Depletion of PI(4,5)P2 from the plasma membrane leads to disassembly of the actomyosin network and arrests plasma membrane invagination. (A–L) Still frames from time-lapse two-photon movies of embryos coexpressing the plasma membrane anchor FRB-CFP (CFP channel is not shown), mRFP-FKBP-5Ptase, and Sqh::GFP. Rapamycin was injected during cycle 13 (E–H) and at the onset of cellularization (I–L). (A–D) In control (DMSO)-injected embryos the 5Ptase remains cytosolic (A) and furrows ingress without any visible alternation (B–D). (E–H) Injection of rapamycin during cycle 13. Upon recruitment of the 5Ptase to the plasma membrane (E, red arrowheads), Sqh::GFP is progressively disassembled over the course of cellularization (F and G). Apical view illustrating the disorganization of the actomyosin network during fast phase (H). (I–L) Rapamycin injection at the onset of cellularization. The 5Ptase is recruited to the plasma membrane (I, red arrowheads), and furrows are assembled (J) and ingress normally (K). Apical view: during the fast phase the area of the actomyosin rings in rapamycin-injected embryos (L) is double the area of similar stage control-injected embryos (D; control = 11.96 ± 1.34 µm2, n = 5; rapamycin = 22.76 ± 7.11 µm2, n = 3; Student’s t test, P = 2.74 × 10−8). (M–P) Injection of rapamycin in control embryos expressing the plasma membrane anchor FRB-CFP (CFP channel is not shown) and the FKBP construct lacking the 5Ptase catalytic domain (M) demonstrates that the morphology and dynamics of the actomyosin network is not altered upon rapamycin injection (N–P). Bar, 10 µm. See also Fig. S2.
