Fig. 1
Collective extrusion triggered by use of light-inducible Src. a We use cryptochrome 2 (CRY2) and the truncated cryptochrome-interacting basic-helix-loop-helix (CIBN) light-gated heterodimerizer system to create the optoSrc. In response to blue light, the cytosolic optoSrc relocates to the membrane where it can phosphorylate its substrates. Note that the endogenous Src is present in all the optoSrc cells. b By illuminating a monolayer of optoSrc cells with patterned (blue disk) and intermittent (pulses separated by an interval Δt) blue light, we select in time and space the cells to transform. c, d A monolayer of Madin-Darby Canine Kidney (MDCK) optoSrc cells is intermittently exposed to blue light (~470 nm pulse of duration 200 ms every Δt = 5 min) in a circular domain of diameter 70 μm. c Only blue-light illuminated cells are displaying CIBN-GFP-Caax. d Phase contrast images of the monolayer of MDCK optoSrc cells at different time-points during the blue-light illumination. Time after the start of illumination is indicated in hours and blue circles delineate the region of illumination. At 30 h, the illuminated cells have started to collectively extrude from the monolayer. e Number of 3D aggregates formed, in percentage of attempt, depending on the optoSrc construct used, in presence or not of Src inhibitors (PP2 and Src inhibitor #5) for the same conditions of blue-light illumination. The number of attempts n is indicated above each bar, coming from at least 3 different experiments for each conditions, except for Src inhibitor #5 (1 experiment). The error bars represent the uncertainty as described in the Methods. f, g Confocal images acquired after 60 h of blue-light stimulation. Green: membranes (CIBN-GFP-Caax). Blue: nuclei (Hoechst). f Reconstructed side view reveals that extruded cells form a three-dimensional multi-cellular aggregate whose height reached 40 μm. Shaded blue area indicates the original region of illumination. g Single confocal slices at the level of the monolayer (left, orange dashed line in (f)) and 25 μm above (right, pink dashed line in (f)). Scale bars: 50 μm
