Myosin up-regulation leads to furrow mispositioning. (A–D) Furrow and spindle positions are coupled when myosin is inhibited in ani-1(RNAi); par-4(it47) embryos (A and C) and uncoupled when myosin is hyperactivated upon rga-3/4(RNAi) (B and D). Superposed confocal sections and DIC images (A and B) and quantification of furrow/spindle coupling (C and D) in dividing one-cell embryos of the indicated genotypes (all strains also express an α-tub::YFP transgene [green]). Furrow position was measured when the furrow reached its most anterior position. Green dashed lines correspond to the spindle center and arrowheads to furrow position. Bars, 10 µm. P-values from Student’s t test. See also Videos 9 and 10. (E) A novel pathway regulating furrow positioning: PAR-4 and CUL-5 redundantly prevent ANI-2 accumulation at the cortex; ANI-2 in turns inhibits ANI-1. PAR-4 also regulates an anillin-independent pathway that involves the PIG-1/MELK kinase. Together, these two pathways prevent the cortical accumulation of activated myosin, thereby ensuring that myosin does not shift the furrow toward the anterior. (F) A model to explain furrow mispositioning in ani-1; par-4 and ani-1; pig-1 embryos. In wild-type as well as ani-1; par-4 or ani-1; pig-1 embryos, centralspindlin and astral microtubules induce furrowing in the vicinity of the spindle midzone. In wild-type embryos, PAR-4, PIG-1, and ANI-1 keep myosin accumulation and activity at the anterior cortex at a low level during cytokinesis, thereby preventing furrow displacement. In the absence of ANI-1 and PAR-4 or PIG-1, myosin accumulation at the anterior cortex increases during cytokinesis and excess of myosin activity drives the furrow toward the anterior pole of the embryo.