Securin regulates the spatiotemporal dynamics of separase

Abstract

Separase regulates multiple aspects of the metaphase-to-anaphase transition. Separase cleaves cohesin to allow chromosome segregation and localizes to vesicles to promote exocytosis. The anaphase-promoting complex/cyclosome (APC/C) activates separase by ubiquitinating its inhibitory chaperone, securin, triggering its degradation. How this pathway controls the exocytic function of separase is unknown. During meiosis I, securin is degraded over several minutes, while separase rapidly relocalizes from kinetochore structures at the spindle and cortex to sites of action on chromosomes and vesicles at anaphase onset. The loss of cohesin coincides with the relocalization of separase to the chromosome midbivalent at anaphase onset. APC/C depletion prevents separase relocalization, while securin depletion causes precocious separase relocalization. Expression of non-degradable securin inhibits chromosome segregation, exocytosis, and separase localization to vesicles but not to the anaphase spindle. We conclude that APC/C-mediated securin degradation controls separase localization. This spatiotemporal regulation will impact the effective local concentration of separase for more precise targeting of substrates in anaphase.

Figure 1.

Separase and securin dynamics in meiosis I. (A) Diagram of meiosis I showing SEP-1 (green), chromosomes (blue), and cortical granules (magenta). Insets emphasize SEP-1 at the spindle (left) and cortex (right) during prometaphase I. (B–D) SEP-1::GFP (green) with H2B::mCherry (magenta) during meiosis I. (B) During prophase I, SEP-1::GFP is cytoplasmic and excluded from the nucleus. (C) At NEBD, SEP-1::GFP accumulates on chromosomes in the nucleus (caret) and on cytoplasmic kinetochore linear elements in the cortex (white arrows), where it remains throughout prometaphase (D). (E) By mid-anaphase, separase localizes between separating chromosomes (caret) and vesicles (arrowheads). (F–I) IFY-1^WT::GFP (green) and H2B::mCherry (magenta) in meiosis I. (F) IFY-1^WT::GFP is present in both the cytoplasm and the nucleus in prophase. (G and H) IFY-1^WT::GFP displays identical localization patterns as separase at NEBD and through prometaphase I. (I) In anaphase I, IFY-1^WT::GFP is mostly degraded and is not observed on vesicles. (J–M) Endogenously tagged SEP-1::mScarlet (green) and IFY-1::GFP (magenta) colocalize at different stages as described above. (N) Quantification of IFY-1^WT::GFP spindle-associated and cytoplasmic signal showing rapid degradation (t = 0 is chromosome separation at anaphase onset). (O) Quantification of endogenously tagged SEP-1::mCherry and IFY-1^WT::GFP localized to linear elements and cytoplasm in the cortex, (t = 0 is separase localization to vesicles). Securin levels equilibrate with cytoplasmic signal before separase leaves linear elements and appears on vesicles. Scale bar: 10 µm.

Figure S1.

Dynamics of separase and securin during meiosis I. (A) Endogenously tagged SEP-1::mScarlet (green) and IFY-1::GFP (magenta) colocalize on the spindle at the metaphase to anaphase transition. Securin is rapidly degraded while separase relocalizes to the midbivalent (yellow caret) at anaphase onset (time shown in seconds, t = 0 is anaphase onset). (B) Cortical images of SEP-1::mScarlet (magenta) and IFY-1::GFP (green). IFY-1::GFP colocalizes with SEP-1::mScarlet on linear elements (yellow arrow) until it is degraded before separase relocalizes to vesicles (time shown in seconds, t = 0 marks vesicle localization). (C) Quantification of cytoplasmic securin levels in different WT or mutant securin GFP lines (t = 0 denotes anaphase onset). (C′) Cytoplasmic GFP levels measured in embryos 300 s before anaphase showing similar expression in WT1 and DM1 lines used throughout the manuscript. (D) Images of endogenously tagged SEP-1::mScarlet (green) and Tubulin::mCherry (magenta) show polar enriched separase within the tubulin cage and midbivalent separase (yellow caret) between microtubule bundles. (E–H) Endogenously tagged SEP-1::mScarlet (green) colocalizes with CZW-1::GFP (magenta, Yamamoto et al., 2008) at kinetochores during prometaphase I, partially colocalizes with CZW-1::GFP at the spindle pole (yellow caret) before midbivalent localization in anaphase. Scale bars: 5 µm.

Figure 2.

Dynamics of separase, securin, and cohesin at anaphase onset. (A) SEP-1::GFP (green) co-expressed with H2B::mCherry (magenta) at the spindle in meiosis I. In prometaphase I, spindle SEP-1::GFP localizes to kinetochore cups around the bivalents (kinetochore). Just before anaphase onset, SEP-1::GFP accumulates on spindle poles, then invades the midbivalent region (yellow caret), where it accumulates when chromosomes move apart. (B) Montage showing rapid SEP-1::mScarlet (green) relocalization to the midbivalent just before the abrupt loss of COH-3::GFP and the poleward movement of chromosomes (magenta, time in seconds relative to t = 0, anaphase onset). (C) Montage showing the dynamics of endogenous IFY-1^WT::GFP (green) co-expressed with chromosome marker H2B::mCherry (magenta) during the metaphase-to-anaphase I transition. IFY-1^WT::GFP signal is significantly reduced by anaphase onset. (D) In the cortex, SEP-1::GFP relocalizes from linear elements (white arrow) to cortical granules labeled with mScarlet::RAB-11.1 (arrowhead) by 30 s after anaphase onset. Scale bars: 2 µm.

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.

Figure 3.

The APC/C and securin control SEP-1 localization in meiosis I. Prometaphase I embryos within the spermatheca expressing endogenously tagged SEP-1::GFP (green) with chromosome marker H2B::mCherry and cortical granule cargo protein mCherry::CPG-2 (magenta) in control (A–A″), ify-1(RNAi) (B–B″), or apc-2(RNAi) (C–C″). (A) In control embryos, SEP-1::GFP is localized to kinetochore cups and linear elements near the spindle (insets in A, B, and C show SEP-1::GFP on a bivalent in greyscale, white lines indicate the region used to generate a line scan in A′, B,″ and C″). (A′) Line scan showing the SEP-1::GFP and H2B::mCherry fluorescent profiles. (A″) In the cortex, SEP-1::GFP is localized to linear elements (arrow) but not vesicles (arrowhead). (B and B′) Prometaphase I ify-1(RNAi) embryos have reduced SEP-1::GFP signal mislocalized at chromosomes, with some signal at the midbivalent. (B″) SEP-1::GFP precociously localizes to cortical granules (arrowheads) in addition to linear elements (arrow) after securin depletion. (C–C″) In apc-2 (RNAi) prometaphase I embryos, SEP-1::GFP localization is normal on chromosomes and linear elements. Scale bars: 5 µm, except the 1 µm bars for insets found in A, B, and C.

Figure S3.

Meiosis I cohesin dynamics and regulation during meiosis I. (A) Kymographs of anaphase onset in embryos expressing GFP::COH-3 (green) and H2B::mCherry (magenta). Time (s) is relative to anaphase I onset (t = 0). GFP::COH-3 remains high until seconds before anaphase I onset in WT, is reduced before anaphase onset in ify-1(RNAi) embryos, and persists past anaphase I in sep-1(RNAi) embryos. (B) Representative image of GFP::COH-3 (green) and H2B::mCherry (magenta) showing a folded short arm (yellow caret) from different orientations on three bivalents. (B′) Proposed model of GFP::COH-3 at the bivalent short arm adopting a folded configuration based on images from 10 embryos at several stages of meiosis I. (C) In control, GFP::COH-3 midbivalent signal is similar in oocytes (−1) and prometaphase I embryos (labeled 0, white arrows) but is absent from embryos after meiosis I in the uterus (+1, +2). (D) After apc-2 RNAi, GFP::COH-3 levels are retained at the midbivalent in prometaphase I embryos (0) and several arrested embryos in the uterus (+1, +2). (E) GFP::COH-3 midbivalent signal is prematurely reduced after ify-1 RNAi, in prometaphase I embryos (labeled 0) relative to oocytes (−1) and is not observed in older embryos (+1). (F) Quantification of GFP::COH-3 levels relative to H2B from a time series of control or ify-1(RNAi) embryos. Time (s) is relative to anaphase I onset. (G) Quantification of the ratio between the GFP::COH-3 signal at the midbivalent in prometaphase I embryo relative to the −1 oocyte in the same plane. The GFP::COH-3 signal ratio was near 1 in control (N = 10) and apc-2 RNAi (N = 5, P > 0.05) but was significantly reduced (about 50% reduction) after ify-1 RNAi (N = 13, P < 0.05). Scale bars: 5 µm.

Figure 4.

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.

Figure S4.

GFP::IFY-1^DM inhibits chromosome segregation and exocytosis. (A) GFP::IFY-1^WT or (B) GFP::IFY-1^DM (green) with H2B::mCherry (magenta) during meiosis I. Chromosomes move apart quickly in GFP::IFY-1^WT but are severely delayed in GFP::IFY-1^DM embryos. Time (s) is relative to anaphase onset. (C) Average distance between chromosomes during anaphase I is significantly reduced in IFY-1^DM::GFP embryos relative to control (N = 9 for GFP::IFY-1^WT, N > 18 for GFP::IFY-1^DM, asterisks denote a statistically significant difference. P value = <0.05). (D) Frequency and images of chromosome segregation defects (yellow caret) observed in embryos expressing GFP::IFY-1^DM (green, H2B::mCherry in magenta). N = number of embryos scored. (E–K) GFP::IFY-1^DM blocks cortical granule exocytosis. (E) Quantification of cortical granules in a single spindle plane over time after anaphase I onset (N = 6 for GFP::IFY-1^WT, N = 7 for GFP::IFY-1^DM). Asterisks denote a statistically significant difference, P value <0.0001. Error bars represent standard error of the mean. (F–H) Images shown of GFP::IFY-1^WT or (I–K) GFP::IFY-1^DM (green) and mCherry::CPG-2 (magenta, arrowheads) during meiosis I. Time (seconds) is relative to midbivalent localization. In wildtype, most vesicles (arrowheads) undergo exocytosis after 120 s of anaphase onset (G) while mCherry::CPG-2 is extracellular (H, yellow asterisk). (I–K) In contrast, cortical granules (arrowheads) are retained in GFP::IFY-1^DM embryos, even after several minutes. Scalebar: 5 µm (A, B, and D), 10 µm (F–K).

Figure 5.

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.