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Caenorhabditis elegans cyclin B3 is required for multiple mitotic processes including alleviation of a spindle checkpoint-dependent block in anaphase chromosome segregation.

Deyter GM, Furuta T, Kurasawa Y, Schumacher JM - PLoS Genet. (2010)

Bottom Line: Mitotic Cdk1 is activated by A-type, as well as B1- and B2-type, cyclins.Our experiments reveal that the extended metaphase delay in CYB-3-depleted embryos is dependent on an intact spindle assembly checkpoint (SAC) and results in salient defects in the architecture of holocentric metaphase chromosomes.Altogether, these data reveal that CYB-3 plays a unique, essential role in the cell cycle including promoting mitotic dynein functionality and alleviation of a SAC-dependent block in anaphase chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.

ABSTRACT
The master regulators of the cell cycle are cyclin-dependent kinases (Cdks), which influence the function of a myriad of proteins via phosphorylation. Mitotic Cdk1 is activated by A-type, as well as B1- and B2-type, cyclins. However, the role of a third, conserved cyclin B family member, cyclin B3, is less well defined. Here, we show that Caenorhabditis elegans CYB-3 has essential and distinct functions from cyclin B1 and B2 in the early embryo. CYB-3 is required for the timely execution of a number of cell cycle events including completion of the MII meiotic division of the oocyte nucleus, pronuclear migration, centrosome maturation, mitotic chromosome condensation and congression, and, most strikingly, progression through the metaphase-to-anaphase transition. Our experiments reveal that the extended metaphase delay in CYB-3-depleted embryos is dependent on an intact spindle assembly checkpoint (SAC) and results in salient defects in the architecture of holocentric metaphase chromosomes. Furthermore, genetically increasing or decreasing dynein activity results in the respective suppression or enhancement of CYB-3-dependent defects in cell cycle progression. Altogether, these data reveal that CYB-3 plays a unique, essential role in the cell cycle including promoting mitotic dynein functionality and alleviation of a SAC-dependent block in anaphase chromosome segregation.

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Modulation of dynein activity alters cell cycle progression and the rate of spindle pole separation in CYB-3–depleted embryos.A) Selected live images of OD57 embryos treated with the indicated RNAi. 0:00: NEB. A: anaphase entry. Scale Bar: 10 µm. B) Time from NEB-anaphase entry (seconds) in OD57 embryos treated with the indicated RNAi. Error bars: SEM; n =  embryos; control(RNAi), n = 11; dylt-1;control(RNAi), n = 11; dilute cyb-3(RNAi), n = 6; dilute cyb-3;dylt-1(RNAi), n = 7; Scale bar: 10 µm. *: p<0.05 as compared to control embryos. C) Embryos treated with the indicated RNAi conditions were fixed and stained with DAPI (blue), and BUB-1 (red) and HCP-1 (green) antibodies. n =  number of twisted metaphase plates/number of embryos examined. control(RNAi), n = 0/5; dylt-1+control(RNAi), n = 1/5; dilute cyb-3(RNAi) n = 5/5; dilute cyb-3+dylt-1, n = 0/3. Scale bar: 5 µm. D) The centrosome-centrosome distance (µm) in one-cell OD57 embryos treated with the indicated RNAi is plotted with respect to time from NEB (seconds). NEB: 0. Error bars: SEM. *:p<0.05 compared to control(RNAi) embryos at the same time-point. n =  embryos. control(RNAi), n = 8; dylt-1;control(RNAi), n = 5; dilute cyb-3(RNAi), n = 7; dilute cyb-3;dylt-1(RNAi), n = 6.
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pgen-1001218-g006: Modulation of dynein activity alters cell cycle progression and the rate of spindle pole separation in CYB-3–depleted embryos.A) Selected live images of OD57 embryos treated with the indicated RNAi. 0:00: NEB. A: anaphase entry. Scale Bar: 10 µm. B) Time from NEB-anaphase entry (seconds) in OD57 embryos treated with the indicated RNAi. Error bars: SEM; n =  embryos; control(RNAi), n = 11; dylt-1;control(RNAi), n = 11; dilute cyb-3(RNAi), n = 6; dilute cyb-3;dylt-1(RNAi), n = 7; Scale bar: 10 µm. *: p<0.05 as compared to control embryos. C) Embryos treated with the indicated RNAi conditions were fixed and stained with DAPI (blue), and BUB-1 (red) and HCP-1 (green) antibodies. n =  number of twisted metaphase plates/number of embryos examined. control(RNAi), n = 0/5; dylt-1+control(RNAi), n = 1/5; dilute cyb-3(RNAi) n = 5/5; dilute cyb-3+dylt-1, n = 0/3. Scale bar: 5 µm. D) The centrosome-centrosome distance (µm) in one-cell OD57 embryos treated with the indicated RNAi is plotted with respect to time from NEB (seconds). NEB: 0. Error bars: SEM. *:p<0.05 compared to control(RNAi) embryos at the same time-point. n =  embryos. control(RNAi), n = 8; dylt-1;control(RNAi), n = 5; dilute cyb-3(RNAi), n = 7; dilute cyb-3;dylt-1(RNAi), n = 6.

Mentions: Conceived and designed the experiments: GMRD JMS. Performed the experiments: GMRD TF YK. Analyzed the data: GMRD YK JMS. Wrote the paper: GMRD JMS. Designed and performed the bulk of the experiments: GMRD. Performed the dsRNA microinjections and created several strains: TF. Performed the experiments in Table 1 and Figure 6C: YK. Supervised the project, analyzed the data: JMS. Co-wrote the manuscript: GMRD JMS.


Caenorhabditis elegans cyclin B3 is required for multiple mitotic processes including alleviation of a spindle checkpoint-dependent block in anaphase chromosome segregation.

Deyter GM, Furuta T, Kurasawa Y, Schumacher JM - PLoS Genet. (2010)

Modulation of dynein activity alters cell cycle progression and the rate of spindle pole separation in CYB-3–depleted embryos.A) Selected live images of OD57 embryos treated with the indicated RNAi. 0:00: NEB. A: anaphase entry. Scale Bar: 10 µm. B) Time from NEB-anaphase entry (seconds) in OD57 embryos treated with the indicated RNAi. Error bars: SEM; n =  embryos; control(RNAi), n = 11; dylt-1;control(RNAi), n = 11; dilute cyb-3(RNAi), n = 6; dilute cyb-3;dylt-1(RNAi), n = 7; Scale bar: 10 µm. *: p<0.05 as compared to control embryos. C) Embryos treated with the indicated RNAi conditions were fixed and stained with DAPI (blue), and BUB-1 (red) and HCP-1 (green) antibodies. n =  number of twisted metaphase plates/number of embryos examined. control(RNAi), n = 0/5; dylt-1+control(RNAi), n = 1/5; dilute cyb-3(RNAi) n = 5/5; dilute cyb-3+dylt-1, n = 0/3. Scale bar: 5 µm. D) The centrosome-centrosome distance (µm) in one-cell OD57 embryos treated with the indicated RNAi is plotted with respect to time from NEB (seconds). NEB: 0. Error bars: SEM. *:p<0.05 compared to control(RNAi) embryos at the same time-point. n =  embryos. control(RNAi), n = 8; dylt-1;control(RNAi), n = 5; dilute cyb-3(RNAi), n = 7; dilute cyb-3;dylt-1(RNAi), n = 6.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991249&req=5

pgen-1001218-g006: Modulation of dynein activity alters cell cycle progression and the rate of spindle pole separation in CYB-3–depleted embryos.A) Selected live images of OD57 embryos treated with the indicated RNAi. 0:00: NEB. A: anaphase entry. Scale Bar: 10 µm. B) Time from NEB-anaphase entry (seconds) in OD57 embryos treated with the indicated RNAi. Error bars: SEM; n =  embryos; control(RNAi), n = 11; dylt-1;control(RNAi), n = 11; dilute cyb-3(RNAi), n = 6; dilute cyb-3;dylt-1(RNAi), n = 7; Scale bar: 10 µm. *: p<0.05 as compared to control embryos. C) Embryos treated with the indicated RNAi conditions were fixed and stained with DAPI (blue), and BUB-1 (red) and HCP-1 (green) antibodies. n =  number of twisted metaphase plates/number of embryos examined. control(RNAi), n = 0/5; dylt-1+control(RNAi), n = 1/5; dilute cyb-3(RNAi) n = 5/5; dilute cyb-3+dylt-1, n = 0/3. Scale bar: 5 µm. D) The centrosome-centrosome distance (µm) in one-cell OD57 embryos treated with the indicated RNAi is plotted with respect to time from NEB (seconds). NEB: 0. Error bars: SEM. *:p<0.05 compared to control(RNAi) embryos at the same time-point. n =  embryos. control(RNAi), n = 8; dylt-1;control(RNAi), n = 5; dilute cyb-3(RNAi), n = 7; dilute cyb-3;dylt-1(RNAi), n = 6.
Mentions: Conceived and designed the experiments: GMRD JMS. Performed the experiments: GMRD TF YK. Analyzed the data: GMRD YK JMS. Wrote the paper: GMRD JMS. Designed and performed the bulk of the experiments: GMRD. Performed the dsRNA microinjections and created several strains: TF. Performed the experiments in Table 1 and Figure 6C: YK. Supervised the project, analyzed the data: JMS. Co-wrote the manuscript: GMRD JMS.

Bottom Line: Mitotic Cdk1 is activated by A-type, as well as B1- and B2-type, cyclins.Our experiments reveal that the extended metaphase delay in CYB-3-depleted embryos is dependent on an intact spindle assembly checkpoint (SAC) and results in salient defects in the architecture of holocentric metaphase chromosomes.Altogether, these data reveal that CYB-3 plays a unique, essential role in the cell cycle including promoting mitotic dynein functionality and alleviation of a SAC-dependent block in anaphase chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.

ABSTRACT
The master regulators of the cell cycle are cyclin-dependent kinases (Cdks), which influence the function of a myriad of proteins via phosphorylation. Mitotic Cdk1 is activated by A-type, as well as B1- and B2-type, cyclins. However, the role of a third, conserved cyclin B family member, cyclin B3, is less well defined. Here, we show that Caenorhabditis elegans CYB-3 has essential and distinct functions from cyclin B1 and B2 in the early embryo. CYB-3 is required for the timely execution of a number of cell cycle events including completion of the MII meiotic division of the oocyte nucleus, pronuclear migration, centrosome maturation, mitotic chromosome condensation and congression, and, most strikingly, progression through the metaphase-to-anaphase transition. Our experiments reveal that the extended metaphase delay in CYB-3-depleted embryos is dependent on an intact spindle assembly checkpoint (SAC) and results in salient defects in the architecture of holocentric metaphase chromosomes. Furthermore, genetically increasing or decreasing dynein activity results in the respective suppression or enhancement of CYB-3-dependent defects in cell cycle progression. Altogether, these data reveal that CYB-3 plays a unique, essential role in the cell cycle including promoting mitotic dynein functionality and alleviation of a SAC-dependent block in anaphase chromosome segregation.

Show MeSH
Related in: MedlinePlus