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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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Depletion of C. elegans Cyclin B3 leads to defects in pronuclear migration and synchrony of chromosome condensation.Embryos from TH32 (GFP::Histone H2B;GFP::γ-tubulin) and OD57 (mCherry::Histone H2B; GFP:: α-tubulin) hermaphrodites fed control or cyb-3 dsRNA were subjected to live imaging. A) The rate of pronuclear migration was calculated by measuring the distance (µm) between the maternal and paternal pronuclei with respect to time (seconds). Slow: slow phase (0–40% EL); Fast: fast phase (>40% EL); 0%: anterior end; control(RNAi), n = 5; cyb-3(RNAi), n = 7; Error bars: standard error of the means (SEM). B) The position of PNM was measured as the distance from the position of PNM to the anterior end and is displayed as % EL. control(RNAi), n = 12; cyb-3(RNAi), n = 14; Error bars: SEM; p = 0.0007. C) Centrosome size in TH32 embryos treated with control and cyb-3(RNAi) was measured at PNM and NEB. n =  centrosomes; control(RNAi), PNM: n = 17, NEB: n = 17; cyb-3(RNAi), PNM: n = 11, NEB: n = 13; Error bars: SEM; PNM: p<0.0001; NEB: p = 0.9. D) Selected images from Videos S3 and S4: TH32 embryos treated with control and cyb-3(RNAi) at PNM and NEB. Anterior is to the left in all images. Scale bar: 10 µm.
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pgen-1001218-g001: Depletion of C. elegans Cyclin B3 leads to defects in pronuclear migration and synchrony of chromosome condensation.Embryos from TH32 (GFP::Histone H2B;GFP::γ-tubulin) and OD57 (mCherry::Histone H2B; GFP:: α-tubulin) hermaphrodites fed control or cyb-3 dsRNA were subjected to live imaging. A) The rate of pronuclear migration was calculated by measuring the distance (µm) between the maternal and paternal pronuclei with respect to time (seconds). Slow: slow phase (0–40% EL); Fast: fast phase (>40% EL); 0%: anterior end; control(RNAi), n = 5; cyb-3(RNAi), n = 7; Error bars: standard error of the means (SEM). B) The position of PNM was measured as the distance from the position of PNM to the anterior end and is displayed as % EL. control(RNAi), n = 12; cyb-3(RNAi), n = 14; Error bars: SEM; p = 0.0007. C) Centrosome size in TH32 embryos treated with control and cyb-3(RNAi) was measured at PNM and NEB. n =  centrosomes; control(RNAi), PNM: n = 17, NEB: n = 17; cyb-3(RNAi), PNM: n = 11, NEB: n = 13; Error bars: SEM; PNM: p<0.0001; NEB: p = 0.9. D) Selected images from Videos S3 and S4: TH32 embryos treated with control and cyb-3(RNAi) at PNM and NEB. Anterior is to the left in all images. Scale bar: 10 µm.

Mentions: Upon completion of the two meiotic divisions in wild-type cells, the maternal pronucleus migrates toward the male pronucleus, which is positioned at the posterior end of the embryo. The maternal pronucleus migrates in two distinct phases, with an initial slow velocity until it reaches approximately 40% of embryo length (EL) from the anterior end (Anterior: 0%; Posterior: 100%) [22], [37]. The migration rate then increases significantly (fast phase). Since the paternal pronucleus migrates toward the anterior, the two pronuclei meet at approximately 70% of EL [37]. Compared to control, both phases of maternal pronuclear migration were approximately two-fold slower in cyb-3(RNAi) embryos (Figure 1A; Videos S3, S4, S5, S6). In addition, paternal pronuclear migration toward the anterior was greatly reduced, resulting in pronuclear meeting (PNM) occurring significantly closer to the embryo posterior (Figure 1B, 1D, and Figure 3C; Videos S4, S6).


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)

Depletion of C. elegans Cyclin B3 leads to defects in pronuclear migration and synchrony of chromosome condensation.Embryos from TH32 (GFP::Histone H2B;GFP::γ-tubulin) and OD57 (mCherry::Histone H2B; GFP:: α-tubulin) hermaphrodites fed control or cyb-3 dsRNA were subjected to live imaging. A) The rate of pronuclear migration was calculated by measuring the distance (µm) between the maternal and paternal pronuclei with respect to time (seconds). Slow: slow phase (0–40% EL); Fast: fast phase (>40% EL); 0%: anterior end; control(RNAi), n = 5; cyb-3(RNAi), n = 7; Error bars: standard error of the means (SEM). B) The position of PNM was measured as the distance from the position of PNM to the anterior end and is displayed as % EL. control(RNAi), n = 12; cyb-3(RNAi), n = 14; Error bars: SEM; p = 0.0007. C) Centrosome size in TH32 embryos treated with control and cyb-3(RNAi) was measured at PNM and NEB. n =  centrosomes; control(RNAi), PNM: n = 17, NEB: n = 17; cyb-3(RNAi), PNM: n = 11, NEB: n = 13; Error bars: SEM; PNM: p<0.0001; NEB: p = 0.9. D) Selected images from Videos S3 and S4: TH32 embryos treated with control and cyb-3(RNAi) at PNM and NEB. Anterior is to the left in all images. Scale bar: 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991249&req=5

pgen-1001218-g001: Depletion of C. elegans Cyclin B3 leads to defects in pronuclear migration and synchrony of chromosome condensation.Embryos from TH32 (GFP::Histone H2B;GFP::γ-tubulin) and OD57 (mCherry::Histone H2B; GFP:: α-tubulin) hermaphrodites fed control or cyb-3 dsRNA were subjected to live imaging. A) The rate of pronuclear migration was calculated by measuring the distance (µm) between the maternal and paternal pronuclei with respect to time (seconds). Slow: slow phase (0–40% EL); Fast: fast phase (>40% EL); 0%: anterior end; control(RNAi), n = 5; cyb-3(RNAi), n = 7; Error bars: standard error of the means (SEM). B) The position of PNM was measured as the distance from the position of PNM to the anterior end and is displayed as % EL. control(RNAi), n = 12; cyb-3(RNAi), n = 14; Error bars: SEM; p = 0.0007. C) Centrosome size in TH32 embryos treated with control and cyb-3(RNAi) was measured at PNM and NEB. n =  centrosomes; control(RNAi), PNM: n = 17, NEB: n = 17; cyb-3(RNAi), PNM: n = 11, NEB: n = 13; Error bars: SEM; PNM: p<0.0001; NEB: p = 0.9. D) Selected images from Videos S3 and S4: TH32 embryos treated with control and cyb-3(RNAi) at PNM and NEB. Anterior is to the left in all images. Scale bar: 10 µm.
Mentions: Upon completion of the two meiotic divisions in wild-type cells, the maternal pronucleus migrates toward the male pronucleus, which is positioned at the posterior end of the embryo. The maternal pronucleus migrates in two distinct phases, with an initial slow velocity until it reaches approximately 40% of embryo length (EL) from the anterior end (Anterior: 0%; Posterior: 100%) [22], [37]. The migration rate then increases significantly (fast phase). Since the paternal pronucleus migrates toward the anterior, the two pronuclei meet at approximately 70% of EL [37]. Compared to control, both phases of maternal pronuclear migration were approximately two-fold slower in cyb-3(RNAi) embryos (Figure 1A; Videos S3, S4, S5, S6). In addition, paternal pronuclear migration toward the anterior was greatly reduced, resulting in pronuclear meeting (PNM) occurring significantly closer to the embryo posterior (Figure 1B, 1D, and Figure 3C; Videos S4, S6).

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