Generation and characterization of GFP::IFY-1 ^DM in C. elegans. (A) Schematic of IFY-1 indicating the APC/C recognition motif (destruction box) and mutated residues within the unstructured N-terminus. (B) Embryonic lethality of different GFP::IFY-1^WT and GFP::IFY-1^DM lines at 20°C and 25°C in homozygous or heterozygous animals. (C–F) IFY-1^DM::GFP (green) and H2B::mCherry (magenta) localization in meiosis I. GFP::IFY-1^DM has identical localization patterns as separase and wild type securin from prophase arrest through prometaphase I. (F) GFP::IFY-1^DM is not degraded in anaphase and accumulates on chromosomes (asterisk) and the anaphase I spindle (caret) but not on cortical granules. (G) In GFP::IFY-1^WT, two-celled embryos always have two small polar bodies (22/22 embryos) while GFP::IFY-1^DM embryos have high rates of polar body extrusion defects. (H) GFP::IFY-1^WT multicellular embryos are not permeable, while GFP::IFY-1^DM embryos all shrink in hyperosmotic solution, indicating permeability barrier defects. N = number of embryos scored. Scale bars: 10 µm.