Figure 1 (A) Schematic representation of the RhoGEF2-CRY2/CIBN optogenetic system employed to control myosin-II activity during early Drosophila embryogenesis. The photosensitive domain of CRY2 is fused to the catalytic domain of the GTP Exchange factor RhoGEF2, while CIBN is anchored at the plasma membrane. In the dark RhoGEF2-CRY2 is present in the cytoplasm (left). Blue light illumination triggers the CRY2/CIBN interaction and causes the translocation of RhoGEF2-CRY2 to the plasma membrane, where it activates endogenous Rho1 signaling (right), and myosin-II  (B) Multiphoton-microscopy (λ=950 nm) enables the selective illumination of the basal surface of the cells at a tissue depth > 30 µm with subcellular precision  (C) Still frames from time-lapse recordings of an embryo expressing a myosin-II probe (Sqh::GFP). Embryos were mounted vertically to image the transverse cross-section using two-photon microscopy. At the onset of gastrulation, myosin-II localized to ring structures representing the leading edge of the cellularization front (lower arrow). During ventral furrow formation (torques open rectangle), myosin-II accumulated at the apical side (upper arrow) of the cells that invaginate and the basal pool was progressively depleted. Scale bar, 40 µm  (D) Quantification of basal myosin-II levels (N=3 embryos) in ectodermal and mesodermal cells. In the mesoderm, basal myosin-II levels decreasd 5-fold over the course of internalization, while in the ectoderm only ~3-fold. Additionally, myosin-II depletion appeared to be accelerated in the mesodermal cells as indicated by the steeper slope of the polynomial fit (3rd degree with r2Ectoderm: 0.98 and r2Mesoderm: 0.87). Note the logarithmic y-axis  (E-H) Embryos at the end of cellularization co-expressing RhoGEF2-CRY2; CIBN::pmGFP, and Sqh::mCherry were mounted with the dorsal epithelium facing the objective and a region of interest (red dashed line) was defined to specifically illuminate the cell base using two-photon microscopy. A representative embryo (N=3) showing basal myosin-II from top view (sum-of-slice projection of 6 focal planes) at the initial stage of the experiment (E) and 6 min after activation (F). Z-projection of the same embryo showing the cross section of the activated region (AR) (G) and non-activated region (NR) (H) with the superimposed plasma membrane signal (CIBN::GFPpm in magenta) recorded immediately after the final Sqh::mCherry acquisition (displayed in white). The contrast has been adjusted to better visualize the distribution of myosin-II along the apicobasal axis in the respective area of the tissue. Scale bars, 20 µm  (I) Quantification of myosin-II levels (N=3) within the region of activation (AR, red) relative to the non-activated region (NR, green) over the course of 6 min. The y-axis represents the fold-change of myosin-II signal intensity in the activated region versus non-activated region. A polynomial function (2nd degree, r2AR = 0.88) was fitted to the data (straight line) indicating the level of saturation that is reached at about 1.6-fold change of myosin-II in the activated area after 6 min  (J Analysis of the extent of signal spreading in the z-dimension (apico-basla) of basal myosin-II. Myosin-II signal intensity was plotted along the apicobasal axis and a Gaussian curve fitted to the basal peak. The width of the Gaussian fit was used as a parameter to estimate myosin-II signal spreading in the z-dimension, which was limited to 4 µm (N=3). In each box plot, the central mark, the bottom, and the top edge of each box indicates the median, the 25th and 75th percentile, respectively. Whiskers extent to the most extreme data point  (K) Quantification of the diameter of basal actomyosin rings in the non-activated region (NR, green) and activated region (AR, red) showing increased speed (4.8-fold change) and extent of ring constrictions in the photo-activated region (n= >41 cells). The ring diameter was normalized to the mean value of the first time point. The constriction speed was estimated by the slope of a fitted linear function to the data. (L) Light activation of the cell base resulted in a compaction of the tissue in the activated (AR) compared to the non-activated region (NR). Actomyosin rings were counted and normalized to the analyzed area to score ring density, which increased 1.35 times in the activated region after 5 min of photo-activation, showing that cells were still interconnected at the base and that photo-activation did not affect tissue integrity (N = 3 embryos; nNR: 431, nAR: 302). In each box plot, the central mark, the bottom, and the top edge of each box indicates the median, the 25th and 75th percentile, respectively. Whiskers extent to the most extreme data point