ANI-1 limits Formin ^CYK-1 ::GFP accumulation to the cell cortex during ring assembly. (A) Representative intensity graph of NMY-2::mRFP line scan from anterior to the posterior pole of the embryo. For the equatorial intensity, the mean fluorescence intensity ±5% embryo length at the maximum intensity was calculated. (B) Mean normalized fluorescence intensity of NMY-2::mRFP and GFP::ROK^LET-502 at the cell equator with and without ani-1(RNAi). (C) During cytokinesis, active RhoA activates the effectors ROK^LET-502 and Formin^CYK-1. Activated ROK^LET-502 activates myosin II and fomin^CYK-1 polymerizes the long F-actin of the contractile ring. (D) Single z-plane images of the cell cortex of one-cell C. elegans embryos expressing Formin^CYK-1::GFP treated with or without ani-1(RNAi). A zoom-in of the equatorial region for the indicated time points is shown on the right in gray and fire scaling. (E) Normalized cortical fluorescence intensity of Formin^CYK-1::GFP from the anterior (0%) to the posterior (100%) pole for control and ani-1(RNAi) embryos. (F) Mean normalized fluorescence intensity of Formin^CYK-1::GFP at the cell equator with and without ani-1(RNAi) at 180 s after NEBD. For all, time in seconds (s) after NEBD is indicated, scale bars are 5 µm, error bars SEM, and n = number of embryos analyzed. P values were calculated using two-tailed Student’s t test or Mann–Whitney U test and are *P < 0.05, **P < 0.01.