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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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Mitotic chromosome condensation is delayed in cyb-3(RNAi) embryos.Embryos from TH32 (GFP::Histone H2B;GFP::γ-tubulin) hermaphrodites treated with A) control(RNAi), B) cyb-3(RNAi), or C) smc-4(RNAi) were subjected to live imaging. Condensation of the paternal pronucleus was measured as described in Materials and Methods. Condensation parameters (% pixels below threshold) are plotted for four thresholds (20, 35, 50, and 65%) with respect to time from NEB (t = 0) [87]. PNM: Pronuclear meeting. SMC-4-depleted embryos were used as a control for loss of condensin complexes and mitotic chromosome condensation [51]. The control(RNAi) results are overlaid on the cyb-3(RNAi) panel to assist in direct comparison. n =  embryos; control(RNAi), n = 6; cyb-3(RNAi), n = 7; smc-4(RNAi), n = 8; Error bars: SEM.
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pgen-1001218-g002: Mitotic chromosome condensation is delayed in cyb-3(RNAi) embryos.Embryos from TH32 (GFP::Histone H2B;GFP::γ-tubulin) hermaphrodites treated with A) control(RNAi), B) cyb-3(RNAi), or C) smc-4(RNAi) were subjected to live imaging. Condensation of the paternal pronucleus was measured as described in Materials and Methods. Condensation parameters (% pixels below threshold) are plotted for four thresholds (20, 35, 50, and 65%) with respect to time from NEB (t = 0) [87]. PNM: Pronuclear meeting. SMC-4-depleted embryos were used as a control for loss of condensin complexes and mitotic chromosome condensation [51]. The control(RNAi) results are overlaid on the cyb-3(RNAi) panel to assist in direct comparison. n =  embryos; control(RNAi), n = 6; cyb-3(RNAi), n = 7; smc-4(RNAi), n = 8; Error bars: SEM.

Mentions: C. elegans oocytes are devoid of centrioles and centrosomes [38]. Therefore, the centriole donated by the sperm is the sole mitotic organizing center (MTOC) in the newly fertilized one-cell embryo [39]. The paternal centriole duplicates upon completion of the meiotic divisions of the oocyte nucleus. As the maternal pronucleus migrates, the centrioles recruit pericentriolar material and separate away from one another along the surface of the paternal pronucleus. Concurrently, condensation of the maternal and paternal pronuclei occurs in a synchronous manner. We noted that the maturing centrosomes in cyb-3(RNAi) embryos were much smaller compared to controls. At the time of PNM, cyb-3(RNAi) centrosomes were approximately two-fold smaller than control centrosomes (Figure 1C, 1D; Videos S3, S4). However, by nuclear envelope breakdown (NEB), there was no appreciable difference in centrosome size between CYB-3-depleted embryos and controls. Curiously, condensation of the paternal and maternal pronuclei was asynchronous in cyb-3(RNAi) embryos; condensation of the paternal pronucleus was significantly delayed with respect to the maternal pronucleus (Figure 1D; Videos S3, S4). However, as with centrosome size, condensation of the paternal pronucleus also “caught up” to control levels by NEB (Figure 1D, Figure 2, and Figure S2; Videos S3, S4). These defects are not likely to be secondary consequences of a failure to undergo MII anaphase since other MII defective mutants do not display these phenotypes [40].


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)

Mitotic chromosome condensation is delayed in cyb-3(RNAi) embryos.Embryos from TH32 (GFP::Histone H2B;GFP::γ-tubulin) hermaphrodites treated with A) control(RNAi), B) cyb-3(RNAi), or C) smc-4(RNAi) were subjected to live imaging. Condensation of the paternal pronucleus was measured as described in Materials and Methods. Condensation parameters (% pixels below threshold) are plotted for four thresholds (20, 35, 50, and 65%) with respect to time from NEB (t = 0) [87]. PNM: Pronuclear meeting. SMC-4-depleted embryos were used as a control for loss of condensin complexes and mitotic chromosome condensation [51]. The control(RNAi) results are overlaid on the cyb-3(RNAi) panel to assist in direct comparison. n =  embryos; control(RNAi), n = 6; cyb-3(RNAi), n = 7; smc-4(RNAi), n = 8; Error bars: SEM.
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Related In: Results  -  Collection

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pgen-1001218-g002: Mitotic chromosome condensation is delayed in cyb-3(RNAi) embryos.Embryos from TH32 (GFP::Histone H2B;GFP::γ-tubulin) hermaphrodites treated with A) control(RNAi), B) cyb-3(RNAi), or C) smc-4(RNAi) were subjected to live imaging. Condensation of the paternal pronucleus was measured as described in Materials and Methods. Condensation parameters (% pixels below threshold) are plotted for four thresholds (20, 35, 50, and 65%) with respect to time from NEB (t = 0) [87]. PNM: Pronuclear meeting. SMC-4-depleted embryos were used as a control for loss of condensin complexes and mitotic chromosome condensation [51]. The control(RNAi) results are overlaid on the cyb-3(RNAi) panel to assist in direct comparison. n =  embryos; control(RNAi), n = 6; cyb-3(RNAi), n = 7; smc-4(RNAi), n = 8; Error bars: SEM.
Mentions: C. elegans oocytes are devoid of centrioles and centrosomes [38]. Therefore, the centriole donated by the sperm is the sole mitotic organizing center (MTOC) in the newly fertilized one-cell embryo [39]. The paternal centriole duplicates upon completion of the meiotic divisions of the oocyte nucleus. As the maternal pronucleus migrates, the centrioles recruit pericentriolar material and separate away from one another along the surface of the paternal pronucleus. Concurrently, condensation of the maternal and paternal pronuclei occurs in a synchronous manner. We noted that the maturing centrosomes in cyb-3(RNAi) embryos were much smaller compared to controls. At the time of PNM, cyb-3(RNAi) centrosomes were approximately two-fold smaller than control centrosomes (Figure 1C, 1D; Videos S3, S4). However, by nuclear envelope breakdown (NEB), there was no appreciable difference in centrosome size between CYB-3-depleted embryos and controls. Curiously, condensation of the paternal and maternal pronuclei was asynchronous in cyb-3(RNAi) embryos; condensation of the paternal pronucleus was significantly delayed with respect to the maternal pronucleus (Figure 1D; Videos S3, S4). However, as with centrosome size, condensation of the paternal pronucleus also “caught up” to control levels by NEB (Figure 1D, Figure 2, and Figure S2; Videos S3, S4). These defects are not likely to be secondary consequences of a failure to undergo MII anaphase since other MII defective mutants do not display these phenotypes [40].

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