C-GAP is required for normal myosin pulsing. (A) C-GAP levels influence the amount of apical myosin that persists between pulses. Plots represent apical area and apical myosin intensity quantified for individual cells. Shaded region indicates time window used in montages on the right. Time-lapse images in montages from live embryos expressing myosin::GFP and membrane::RFP. Arrows indicate myosin pulse. (B and C) C-GAP shRNA disrupts contractile pulses. (B) Quantification of rapid constriction events in different genetic backgrounds. C-GAP shRNA has significantly lower pulse frequency (rapid constriction events per minute, 0.2164 ± 0.1 min⁻¹, two embryos, n = 123 cells) than WT (0.4815 ± 0.01 min⁻¹, two embryos, n = 181 cells) or C-GAP overexpression (O.E., 0.462 ± 0.08 min⁻¹, 2 embryos, n = 110 cells; ***, P < 0.0001; n.s., not significant). (C) Quantification of maximum constriction rate for individual cells in shows C-GAP shRNA has significantly lower constriction rate (0.228 ± 0.01 µm min⁻¹, three embryos, n = 190 cells) than WT (0.345 ± 0.03 µm min⁻¹, three embryos, n = 246 cells) or C-GAP OE (0.433 ± 0.02 µm min⁻¹, three embryos, n = 161 cells; ***, P < 0.0001; *, P < 0.05). Error bars represent SEM. (D) Myosin persistence measured for all WT (five embryos, 227 cells, 822 pulses) and C-GAP overexpression (three embryos, 145 cells, 447 pulses) pulses reveal significantly lower persistent myosin intensity in C-GAP overexpression embryos (***, P < 0.001; Komolgorov–Smirnov test and t test). Red bars represent sample medians, and boxes demarcate the 25th and 75th percentiles. (E) RhoA pathway leading to myosin activation. (F) ROCK pulsing occurs in the absence of myosin pulsing. Time-lapse images are from either Control or MBS shRNA (MBS of myosin phosphatase) embryos expressing GFP::ROCK and either membrane::RFP. In MBS shRNA embryos, ROCK increases and decreases or pulses (arrows). (G) Quantification of ROCK pulse frequency in Control (n = 104 cells, two embryos), C-GAP (n = 89 cells, two embryos), and MBS shRNA (n = 92 cells, two embryos) embryos shows that only loss of C-GAP results in fewer ROCK pulses (***, P < 0.0001; *, P < 0.05). Error bars represent SD. Bars, 5 µm.