SummaryContact inhibition of locomotion (CIL) is a multifaceted process that causes many cell types to repel each other upon collision. During development, this seemingly uncoordinated reaction is a critical driver of cellular dispersion within embryonic tissues. Here, we show that Drosophila hemocytes require a precisely orchestrated CIL response for their developmental dispersal. Hemocyte collision and subsequent repulsion involves a stereotyped sequence of kinematic stages that are modulated by global changes in cytoskeletal dynamics. Tracking actin retrograde flow within hemocytes in vivo reveals synchronous reorganization of colliding actin networks through engagement of an inter-cellular adhesion. This inter-cellular actin-clutch leads to a subsequent build-up in lamellar tension, triggering the development of a transient stress fiber, which orchestrates cellular repulsion. Our findings reveal that the physical coupling of the flowing actin networks during CIL acts as a mechanotransducer, allowing cells to haptically sense each other and coordinate their behaviors.

Cell migration is hypothesised to involve a cycle of behaviours beginning with leading edge extension. However, recent evidence suggests that the leading edge may be dispensable for migration, raising the question of what actually controls cell directionality. Here we exploit the embryonic migration of Drosophila macrophages to bridge the different temporal scales of the behaviours controlling motility. This reveals that edge fluctuations during random motility are impersistent and weakly correlated with motion. In contrast, flow of the actin network behind the leading edge is highly persistent. Quantification of actin flow structure during migration reveals a stable organisation and asymmetry in the cell-wide flowfield that strongly correlates with cell Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Interactions between different cell types can induce distinct contact inhibition of locomotion (CIL) responses that are hypothesised to control population-wide behaviours during embryogenesis. However, our understanding of the signals that lead to cell-type specific repulsion and the precise capacity of heterotypic CIL responses to drive emergent behaviours is lacking. Using a new model of heterotypic CIL, we show that fibrosarcoma cells, but not fibroblasts, are actively repelled by epithelial cells in culture. We show that knocking down EphB2 or ERK in fibrosarcoma cells specifically leads to disruption of the repulsion phase of CIL in response to interactions with epithelial cells. We also examine the population-wide effects when these various cell combinations are allowed to interact in culture. Unlike fibroblasts, fibrosarcoma cells completely segregate from epithelial cells and inhibiting their distinct CIL response by knocking down EphB2 or ERK family proteins also disrupts this emergent sorting behaviour. These data suggest that heterotypic CIL responses, in conjunction with processes such as differential adhesion, may aid the sorting of cell populations.

SummaryInteractions between different cell-types can induce distinct contact inhibition of 1 locomotion (CIL) responses that are hypothesized to control population-wide 2 behaviors during embryogenesis [1, 2]. However, our understanding of the signals 3 that lead to cell-type specific repulsion, and the precise capacity of heterotypic CIL 4 responses to drive emergent behaviors is lacking. Using a new in vitro model of 5 heterotypic CIL between epithelial and mesenchymal cells, we show that 6 fibrosarcoma cells (HT1080), but not fibroblasts (NIH3T3), are actively repelled by 7 epithelial cells in culture. We show that knocking down EphB2 in fibrosarcoma cells 8 specifically leads to disruption of the repulsion phase of CIL in response to 9 interactions with epithelial cells. Furthermore, this heterotypic interaction requires 10 ERK activation, downstream of EphB2 signaling. We also examine the population-11 wide effects when these various cell combinations, and their specific heterotypic CIL 12 responses, are allowed to interact in culture. Mixtures of fibrosarcoma and epithelial 13 cells -unlike fibroblasts and epithelial cells -lead to complete sorting and 14 segregation of the two populations, and inhibiting their distinct CIL response by 15 knocking down EphB2 or ERK in fibrosarcoma cells disrupts this emergent sorting 16 behavior. Our understanding of the mechanisms underlying developmental 17All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/373696 doi: bioRxiv preprint first posted online Jul. 20, 2018; 2 behaviors such as cell sorting is lacking as predominant sorting hypotheses, such as 18 differential adhesion, have recently been found inadequate in predicting the sorting 19 of mesenchymal cells [3, 4]. These data suggest that heterotypic CIL responses, in 20 conjunction with processes such as differential adhesion, may aid the sorting of cell 21 populations during embryogenesis. 22 23 Results and Discussion 24 25 Fibroblasts and fibrosarcoma cells exhibit distinct responses upon collision 26with an epithelial cell monolayer 27To study heterotypic cell-cell collisions, we developed a confrontation assay 28 whereby two different cell-types are separated by a barrier, which upon removal, 29 creates a uniform gap into which the different cell populations migrate and collide. 30Following a screen of a range of different epithelial versus mesenchymal cell-types, 31 an interesting and unexpected phenomenon was revealed. When a population of 32 migrating epithelial cells (HaCaT) encountered a population of migrating fibroblasts 33 (NIH3T3), both populations ceased their forward migration, forming a sharp border 34 ( Figure 1A, B and Video S1). This is in stark contrast to fibrosarcoma cells (HT1080) 35 which, upon collision with epithelial cells, seemed to undergo a complete repulsion 36 ( Figure 1A, B and Video ...