Separase localization to vesicles is reduced by GFP::IFY-1 ^DM . (A) Spindle localization of endogenously tagged SEP-1::mScarlet (green) co-expressed with GFP::IFY-1^WT (magenta). Securin is rapidly degraded while separase moves from kinetochore cups to the midbivalent (yellow caret). (B) GFP::IFY-1^DM is stable in anaphase and colocalizes with separase in a similar pattern to wildtype. (C) Max projections of SEP-1::mScarlet (green) with vesicle marker CAV-1::GFP (magenta) and either GFP::IFY-1^WT or GFP::IFY-1^DM (magenta). In prometaphase I, SEP-1 localizes to linear elements (white arrow) but not vesicles (white arrowheads) in both conditions. At anaphase I onset, SEP-1::mScarlet begins to enrich on vesicles (yellow arrowhead) in GFP::IFY-1^WT but not GFP::IFY-1^DM embryos. By mid-anaphase I, SEP-1::mScarlet is fully enriched on vesicles in GFP::IFY-1^WT embryos, but not in GFP::IFY-1^DM embryos. (D) Magnified images of SEP-1::mScarlet vesicle localization from the 5 µm² regions indicated in C at mid-anaphase I in wildtype and mutant. SEP-1::mScarlet has only partial vesicle localization when GFP::IFY-1^DM is expressed (yellow arrowhead). (E) Quantification of vesicle-associated SEP-1::mScarlet signal in cortical planes at mid-anaphase I in GFP::IFY-1^WT (N = 11) and GFP::IFY-1^DM (N = 12) embryos. The asterisk denotes a statistically significant difference, P value <0.0001. Error bars represent the standard error of the mean. Scale bars: 5 µm.