Figure 1. Light-dependent membrane recruitment of CRY2-Rho1DN results in rapid myosin inactivation.

(a) Drosophila mesoderm invagination occurs in distinct lengthening and shortening phases. Ventral side is up (same for all cross-section images in this work unless otherwise stated). A single ventral cell undergoing apical constriction is outlined in green. In this study, we sought to test whether myosin contractility is required throughout the folding process by acute, stage-specific inhibition of Rho1 (arrowheads). (b) Cartoon depicting the principle of the optogenetic tool used in this study. Upon blue light stimulation, CRY2-Rho1DN is translocated from the cytosol to the plasma membrane through the interaction between CRY2 and membrane anchored CIBN. (c) Confocal images showing the rapid membrane recruitment of CRY2-Rho1DN upon blue light illumination. (d) Relative abundance of membrane recruited CRY2-Rho1DN over time after blue light stimulation. Error bar: s.d., N=6 embryos. (e) A wild-type embryo expressing Sqh-GFP showing apical myosin accumulation during ventral furrow formation. N=4 embryos. (f) Activation of Opto-Rho1DN results in rapid dissociation of myosin from the ventral cell cortex (arrow) in a gastrulating embryo. N=8 embryos. (g) Confocal images showing the confined membrane recruitment of CRY2-Rho1DN within a region of interest (ROI, red box) that has been scanned by a focused beam of blue laser. N=6 embryos. All scale bars=20 μm.

Figure 1—figure supplement 1. Activation of Opto-Rho1DN inhibits apical constriction during gastrulation.

Confocal images showing the effect of activating Opto-Rho1DN on apical constriction during ventral furrow formation. Time zero corresponds to the time of stimulation. N≥2 embryos were tested for each condition. (a) Activating Opto-Rho1DN before the onset of gastrulation blocks apical constriction and ventral furrow formation. In the stimulated embryo, only a small set of cells scattered across the ventral domain undergo limited apical constriction over the course of 15 min. This is in contrast to the wild-type embryo where mesoderm invagination completes in 15–20 min. (b) Activating Opto-Rho1DN during apical constriction results in immediate relaxation of the constricted cells (cyan arrows). (c) Spatially confined activation of Opto-Rho1DN (red box) before gastrulation blocks apical constriction specifically in the stimulated region. Ventral furrow still forms in the unstimulated region of the embryo (magenta arrows). (d) Spatially confined activation of Opto-Rho1DN (red box) during apical constriction results in an immediate relaxation of the constricted cells specifically in the stimulated region (cyan arrows). Ventral furrow continues to form in the unstimulated region (magenta arrows). Scale bars: 50 μm.

Figure 1—video 1. Rapid membrane recruitment of CRY2-Rho1DN upon blue light irradiation.

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Shown is an embryo at late cellularization stage containing CIBNpm-GFP (magenta) and CRY2-Rho1DN-mCherry (green). Stimulation starts when 488 nm laser is turned on at T=00:00 (mm:ss, same format for all movies). Rapid membrane recruitment of CRY2-Rho1DN-mCherry appears within 10 s and maximum recruitment is achieved at around 30 s.

Figure 1—video 2. Loss of apical myosin during apical constriction upon activation of Opto-Rho1DN.

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Shown is an embryo at early gastrulation stage expressing CIBNpm, CRY2-Rho1DN-mCherry (green), and Sqh-GFP (magenta). Myosin disappears from the apical cortex of the ventral cells within 1 min after 488 nm laser illumination.