ANI-1 binding to RHO-1 via the RBD limits cortical NMY-2::mKate accumulation during ring assembly. (A) Schematic representation of the used GFP-tagged ANI-1 mutant variants. (B) Single z-plane NMY-2::mKate images of the cell cortex of embryos expressing indicated GFP::ANI-1 transgenes and treated with or without ani-1(RNAi). A zoom-in of the equatorial region for the indicated time points is shown on the right. Scale bars are 5 µm. (C) Normalized cortical fluorescence intensity of NMY-2::mKate from the anterior (0%) to the posterior (100%) pole for control and ani-1(RNAi) embryos expressing indicated GFP-tagged ANI-1 variants at 180 s after NEBD. (D) Mean normalized fluorescence intensity of NMY-2::mKate at the cell equator for indicated conditions at 180 s after NEBD. Error bars are SEM and P values were calculated using two-tailed Student’s t test and are n.s. P > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001. For all, time in seconds (s) after NEBD is indicated.