Figure S2.
Cyclin B phenotypes in meiosis and spindle elongation and analysis of cyclin B degradation kinetics in mitosis. (A) Time-lapse sequences of embryos undergoing meiosis I under the specified conditions. Quantification of polar body extrusion rates is included below each sequence. We note the presence of defects in spindle assembly and chromosome segregation in CYB-3–only, but not CYB-1/2–only embryos. (B) Example images of embryos at pronuclear meeting (75–80 s before NEBD), under the specified conditions. Arrowheads indicate extra chromosomal material signifying meiotic defects (see panel A). Numbers indicate quantification of embryos with excess chromatin for the specified conditions. This analysis indicates that CYB-1 and CYB-2 function redundantly during the meiotic divisions that precede the first embryonic mitosis (van der Voet et al., 2009). (C) Measurement of spindle distance over time for the indicated conditions. Whereas CYB-3–only embryos present shortening of the mitotic spindle after NEBD, CYB-1/2 embryos elongate their embryos prematurely (see also Fig. 2 A). (D) Example images of embryos expressing in situ–tagged cyclin B isoforms and imaged through time-lapse microscopy. (E) Top: Methodology used for the quantification of raw embryonic fluorescence intensity levels. Bottom: Raw fluorescence intensity levels for the specified conditions. The autofluorescence average was calculated in a strain expressing no green fluorophores and was subtracted from the raw values to generate the graphs in Fig. 2, G and H. (F and H) Rates of CYB-1 degradation, estimated by measuring the slope of the curves in Fig. 3, E and F, respectively. This analysis revealed that both loss of CYB-3 (F) and direct inhibition of the APC/C through Apc4ts (G) similarly delayed the onset of CYB-1 degradation (the point at which the slopes become negative) and reduced the maximum rate of degradation (minimal slope value). (G) Quantification of the NEBD-to-anaphase interval in embryos expressing either WT or T32A versions of CDC-20 in a cdc-20∆ background. The “All CYBs” conditions are the same as for Fig. 5 E. Scale bars are 5 µm (panels A and B) and 10 µm (panels D and E). Error bars are 95% confidence intervals. n is the number of embryos scored per condition. **** represents P < 0.0001 from Mann-Whitney tests; non-significant (n.s.) is P > 0.05.

Cyclin B phenotypes in meiosis and spindle elongation and analysis of cyclin B degradation kinetics in mitosis. (A) Time-lapse sequences of embryos undergoing meiosis I under the specified conditions. Quantification of polar body extrusion rates is included below each sequence. We note the presence of defects in spindle assembly and chromosome segregation in CYB-3–only, but not CYB-1/2–only embryos. (B) Example images of embryos at pronuclear meeting (75–80 s before NEBD), under the specified conditions. Arrowheads indicate extra chromosomal material signifying meiotic defects (see panel A). Numbers indicate quantification of embryos with excess chromatin for the specified conditions. This analysis indicates that CYB-1 and CYB-2 function redundantly during the meiotic divisions that precede the first embryonic mitosis (van der Voet et al., 2009). (C) Measurement of spindle distance over time for the indicated conditions. Whereas CYB-3–only embryos present shortening of the mitotic spindle after NEBD, CYB-1/2 embryos elongate their embryos prematurely (see also Fig. 2 A). (D) Example images of embryos expressing in situ–tagged cyclin B isoforms and imaged through time-lapse microscopy. (E) Top: Methodology used for the quantification of raw embryonic fluorescence intensity levels. Bottom: Raw fluorescence intensity levels for the specified conditions. The autofluorescence average was calculated in a strain expressing no green fluorophores and was subtracted from the raw values to generate the graphs in Fig. 2, G and H. (F and H) Rates of CYB-1 degradation, estimated by measuring the slope of the curves in Fig. 3, E and F, respectively. This analysis revealed that both loss of CYB-3 (F) and direct inhibition of the APC/C through Apc4ts (G) similarly delayed the onset of CYB-1 degradation (the point at which the slopes become negative) and reduced the maximum rate of degradation (minimal slope value). (G) Quantification of the NEBD-to-anaphase interval in embryos expressing either WT or T32A versions of CDC-20 in a cdc-20∆ background. The “All CYBs” conditions are the same as for Fig. 5 E. Scale bars are 5 µm (panels A and B) and 10 µm (panels D and E). Error bars are 95% confidence intervals. n is the number of embryos scored per condition. **** represents P < 0.0001 from Mann-Whitney tests; non-significant (n.s.) is P > 0.05.

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