Chitin synthase is internalized concurrent with cortical granule exocytosis, facilitating the deposition of chitin and CPG-1/2 in sequential layers. (A) Confocal images of a two-cell stage embryo expressing a GFP-labeled plasma membrane probe (left) and mCherry::CPG-1 (middle). (B) Confocal images of two-cell stage control (top) and sqv-5(RNAi) (bottom) embryos expressing a GFP-labeled plasma membrane probe (left) and mCherry::CPG-2 (middle). (C) Time-lapse confocal images of a one-cell stage embryo expressing GFP::CHS-1 (left) and mCherry::CPG-2 (middle). Times are minutes and seconds after anaphase I. Embryo anterior is on the left. (D) Higher magnification view of the anterior of embryo in C. (E) Confocal images of two control (left) and two cpg-1/2(RNAi) (right) embryos expressing GFP::CHS-1. Times are minutes and seconds past anaphase I, when CHS-1 normally starts to internalize from the plasma membrane into subcortical puncta. Boxed regions are magnified below. Schematics highlight GFP::CHS-1 localization. By 2 min after anaphase I, the majority of GFP::CHS-1 in control embryos has been internalized into puncta (red spheres). In cpg-1/2(RNAi) embryos at a later time point, ∼4 min after anaphase I, some GFP::CHS-1 have internalized into puncta, but some also remain associated with the plasma membrane (red line). Bars, 10 µm. n = number of imaged embryos.