Injection of plasma membrane phosphoinositides causes premature constriction (PI(4,5)P2/AM) and disassembly (PI(3,4,5)P3/AM) of the actomyosin network. (A and B) Schematic summarizing the progression of cellularization in an optical cross section (A) and from a surface view at the plane of the actomyosin network (B). Note the changes in myosin organization (green) from the slow phase (left) to the fast phase/end of cellularization (right). See Introduction for details. (C–E″) Still frames from time-lapse two-photon movies of a cellularizing embryo expressing Sqh::GFP injected before the beginning of cellularization with: carrier (C, C′, and C″), PI(4,5)P2/AM to a final concentration of 50 µM (D, D′, and D″), and PI(3,4,5)P3/AM to a final concentration of 20 µM (E, E′, and E″). Three different time points are shown: 0 min (C, D, and E), 30 min (C′, D′, and E′), and 50 min (C″, D″, and E″). In control-injected embryos cleavage furrows are assembled and ingress normally (C, C′, and C″). In PI(4,5)P2/AM-injected embryos nuclei remain trapped in hyper-constricted actomyosin rings and are pushed away from the epithelium into the interior of the embryo (D, D′, and D″). In PI(3,4,5)P3/AM-injected embryos furrows are progressively disassembled, resulting in the formation of multinucleated cells and multi-layering (E, E′, and E″). In C–E″ some nuclei have been labeled in red to show their position with respect to the Sqh::GFP invaginating furrows. Insets in C″–E″ represent the magnified area of corresponding boxes. Bars, 10 µm. (F–H) Apical view of a cellularizing embryo expressing Sqh::GFP injected with: carrier (F), PI(4,5)P2/AM (G), or PI(3,4,5)P3/AM (H) taken 30 min (F and G) and 50 min (H) after the beginning of cellularization. Arrows show actomyosin hyper-constricted rings upon PI(4,5)P2/AM injection. Bar, 10 µm.
