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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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CYB-3 depletion leads to altered metaphase kinetochore geometry and premature spindle pole separation.A) Control and cyb-3(RNAi) embryos were fixed and stained with DAPI and antibodies recognizing the kinetochore proteins HCP-1 (green) and BUB-1 (red). Arrowheads: resolved sister chromatids (prophase). Arrows: K-Mt immunostaining. B) Embryos treated as in (A) were stained with DAPI and antibodies recognizing HCP-3 (green) and BUB-1 (red). Arrowheads: resolved sister chromatids. C) Embryos treated with the indicated RNAi were fixed and stained with DAPI, and BUB-1 (red) and tubulin (green) antibodies. Arrowheads: pinched spindle poles. Scale bars: 10 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 = 7; cyb-3;control(RNAi), n = 8; mdf-1;control(RNAi), n = 5; cyb-3;mdf-1(RNAi), n = 7; smc-4(RNAi), n = 7; knl-1(RNAi), n = 4.
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pgen-1001218-g004: CYB-3 depletion leads to altered metaphase kinetochore geometry and premature spindle pole separation.A) Control and cyb-3(RNAi) embryos were fixed and stained with DAPI and antibodies recognizing the kinetochore proteins HCP-1 (green) and BUB-1 (red). Arrowheads: resolved sister chromatids (prophase). Arrows: K-Mt immunostaining. B) Embryos treated as in (A) were stained with DAPI and antibodies recognizing HCP-3 (green) and BUB-1 (red). Arrowheads: resolved sister chromatids. C) Embryos treated with the indicated RNAi were fixed and stained with DAPI, and BUB-1 (red) and tubulin (green) antibodies. Arrowheads: pinched spindle poles. Scale bars: 10 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 = 7; cyb-3;control(RNAi), n = 8; mdf-1;control(RNAi), n = 5; cyb-3;mdf-1(RNAi), n = 7; smc-4(RNAi), n = 7; knl-1(RNAi), n = 4.

Mentions: C. elegans chromosomes are holocentric, providing a large centromere advantageous for studying changes in kinetochore structure and centromere resolution [52]. Given that the organization of kinetochore microtubules was altered in cyb-3(RNAi) embryos, we tested whether kinetochore architecture was also changed. Hence, control and cyb-3(RNAi) embryos were fixed and co-stained with antibodies recognizing two kinetochore proteins, CeCENP-F/HCP-1 [53] and CeBub1/BUB-1 [10] (Figure 4A). In wild-type cells, sister chromatids are resolved from one another in prophase, resulting in paired kinetochores oriented to opposite spindle poles [54]. This geometry lessens the probability of kinetochores interacting with microtubules emanating from the wrong spindle pole. Sister chromatid resolution occurred in both control and CYB-3-depleted embryos, as evidenced by parallel stripes of BUB-1 and HCP-1 staining on prophase chromosomes (Figure 4A, arrowheads). This kinetochore geometry was maintained in both types of embryos through prometaphase. At metaphase, 100% of control embryos had two clearly defined stripes of BUB-1 and HCP-1 staining, as well as kinetochore microtubule (K-Mt) staining (Figure 4A, arrows). However, the majority (>80%) of cyb-3(RNAi) embryos had no clear BUB-1 or HCP-1 kinetochore stripes, and no BUB-1 or HCP-1 localization to metaphase K-Mts (Figure 4A). Rather, BUB-1 and HCP-1 staining appeared to be “twisted” and was coincident with the body of the metaphase chromosomes (Figure 4A). Immunostaining with additional kinetochore-specific antibodies (e.g., α-KNL-2) [55], as well as live imaging of GFP::KBP-4Ndc80 (strain OD11) transgenic embryos [56], confirmed these results (data not shown).


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)

CYB-3 depletion leads to altered metaphase kinetochore geometry and premature spindle pole separation.A) Control and cyb-3(RNAi) embryos were fixed and stained with DAPI and antibodies recognizing the kinetochore proteins HCP-1 (green) and BUB-1 (red). Arrowheads: resolved sister chromatids (prophase). Arrows: K-Mt immunostaining. B) Embryos treated as in (A) were stained with DAPI and antibodies recognizing HCP-3 (green) and BUB-1 (red). Arrowheads: resolved sister chromatids. C) Embryos treated with the indicated RNAi were fixed and stained with DAPI, and BUB-1 (red) and tubulin (green) antibodies. Arrowheads: pinched spindle poles. Scale bars: 10 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 = 7; cyb-3;control(RNAi), n = 8; mdf-1;control(RNAi), n = 5; cyb-3;mdf-1(RNAi), n = 7; smc-4(RNAi), n = 7; knl-1(RNAi), n = 4.
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getmorefigures.php?uid=PMC2991249&req=5

pgen-1001218-g004: CYB-3 depletion leads to altered metaphase kinetochore geometry and premature spindle pole separation.A) Control and cyb-3(RNAi) embryos were fixed and stained with DAPI and antibodies recognizing the kinetochore proteins HCP-1 (green) and BUB-1 (red). Arrowheads: resolved sister chromatids (prophase). Arrows: K-Mt immunostaining. B) Embryos treated as in (A) were stained with DAPI and antibodies recognizing HCP-3 (green) and BUB-1 (red). Arrowheads: resolved sister chromatids. C) Embryos treated with the indicated RNAi were fixed and stained with DAPI, and BUB-1 (red) and tubulin (green) antibodies. Arrowheads: pinched spindle poles. Scale bars: 10 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 = 7; cyb-3;control(RNAi), n = 8; mdf-1;control(RNAi), n = 5; cyb-3;mdf-1(RNAi), n = 7; smc-4(RNAi), n = 7; knl-1(RNAi), n = 4.
Mentions: C. elegans chromosomes are holocentric, providing a large centromere advantageous for studying changes in kinetochore structure and centromere resolution [52]. Given that the organization of kinetochore microtubules was altered in cyb-3(RNAi) embryos, we tested whether kinetochore architecture was also changed. Hence, control and cyb-3(RNAi) embryos were fixed and co-stained with antibodies recognizing two kinetochore proteins, CeCENP-F/HCP-1 [53] and CeBub1/BUB-1 [10] (Figure 4A). In wild-type cells, sister chromatids are resolved from one another in prophase, resulting in paired kinetochores oriented to opposite spindle poles [54]. This geometry lessens the probability of kinetochores interacting with microtubules emanating from the wrong spindle pole. Sister chromatid resolution occurred in both control and CYB-3-depleted embryos, as evidenced by parallel stripes of BUB-1 and HCP-1 staining on prophase chromosomes (Figure 4A, arrowheads). This kinetochore geometry was maintained in both types of embryos through prometaphase. At metaphase, 100% of control embryos had two clearly defined stripes of BUB-1 and HCP-1 staining, as well as kinetochore microtubule (K-Mt) staining (Figure 4A, arrows). However, the majority (>80%) of cyb-3(RNAi) embryos had no clear BUB-1 or HCP-1 kinetochore stripes, and no BUB-1 or HCP-1 localization to metaphase K-Mts (Figure 4A). Rather, BUB-1 and HCP-1 staining appeared to be “twisted” and was coincident with the body of the metaphase chromosomes (Figure 4A). Immunostaining with additional kinetochore-specific antibodies (e.g., α-KNL-2) [55], as well as live imaging of GFP::KBP-4Ndc80 (strain OD11) transgenic embryos [56], confirmed these results (data not shown).

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