) as indicated. (Right) Zoomed image of a simulated monolayer with an excluded boundary cell highlighted in yellow. (D) Number of boundary cells surrounding a small lesion plotted as a function of time for different steering conditions, suggesting that drag helps to reduce the number of cells bordering small lesions. All measurements represent the averages of three replicates, with error bars indicating standard errors. (E) Differential migration into small and large open spaces. Cells were assayed as in Fig. 1D in the presence (left) and absence (right) of FGF. Merge, merged image of the monolayer before (red) and after (green) a 12-hour migration period, showing that small lesions can close in the absence of directed migration. The perimeter of the small wound and edge of the sheet boundary are marked with yellow dashed lines. (F) Phalloidin staining for polymerized actin in endothelial cells surrounding a small lesion showing no apparent actomyosin ring, supporting a migration- over a constriction-based closure mechanism. Bars, 150 μm.

FIG. 5.

Four steering mechanisms are necessary and sufficient to generate sheet cohesion; elasticity; and collective, directed migration. Shown is a schematic representation of emerging monolayer functions. The dominant steering terms are indicated in boldface.