Contact inhibition of locomotion probabilities drive solitary versus collective cell migration

Abstract

Contact inhibition of locomotion (CIL) is the process whereby cells collide, cease migrating in the direction of the collision, and repolarize their migration machinery away from the collision. Quantitative analysis of CIL has remained elusive because cell-to-cell collisions are infrequent in traditional cell culture. Moreover, whereas CIL predicts mutual cell repulsion and 'scattering' of cells, the same cells in vivo are observed to undergo CIL at some developmental times and collective cell migration at others. It remains unclear whether CIL is simply absent during collective cell migration, or if the two processes coexist and are perhaps even related. Here, we used micropatterned stripes of extracellular matrix to restrict cell migration to linear paths such that cells polarized in one of two directions and collisions between cells occurred frequently and consistently, permitting quantitative and unbiased analysis of CIL. Observing repolarization events in different contexts, including head-to-head collision, head-to-tail collision, collision with an inert barrier, or no collision, and describing polarization as a two-state transition indicated that CIL occurs probabilistically, and most strongly upon head-to-head collisions. In addition to strong CIL, we also observed 'trains' of cells moving collectively with high persistence that appeared to emerge from single cells. To reconcile these seemingly conflicting observations of CIL and collective cell migration, we constructed an agent-based model to simulate our experiments. Our model quantitatively predicted the emergence of collective migration, and demonstrated the sensitivity of such emergence to the probability of CIL. Thus CIL and collective migration can coexist, and in fact a shift in CIL probabilities may underlie transitions between solitary cell migration and collective cell migration. Taken together, our data demonstrate the emergence of persistently polarized, collective cell movement arising from CIL between colliding cells.

Keywords: cell migration; cell polarization; cell–cell adhesion; contact inhibition of locomotion; micropatterning.

Figure 1.

Patterned culture reveals cell repolarization. (a) Time-lapse microscopy of cells 6–20 h after plating, and trajectories of 10 representative cells (lower right) 30 min before (blue) and after (red) the collision. In phase contrast micrographs, blue arrowheads indicate cell migration direction 1 h before the collision, yellow asterisk indicates the site of the collision at 0 h, and red arrowheads indicate the direction of cell migration 1 h after the collision. (b) X- and y-velocities of cells 30 min before (blue) and after (red) the collision. Means ± s.e. from three independent experiments. (c) Cells were fixed and immunostained 20 h after plating on micropatterns. (d,e) Time-lapse sequence, trajectories and velocities were measured as in (a,b) from cells on micropatterns. In (d), micropatterned stripes are indicated by purple shading. All scale bars, 25 μm.

Figure 2.

CIL is triggered upon head-to-head collision between cells. (a) Time-lapse microscopy of cells 6–20 h after plating, showing (i) persistent migration of a single cell, (ii) head-to-head collision between cells, (iii) head-to-tail collision between cells in which the rear cell repolarizes and the (iv) lead cell remains persistent, and (v) collision of a cell with an inert barrier. In (v), transmitted light optics were poor, so the cell is expressing GFP (green), is migrating along a fluorescent, micropatterned stripe (red), and collides with an inert topographical barrier (dashed line). In (ii–v), the collision occurs at 0 min. All scale bars, 25 µm. (b) The probability of repolarizing within 2 h of (i) beginning a persistent migration, or (ii–v) colliding with another cell or barrier as indicated in (a). Arrowheads in (a) indicate the cell (rear or lead) whose probability was measured for (b). In (ii), either cell of the head-to-head collision was measured. (c) For head-to-head collisions between single cells (situation ii), 58% of total cells had repolarized within 40 min of the collision.

Figure 3.

CIL operates statistically independently. (a) Upon a head-to-head collision between cells, (i) both, (ii) one or (iii) no cells can repolarize. (b) Expected (white bars) and experimentally observed (black bars) probabilities of the outcomes diagrammed in (a). The expected probability was computed using an CIL probability of 0.87 for each cell, and assumed cells repolarized statistically independently. Experimental measures in (b) represent the mean ± s.e. from three independent experiments.

Figure 4.

Trains behave as a coherent unit, and repolarize in a length-dependent manner. (a) Time-lapse microscopy of cells approximately 12–16 h after plating, showing a collective, persistent ‘train’ of seven cells. Panels with numbers are stills taken from a time-lapse sequence (see electronic supplementary material, movie S8). Bottom panel is a kymograph of time versus space, taken from the time-lapse sequence along the dashed yellow line drawn in the top panel. Numbers, time in minutes. Horizontal scale bars, 25 µm. Vertical scale bar, 1 h. (b) Immunofluorescent micrographs showing localization of E-cadherin (left) and microtubules and actin (right) in trains of cells on stripes of fibronectin. (c) Probability of repolarization of a train within 40 min of a head-to-head collision with a single cell versus number of cells in the train. Probabilities from at least three independent experiments (circles), and a curve fit of the data (grey). The curve fit equation is P(n) = exp(−[(n − 1) − β]), where P is probability, n is number of cells, and the fit parameter β = 0.546. See text for details.

Figure 5.

Collective, persistent trains of cells emerge from CIL between cells. (a) Time-lapse microscopy of cells approximately 12–16 h after plating, showing an example of multiple, single cells becoming entrained. Arrows indicate direction of train migration, and arrowheads indicate direction of migration of single cells at t = 0 min. Note that the train remains persistent throughout the sequence, whereas the single cells repolarize. Scale bar, 25 µm. (b) The proportion of single cells (grey) decreases with time and the proportion of cells in trains (black) increases with time. By 9 h, 88% of cells are in trains, and by 14 h, 93% of cells are in trains.

Figure 6.

An agent-based model predicts the emergence of trains. (a) A head-to-head collision between a single cell and a train of cells can result in four outcomes. Entrainment, in which the single cell repolarizes but the train remains persistent, is indicated by the black arrow. The rate of this transition from a head-to-head collision to entrainment is indicated by k. (b) Schematic of the model, in which a collision triggers a non-zero rate of repolarization. The magnitude of the rate of repolarization is given by length-dependent repolarization as measured in figure 4c. The duration of the rate of polarization (τ) is systematically varied in model simulations. At the end of the simulation, the rate of entrainment k is measured. (c) Entrainment rate k versus train length, as predicted by the model for τ = 80 min (grey line) and measured experimentally (black line). Simulated rates are based on 1000 simulations. Experimental rates are based on at least three independent experiments. (d) R², a measure of experimental data predicted by the model, versus τ was computed to assess the ideal value of τ. Note that R² is maximum at τ = 80 min. (e) The time for one cell to, through a series of head-to-head collisions and entrainments, give rise to a seven-cell train was computed based on model predictions versus single cell repolarization rate. Arrowhead, experimentally observed rate (2.2×10⁻² min⁻¹).