Injection of wortmannin causes premature constriction of the actomyosin rings. (A) Still frames from a time-lapse two-photon movie of a cellularizing embryo showing the localization of the PI(3,4,5)P3 sensor GFP::AKT-PH. The dashed line indicates the position of the furrow; GFP::AKT-PH is present at the furrows at early time points (0 and 30 min) and disappears at later stages (40 and 60 min). Insets show higher magnification of boxed areas. Grayscale 8-bit still images were pseudo-colored with the rainbow LookUp Table (LUT; ImageJ software) to produce false-color images. Pixels with a value of 0 are black and pixels with a value of 255 are red. Bars, 10 µm. (B–E) Still frame from a two-photon movie of a control (DMSO)-injected embryo taken after 20 min from the onset of cellularization (B) and of an embryo injected with wortmannin (C). (D and E) Apical view of the same control- and wortmannin-injected embryos. Wortmannin injection causes the premature conversion of the hexagonal actomyosin arrays into contractile rings recapitulating the PI(4,5)P2-injected and bottleneck mutant phenotypes. Bars, 10 µm. (F) Still frame from a two-photon movie of an embryo expressing Sqh::GFP injected with siRNA against Bnk during cycle 10, when the protein starts to be expressed. Bottleneck depletion causes premature contraction of the actomyosin network during early cellularization, resulting in a phenotype that is very similar to the PI(4,5)P2/AM/wortmannin-induced phenotype. Bar, 10 µm. (G) Apical view of the same embryo. Bar, 10 µm.
