Figure S2.
Regulation of separase localization. (A–D) Germline images of worms expressing endogenously tagged SEP-1::GFP (green), H2B::mCherry, and mCherry::CPG-2 (magenta). Numbers indicate embryo position in the uterus, corresponding to increasing age. (A and A′) In control animals, prometaphase I embryos in the spermatheca (embryo 0) have mCherry::CPG-2 in vesicles (white arrowhead), while older embryos (+1-+3) have mCherry::CPG-2 in the eggshell (yellow asterisk). SEP-1::GFP localizes to linear elements (white arrow). (A′) Montage of a time series of a prometaphase I oocyte/embryo from ovulation (t = 0) shows SEP-1::GFP localized to linear elements (white arrows). (B) Intermediate ify-1(RNAi) embryos in the spermatheca (0), and multiple older embryos (+1 - +2) have SEP-1::GFP abnormally localized to cortical granules (yellow arrowheads). Eventually, mCherry::CPG-2 signal appears in the eggshell of older embryos (+3, asterisk), suggesting delayed and reduced secretion. (B′) Montage of a prometaphase ify-1(RNAi) oocyte/embryo beginning at ovulation shows premature SEP-1::GFP localized to cortical granules (yellow arrowheads). (C) Severe ify-1(RNAi) prometaphase I embryos in the spermatheca (0) and multiple older embryos (+1 - +2) have SEP-1::GFP abnormally localized to cortical granules (yellow arrowheads), while mCherry::CPG-2 signal does not accumulate in the eggshell. (D) In apc/c(RNAi) embryos in the spermatheca (embryo 0) and numerous arrested embryos, SEP-1::GFP remains localized to linear elements (arrow) but not vesicles (arrowhead), with mCherry::CPG-2 remaining in cortical granules (yellow arrowheads). (E) SEP-1::mScarlet (green) colocalizes with IFY-1::GFP (magenta) on linear elements (white arrows) in multiple embryos after apc-2 RNAi. (F) Quantification of SEP-1::GFP associated with kinetochores during prometaphase I after control (N = 5), apc-2 (N = 5), or ify-1 (N = 13) RNAi. (G) Quantification of SEP-1::GFP signal on the kinetochore relative to the midbivalent region in prometaphase I after control (n = 15), apc-2 (n = 19), or ify-1 (n = 8) RNAi. Asterisks denote a statistically significant difference, P value <1 × 10−6. N = spindles scored, n = optimal side-view images of homologs scored. For montages (A′ and B′) time is in seconds, t = 0 is ovulation. Scale bars: 10 µm in all except A′ and B′, which are 5 µm.

Regulation of separase localization. (A–D) Germline images of worms expressing endogenously tagged SEP-1::GFP (green), H2B::mCherry, and mCherry::CPG-2 (magenta). Numbers indicate embryo position in the uterus, corresponding to increasing age. (A and A′) In control animals, prometaphase I embryos in the spermatheca (embryo 0) have mCherry::CPG-2 in vesicles (white arrowhead), while older embryos (+1-+3) have mCherry::CPG-2 in the eggshell (yellow asterisk). SEP-1::GFP localizes to linear elements (white arrow). (A′) Montage of a time series of a prometaphase I oocyte/embryo from ovulation (t = 0) shows SEP-1::GFP localized to linear elements (white arrows). (B) Intermediate ify-1(RNAi) embryos in the spermatheca (0), and multiple older embryos (+1 - +2) have SEP-1::GFP abnormally localized to cortical granules (yellow arrowheads). Eventually, mCherry::CPG-2 signal appears in the eggshell of older embryos (+3, asterisk), suggesting delayed and reduced secretion. (B′) Montage of a prometaphase ify-1(RNAi) oocyte/embryo beginning at ovulation shows premature SEP-1::GFP localized to cortical granules (yellow arrowheads). (C) Severe ify-1(RNAi) prometaphase I embryos in the spermatheca (0) and multiple older embryos (+1 - +2) have SEP-1::GFP abnormally localized to cortical granules (yellow arrowheads), while mCherry::CPG-2 signal does not accumulate in the eggshell. (D) In apc/c(RNAi) embryos in the spermatheca (embryo 0) and numerous arrested embryos, SEP-1::GFP remains localized to linear elements (arrow) but not vesicles (arrowhead), with mCherry::CPG-2 remaining in cortical granules (yellow arrowheads). (E) SEP-1::mScarlet (green) colocalizes with IFY-1::GFP (magenta) on linear elements (white arrows) in multiple embryos after apc-2 RNAi. (F) Quantification of SEP-1::GFP associated with kinetochores during prometaphase I after control (N = 5), apc-2 (N = 5), or ify-1 (N = 13) RNAi. (G) Quantification of SEP-1::GFP signal on the kinetochore relative to the midbivalent region in prometaphase I after control (n = 15), apc-2 (n = 19), or ify-1 (n = 8) RNAi. Asterisks denote a statistically significant difference, P value <1 × 10−6. N = spindles scored, n = optimal side-view images of homologs scored. For montages (A′ and B′) time is in seconds, t = 0 is ovulation. Scale bars: 10 µm in all except A′ and B′, which are 5 µm.

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