Local light-activation of the Src oncoprotein in an epithelial monolayer promotes collective extrusion

Abstract

Transformed isolated cells are usually extruded from normal epithelia and subsequently eliminated. However, multicellular tumors outcompete healthy cells, highlighting the importance of collective effects. Here, we investigate this situation in vitro by controlling in space and time the activity of the Src oncoprotein within a normal Madin-Darby Canine Kidney (MDCK) epithelial cell monolayer. Using an optogenetics approach with cells expressing a synthetic light-sensitive version of Src (optoSrc), we reversibly trigger the oncogenic activity by exposing monolayers to well-defined light patterns. We show that small populations of activated optoSrc cells embedded in the non-transformed monolayer collectively extrude as a tridimensional aggregate and remain alive, while the surrounding normal cells migrate towards the exposed area. This phenomenon requires an interface between normal and transformed cells and is partially reversible. Traction forces show that Srcactivated cells either actively extrude or are pushed out by the surrounding cells in a nonautonomous way.

Control of SRC molecular dynamics encodes distinct cytoskeletal responses by specifying signaling pathway usage

“…Indeed, OS activation induces dorsal ruffles, as seen previously for activation of a thermo-sensitive SRC mutant (Mettlen et al, 2006) and the activation of chemo-controllable synthetic SRC, containing both functional SH2 and SH3 domains (Gentry et al, 2016;Karginov et al, 2010). OS activation on membrane-induced lamellipodia in epithelial cells, suggesting activation is a partial epithelial-mesenchymal transition process that is in line with a previous report showing that only this mode of activation induced mechanical remodeling of epithelial layers (Moitrier et al, 2019). In MDCK cells, another mode of OS activation even induced invadosome rings, which are typical c-SRC-dependent structures in physiological and pathological conditions, as revealed by the study of c-Src −/− models (Boateng and Huttenlocher, 2012;Luxenburg et al, 2006).…”

Crisscross multilayering of cell sheets

“…They can take the form of aggregates on top of the initial monolayer (for instance neural progenitor cells (NPCs) in vitro ( 8)), or adopt a stratified structure in which the cells differentiate as the multilayered structure develops (for instance skin epidermis or thymus (9)). Of note, transformed epithelial cells that express an oncogene also develop 3D structures after confluence, often coupled with the down-regulation of cell-cell adhesions in the context of the Epithelial-to-Mesenchymal Transition (EMT) (1,2,(10)(11)(12)(13). Because of the physiological importance of this 2D to 3D transition, strategies to include these 3D aspects have been developed when engineering functional tissues in vitro.…”

Light-regulated allosteric switch enables temporal and subcellular control of enzyme activity

“…Several optogenetic approaches for the regulation of protein kinases have been described previously; however, all had specific shortcomings. Several methods regulate the localizationof a kinase rather than its catalytic activity ( Kawano et al, 2015 ; Graziano et al, 2017 ; Mühlhäuser et al, 2019 ; Moitrier et al, 2019 ; Katsura et al, 2015 ; Zhang et al, 2014 ). The method described by Zhou et al provides efficient control of kinases by light-regulated steric hindrance but it does not enable regulation on a subcellular level ( Zhou et al, 2017 ).…”