Fig. 4: OptoShroom3-induced colony folding.
From: Optogenetic control of apical constriction induces synthetic morphogenesis in mammalian tissues
a Protocol for the formation of flat MDCK monolayers in a thick matrigel bed using acetic acid incubation (top) and scheme of measured parameters (bottom). b 3D renders and higher resolution lateral slices of representative colony folding before and after 24 h of stimulation (stimulated cycle: 3 min off, 57 min on; non-stimulated cycle: 3 min on (GFP-NShroom3-iLID image acquisition), 57 min off). GFP-NShroom3-iLID expression. Scale bar = 20 μm. c Skeletons used for curvature measurements of representative examples of non-stimulated (left), stimulated (center), and stimulated C450V mutant (right) colonies. GFP-NShroom3-iLID expression. d Quantification of changes in average curvature of stimulated, stimulated C450V mutant and non-stimulated colonies. Concave (apical) curvature was set as positive (NStimulated = 7, NNon-stimulated = 7, NC450Vmutant = 5, avg ± sd). e Z-projected images of colonies used for area measurements of non-stimulated (left), stimulated (center), and stimulated C450V mutant (right) colonies. GFP-NShroom3-iLID expression. Scale bar = 20 μm. f Quantification of relative changes in the projected area of stimulated, stimulated C450V mutant, and non-stimulated colonies (NStimulated = 7, NNon-stimulated = 7, NC450Vmutant = 5, avg ± sd). g Representative example of irreversible colony folding on matrigel labeled with infra-red fluorescent beads. White arrowheads indicate matrigel breaking points, and yellow arrowhead indicates matrigel being deformed and carried over with the folding colony. Scale bar = 20 μm. h Quantification of relative changes in the projected area of stimulated colonies during and after stimulation (stimulation period in blue) (N = 5, avg ± sd).
