Two-color analysis of Anillin dynamics and contributions to pulsed contractions in one- and two-cell embryos. (A and E) Micrographs of two-cell (A) and one-cell (E) embryos coexpressing GFP::ANI-1 and NMY-2::RFP. White arrowheads indicate individual pulses. (B and F) Expanded views of single pulses illustrating temporal dynamics of GFP::ANI-1 and NMY-2::RFP accumulation. (C and G) Plots of averaged normalized fluorescence intensities for NMY-2::RFP and GFP::ANI-1 from two-color videos, aligned to the time at which NMY-2::RFP reaches 25% threshold. The averaged normalized fluorescence intensity of GFP::AHPH, coaligned by using the NMY-2::RFP signal, is shown for reference. (D and H) Distribution of the delays in the onset of appearance and disappearance of GFP::ANI-1 measured relative to NMY-2::RFP. The onset of appearance and disappearance was measured, respectively, as the time at which the normalized signal rose above 25% or fell below 75% of the maximum value. (I–K) Single frames (top) and kymographs (bottom) comparing NMY-2::RFP and GFP::AHPH dynamics in (I) wild-type, (J) ani-1_M(RNAi), and (K) ani-1_FL(RNAi) zygotes. (L and M) Plots of averaged normalized fluorescence intensities of NMY-2::RFP and GFP::AHPH (total, diffuse, and myosin-colocalized) for individual pulses from two-color videos in (L) spd-5(RNAi) and (M) ani-1_M(RNAi) zygotes, aligned to the time at which NMY-2::RFP reaches 25% threshold. Data in L are identical to those shown in Fig. S4 E. (N) Plots of averaged normalized fluorescence intensities of NMY-2::RFP for individual pulses in ani-1_FL(RNAi) zygotes, aligned to the time at which NMY-2::RFP reaches 25% threshold. Data in C–L were averaged over the following—C and D: 35 pulses in 10 embryos; G and H: 42 pulses in 4 embryos; I and L: 40 pulses in 3 embryos; J and M: 51 pulses in 4 embryos; K and N: 43 pulses in 3 embryos. Shaded areas represent 95% confidence intervals. Bars: (A, E, I, and K) 5 µm; (B and F) 3 µm.