ANI-1 limits the accumulation of NMY-2::mRFP and GFP::ROK ^LET-502 to the cortex during ring assembly. (A) In the one-cell C. elegans zygote, the cleavage furrow ingresses unilateral with the leading edge ingressing before the lagging edge. The cyan and yellow arrowheads mark the leading edge and lagging edge, respectively. After ANI-1 depletion by RNAi, the contractile ring ingresses symmetrically (left). Furrow ingression triggers a cortical actin flow from the poles toward the equator at the leading edge. Anillin limits myosin II accumulation in the ring by an unknown mechanism. (B) In one-cell C. elegans embryos, cortical NMY-2::mRFP fluorescence intensity was measured on single-z-plane images with a line scan from the anterior to the posterior pole. (C) Single z-plane images of the cell cortex of one-cell C. elegans embryos expressing NMY-2::mRFP treated with or without ani-1(RNAi). A zoom-in of the equatorial region for the indicated time points is shown on the right with gray and fire scaling. Cortical patches of NMY-2::mRFP are highlighted by yellow arrowheads. (D) Normalized cortical fluorescence intensity of NMY-2::mRFP from the anterior (0%) to the posterior (100%) pole for control and ani-1(RNAi) embryos. (E) Domain organization of C. elegans ANI-1 highlighting the predicted (grey) and confirmed (black) interaction partners of each domain (MBD—myosin binding domain, ABD—actin-binding domain, RBD—RhoA binding domain, PH—pleckstrin homology). (F) Single z-plane images of the cell cortex of one-cell C. elegans embryos expressing GFP::ROK^LET-502 treated with or without ani-1(RNAi). A zoom-in of the equatorial region for the indicated time points is shown with gray and fire scaling. (G) Normalized cortical fluorescence intensity of GFP::ROK^LET-502 from the anterior (0%) to the posterior (100%) pole for control and ani-1(RNAi) embryos. For all, time in seconds (s) after NEBD is indicated, scale bars are 5 µm, error bars are standard error of the mean (SEM), and n = number of embryos analyzed.