PI(4,5)P2/AM injection increases the stability and recruitment of myosin-II to the actomyosin network. (A) Still frames from a time-lapse two-photon movie of a cellularizing embryo expressing the PI(4,5)P2 sensor PLCδ1-PH::GFP (green) and Sqh::mCherry (red) showing the overlap between PI(4,5)P2 and myosin-II over the course of cellularization (0, 30, 40, and 60 min). Note: the bright fluorescent spots below the cellularization front correspond to autofluorescence of yolk granules. Bar, 10 µm. (B) FRAP analysis in Sqh::GFP-expressing embryos. During the slow phase of cellularization the recovery rate of Sqh::GFP is much faster than during the fast phase, indicating that myosin-II is more stably associated at the furrow during late stages. In PI(4,5)P2/AM-injected embryos the recovery rates during the slow phase are significantly slower than control-injected embryos and are comparable with the recovery rates of late embryos (Student’s t test, P value = 7.76 × 10−15 for wild-type early embryos compared with PI(4,5)P2-injected embryos; n = 3 embryos each condition). Errors bars represent mean ± SD. (C) Quantification of Sqh::GFP fluorescence intensity at the furrow in early embryos, late embryos, and PI(4,5)P2-injected embryos. Sqh::GFP levels increase from early to late stages of cellularization. Injection of PI(4,5)P2/AM causes a twofold increase in Sqh::GFP levels in early embryos (Student’s t test, P = 0.039). Each dot represents an embryo. At least three independent experiments were quantified.
