NMY-2::mKate localization in the ring in embryos expressing different GFP::ANI-1 variants. (A) To generate an end-on view of the contractile ring 21 z-planes were acquired. The equatorial region was cropped, rotated 90°, and a maximum fluorescence intensity projection was performed. End-on reconstruction of the general membrane marker mCherry::PH is shown. Please note that mCherry::PH intensity is less bright at the bottom of the image away from the objective, due to the decay of the fluorescence signal with increasing distance from the objective. (B) End-on reconstruction of GFP::ROK^LET-502 control embryo at the cleavage plane for indicated % of constriction. (C) End-on reconstruction of NMY-2::mKate in embryos expressing different GFP::ANI-1 variants treated with ani-1(RNAi) or NG::ANI-1 at the cleavage plane for indicated % of constriction. White star marks the leading edge. (D and E) Total ring intensity of NMY-2::mKate at 50% ring constriction (D) and ratio of NMY-2::mKate intensity of the leading and lagging edge (E) in embryos expressing different GFP::ANI-1 variants treated with ani-1(RNAi) or NG::ANI-1. Error bars are SEM. P values were calculated using two-tailed Student’s t test or Mann–Whitney U test and are n.s. P > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001. (F) Schematics of the GFP-tagged ANI-1^Linker-CX fragment of ANI-1. All scale bars are 5 µm.