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, 10 (6), 469-75

Collective Cell Guidance by Cooperative Intercellular Forces

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Collective Cell Guidance by Cooperative Intercellular Forces

Dhananjay T Tambe et al. Nat Mater.

Abstract

Cells comprising a tissue migrate as part of a collective. How collective processes are coordinated over large multi-cellular assemblies has remained unclear, however, because mechanical stresses exerted at cell-cell junctions have not been accessible experimentally. We report here maps of these stresses within and between cells comprising a monolayer. Within the cell sheet there arise unanticipated fluctuations of mechanical stress that are severe, emerge spontaneously, and ripple across the monolayer. Within that stress landscape, local cellular migrations follow local orientations of maximal principal stress. Migrations of both endothelial and epithelial monolayers conform to this behaviour, as do breast cancer cell lines before but not after the epithelial-mesenchymal transition. Collective migration in these diverse systems is seen to be governed by a simple but unifying physiological principle: neighbouring cells join forces to transmit appreciable normal stress across the cell-cell junction, but migrate along orientations of minimal intercellular shear stress.

Figures

Figure 1
Figure 1
(a) Simplified representation of the physical relationship between cell-substrate tractions, T, which have been reported previously, and intercellular stresses, σ, which are reported for the first time here. Intercellular stresses arise from the accumulation of unbalanced cell-substrate tractions. At any point within the monolayer (b), the intercellular stresses, defined in laboratory frame (x, y), (c), have shear (σxy, and σyx) and normal (σxx, and σyy) components. This frame can be rotated locally to obtain the principal frame (x', y'), (d), where shear stresses vanish and the resulting normal stresses are called principal stresses (σmax and σmin). The corresponding axes are called maximum, aligned with x', and minimum, aligned with y', principal orientations.
Figure 2
Figure 2. Intercellular stress maps and mechanical guidance of collectively migrating monolayers
(a) Transmitted light image of rat pulmonary microvascular endothelial (RPME) cell monolayer. Corresponding to this image are the maps of average normal stress (b), which is predominately tensile but forms a rugged stress landscape (c), the maximum shear stress (d), principal stress ellipses (blue) and cell velocity vectors (red) (e). The alignment angle, ϕ, between major axis of the principal stress ellipse and direction of the cellular motion (f, inset) shows that the greater the local shear stress the narrower is the distribution of ϕ (f, g, h). The cumulative probability distribution (ϕ) varied strongly and systematically with stress anisotropy (i); curves from blue, to red are in the order of higher quintiles. Comparable maps are found for the Madin-Darby canine kidney (MDCK) cell monolayer (j–n). Note that the average tensile stress (k) increased systematically with increasing distance from the advancing front thus contributing to the state of global tug-of-war. Vertical size of the images of cell monolayer: RPME − 545 µm, MDCK − 410 µm. Each curve in (i) and (n) and distributions in (f), (g), and (h) have more than 8,000 observations.
Figure 3
Figure 3. Stress maps and migration in monolayers of breast-cancer model systems
Phase contrast image of nontransformed human mammary epithelial cell line, MCF10A, control or vector (a), cells overexpressing ErbB2 (b), and 14-3-3ζ (c). Maps of cell-substrate tractions, Tx, (d, e, f), normal stress (g, h, i), and maximum shear stress (j, k, l) corresponding to each of these three mammary epithelial cell lines. (m) Cumulative probability distribution of ϕ for the regions corresponding to highest quintile of the shear stress for five different cell sheets. (t) Distributions corresponding to the curves in (m). Vertical size of the images of monolayer: 410 µm. Each curve in (m) has more than 8,000 observations.
Figure 4
Figure 4. Local cell guidance requires force transmission from cell-to-cell
Time-controls of intercellular stress maps of MCF10A-vector cell monolayers (af). The stress patterns do not change appreciably over a period of 80 minutes. After 10 minutes in presence of the calcium chelator EGTA (4mM), however, cells lose contacts with their neighbors (g, i and m, o). These changes lead to attenuation of intercellular average normal stress (h, j and n, p). After returning to normal growth medium for 80 minutes, the stresses and the cell-cell contacts are largely restored (k, l), but if the growth medium is supplemented with E-cadherin antibody (7 µg/ml) recovery of stresses and cell-cell contact is blocked (q, r). EGTA treatment widens the distribution of angle (ϕ) between local cellular velocity and local maximum principal orientation corresponding to highest of the maximum shear stress quintiles (s, t). The distribution of ϕ is narrowed if calcium is restored (s and t, blue), but widened further if the restoration medium is supplemented with E-cadherin antibody (s and t, red). Together, these data show that local cell guidance along the orientation of maximal principal stress (plithotaxis) requires force transmission across cell-cell junctions. These preferred orientations correspond to those engendering minimal intercellular shear stresses. Increased intensity at cell boundaries in phase contrast images (panels i, o, and q) reveals disruption of cell-cell junctions. Vertical size of the images of monlayer: 410 µm. Each data set in (s and t) has more than 1,500 observations.
Figure 5
Figure 5. Signatures of cooperativity and associated glassy dynamics
Phase contrast images of a monolayer of Madin-Darby canine kidney (MDCK) cells well away from the leading edge at early (a, t=196 min, density=1681±88 cells/mm2) and late (b, t=3196 minutes, density=2487±218 cells/mm2) times. Also shown are corresponding maps of average normal stress (c, d). Note that any contribution to the stress field with a wavelength longer than the size of the field of view is not included in the calculation. Thus a stress build up extending over the entire monolayer as previously reported is absent from this analysis. (e) Time averaged spatial autocorrelation function, C(r), of average normal stress in low density (1681 cells/mm2, blue), and high density (2487 cells/mm2, red) regions. (f) C(r) of high density maximal principal stress resolved into components representing force chains (circles) and force clusters (squares). (g) Variance, χss, of the self-overlap parameter, qs, as a function of time, in early, low denisty (t=1–270 minutes, 1699 ±40 cells/mm2, blue) and late, high density (t= 1800–2070 minutes, 1950±156 cells/mm2, red) intervals. Each curve represents an average over three successive 90 minute windows of similar density. Error bars represent the standard deviation over the square root of the number of windows. Vertical size of the images of monolayer: 480 µm.

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