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. 2013 Sep;12(9):856-63.
doi: 10.1038/nmat3689. Epub 2013 Jun 23.

Propulsion and navigation within the advancing monolayer sheet

Jae Hun Kim  1 , Xavier Serra-Picamal, Dhananjay T Tambe, Enhua H Zhou, Chan Young Park, Monirosadat Sadati, Jin-Ah Park, Ramaswamy Krishnan, Bomi Gweon, Emil Millet, James P Butler, Xavier Trepat, Jeffrey J Fredberg
Affiliations

Propulsion and navigation within the advancing monolayer sheet

Jae Hun Kim et al. Nat Mater. 2013 Sep.

Abstract

As a wound heals, or a body plan forms, or a tumour invades, observed cellular motions within the advancing cell swarm are thought to stem from yet to be observed physical stresses that act in some direct and causal mechanical fashion. Here we show that such a relationship between motion and stress is far from direct. Using monolayer stress microscopy, we probed migration velocities, cellular tractions and intercellular stresses in an epithelial cell sheet advancing towards an island on which cells cannot adhere. We found that cells located near the island exert tractions that pull systematically towards this island regardless of whether the cells approach the island, migrate tangentially along its edge, or paradoxically, recede from it. This unanticipated cell-patterning motif, which we call kenotaxis, represents the robust and systematic mechanical drive of the cellular collective to fill unfilled space.

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Figures

Fig. 1
Fig. 1. Advancing monolayer of MDCK cells encounters and envelops a non-adherent island
A–D: MDCK cells in phase contrast at a sequence of times. In each of these panels, the inset depicts the whole island at the corresponding time point. E–H: Corresponding vectors of instantaneous migration velocities (obtained from PIV) (see Methods). I–L: Migration velocities, <V̄>, averaged over an ensemble of 6 such islands. Three findings are of note. First, fluctuations of velocity are comparable to or exceed local mean values. Second, two points of zero velocity, called stagnation points (red arrows), are evident; the positions of these stagnation points fluctuate in time but reside on average at the equator. Third, as a result, the flow of cells divides into two streams at the upstream stagnation point and merge at the downstream stagnation point. Scale bar in panel (A): 100μm. Velocity scale bars in (E) and (I) applies to (F–H) and (J–L), respectively.
Fig. 2
Fig. 2. Orientations of tractions, velocities, and principal stresses coincide, diverge, and recover
A–D: Color maps of the ensemble averaged tractions exerted between the monolayer and its substrate (see text for sign convention). A,B,D: x-component of traction, <Tx>, west and east of the island. C: y-component, <Ty>, north of the island (the inset shows <Tx> on the north boundary). These components were selected to reflect the directions roughly normal to the island boundaries. Upstream versus downstream (A,D), <Tx>shows large fluctuations but systematic differences. Regardless of position near a frustrated edge, tractions pull toward that edge. E–H: Color maps showing the systematic buildup of tension and velocity fields (black arrows) at the same locations and times as in panels (A–D). (Due to large gradients of accumulated tensions, the color scale for panels (G) and (H) are expanded for clarity.) I–L: Expanded views of two regions from (F) and one each from (G) and (H). Together with tractions (blue arrows) and the velocity field (black arrows), monolayer stresses are depicted by ellipses, with axes and orientations corresponding to the principal stresses, and iso-tension contours by dashed lines in (I) and (J). Stagnation points are shown by red arrows in (J) and (L). Note the coincidence, divergence and recovery of orientations as the monolayer engulfs the island. Scale bar in panel (A): 100μm. Velocity scale bars in (E) and (J) applies to (F–H) and (I,J,J), respectively.
Fig. 3
Fig. 3. Cellular morphology, tight junction structure and actin structure near the island
A: ZO-1 immunofluorescence micrograph at t = 24h when a monolayer of MDCK cells fully enclosed the island. B: Cell boundaries retrieved from ZO-1 micrograph in panel (A). Segmentation was performed using a watershed algorithm. C–F: Eccentricity (C), orientation (D), cell area (E) and major axis length (F) determined from cell boundaries in panel (B). Red arrows depict downstream stagnation points. Scale bar in (A): 200μm. G: All projected actin immunofluorescence micrographs at t=24h when a monolayer of MDCK cells fully enclosed the island at west of the island, at northern pole and at east of the island. Basal actin (H) and ZO-1 (I) immunofluorescence micrographs at the same locations corresponding to locations in panel (G). Scale bars in (G,H,I): 20μm.
Fig. 4
Fig. 4. Kenotactic tractions are evident in human mammary epithelial cells MCF10A vector, but are attenuated in MCF10A 14–3–3ζ, which disrupts adherens junctions
A,E: Phase contrast images of nontransformed human mammary epithelial cell line, MCF10A, vector control (A) and cells overexpressing 14–3–3ζ which have decreased expression of cell-cell junctional markers (E). B,F: Traction vectors, <T⃗>, averaged over an ensemble of 4 monolayers corresponding to cell types in panels (A,E) (see Methods). C,G: Color maps of x-component of tractions,<Tx>. D,H: Color maps of tractions normal to the frustrated edge, <Tn>. In case of nontransformed MCF10A vector cells, tractions near the frustrated edge are largest and oriented toward the edge (B,C,D). In case of MCF10A 14–3–3ζ cells, however, both the magnitude and alignment of tractions near the edge are attenuated (F,G,H). I: Normal component of tractions at the frustrated edge normalized by root-mean-square (RMS) traction across the entire maps, Tnedge/TRMS, for three cell types, MDCK (black), MCF10A vector (blue) and MCF10A 14–3–3ζ cells (red) (see Methods). *: Tnedge/TRMS of 14–3–3ζ transfected MCF10a cells is smaller than that of vector-transfected MCF10A cells or that of MDCK cells (mean +/− standard error of the mean; p< 0.05 by Kruskal-Wallis test). J: The alignment angle, φ, between traction vectors at the frustrated edge and normal vectors to the edge for three cell types in panel (I). MDCK and MCF10A vector cells are seen to exert tractions highly oriented toward the frustrated edge, which are largest at that edge (I, J). In contrast, MCF10A 14–3–3ζ cells exert tractions in smaller extent toward the edge, the alignment angle of which are widely distributed, as if they are not frustrated by the edge (I, J). Scale bar in panels (A,E): 100μm. Each bar in (I) include observations from 6 monolayers of MDCK cells and 4 monolayers per each MCF10A cell type. Distributions in (J) have more than 7,000 observations.
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Comment in

  • Cell migration: Towards the void.
    Dufresne ER, Schwartz MA. Dufresne ER, et al. Nat Mater. 2013 Sep;12(9):783-4. doi: 10.1038/nmat3743. Nat Mater. 2013. PMID: 23966050 No abstract available.

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