Regulated Separation of Sister Centromeres depends on the Spindle Assembly Checkpoint but not on the Anaphase Promoting Complex/Cyclosome

Cyclin B is a regulatory subunit of CDK1 within MPF complex. Degradation of cyclin B via ubiquitin-proteasome pathway seemed to be absolutely required for the M-phase exit. However, inhibition of the proteasome proteolytic activity upon the exit from the meiotic metaphase II-arrest in Xenopus cell-free extract revealed that the proteasome-dependent dissociation of cyclin B from CDK1 is sufficient to inactivate MPF without cyclin B degradation. In this study we analyze whether the same mechanism operates during the exit from mitotic M-phase. We show in Xenopus cell-free extract undergoing the first or the second embryonic mitosis that CDK1 oscillations are not affected by proteasome inhibition with MG132 or ALLN despite effective inhibition of cyclins B degradation. The majority of cyclins B1 and B2 surviving CDK1 inactivation is CDK-free and cyclin B2 becomes resistant to phosphatase λ dephosphorylation. The pool of cyclins B remaining after CDK1 inactivation in the presence of MG132 is mitotically inert, while exogenous or newly synthesized cyclin B activates CDK1. This suggests that cyclins B remain sequestered within the proteasome upon MPF inactivation in the presence of MG132.Comparison of the dynamics of the decline of total and CDK-bound pools of cyclins B1, B2 and B4 upon mitotic exit in absence of protein synthesis reveals that CDK-bound cyclins B diminish clearly faster. Our results thus show that cyclin B dissociation from CDK1 precedes cyclins B degradation upon CDK1 inactivation in mitotic embryo extracts and that proteasome proteolytic activity is dispensable for both activation and inactivation of CDK1 in such extracts.

Section: Discussionmentioning

confidence: 99%

Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos

Chesnel

Bazile²,

Pascal³

et al. 2006

“…Any error in this process may result in aneuploid embryo formation, which causes early embryo death, spontaneous abortion and genetic diseases. 55,56 Central to the accurate segregation is the assembly of a specific site for microtubule attachment, the kinetochore, on each sister chromatid. 57 The centromeric chromatin is essential for the formation of a functional kinetochore in all eukaryotes.…”

Section: Discussionmentioning

confidence: 99%

Histone Phosphorylation and Pericentromeric Histone Modifications in Oocyte Meiosis

Wang

Wang²,

Ai³

et al. 2006

Epigenetic regulation of pericentromeric heterochromatin is crucial for proper interactions between kinetochores and spindle microtubules governing accurate chromosome segregation. Here, we first examined the dynamic distribution of phosphorylated serine 10 and 28 on H3 during mouse oocyte maturation and early embryo development using immunofluorescent staining and confocal microscopy. Our results revealed strong signals of phosphorylated H3/ser10 and 28 in the pericentromeric heterochromatin area and continuous persistent staining of the chromosome periphery, respectively. A panel of specific antibodies against various acetylated lysine, dimethylated lysine or phosphorylated serine residues on histone H3 or H4 were used to investigate the effects of Trichostatin A (TSA), a general inhibitor of histone deacetylases (HDACs), on histone modifications of pericentromeric heterochromatin. Unexpectedly, TSA treatment was unable to alter the acetylation and methylation status of pericentromeric heterochromatin, however, it resulted in significant dephosphorylation of H3/ser10 at this site during mouse oocyte meiosis, which is likely to play a role in the TSA-induced defective chromosome segregation. Furthermore, by using ZM447439, an inhibitor of Aurora kinases, we revealed that Aurora kinases may participate in the regulation of histone phosphorylation during mouse oocyte maturation.

“…4A, bottom), excluding the possibility that Cdc14B Δ/Δ cells initially presented with a mitotic exit defect that was silenced over time through compensatory mechanisms. Because timely completion of anaphase and mitotic exit require the APC-dependent degradation of securin and cyclin B, 2,28 we examined the patterns of securin and cyclin B turnover in wildtype and Cdc14B Δ/Δ cells, as well as auto-cleavage of the cysteine protease separase, which is normally inhibited by securin and cyclin B. All three anaphase-specific proteolytic events occurred normally in Cdc14B Δ/Δ cells (Fig.…”

confidence: 99%

The nucleolar phosphataseCdc14Bis dispensable for chromosome segregation and mitotic exit in human cells

Berdougo¹,

Nachury²,

Jackson³

et al. 2008

In yeast, the protein phosphatase Cdc14 promotes chromosome segregation, mitotic exit, and cytokinesis by reversing M-phase phosphorylations catalyzed by Cdk1. A key feature of Cdc14 regulation is its sequestration within the nucleolus, which restricts its access to potential substrates for much of the cell cycle. Mammals also possess a nucleolar Cdc14 homolog, termed Cdc14B, but its roles during mitosis and cell division remain speculative. Here we analyze Cdc14B's subcellular dynamics during mitosis and rigorously test its functional contributions to cell division through homozygous disruption of the Cdc14B locus in human somatic cells. While Cdc14B is initially released from nucleoli at the start of mitosis, the phosphatase quickly redistributes onto segregating sister chromatids during anaphase. This relocalization is mainly driven by Cdk1 inactivation, as pharmacologic inhibition of Cdk1 in prometaphase cells redirects Cdc14B onto chromosomes. However, in sharp contrast to yeast cdc14 mutants, human Cdc14B Δ/Δ cells were viable and lacked defects in spindle assembly, anaphase progression, mitotic exit, and cytokinesis, and continued to segregate ribosomal DNA repeats with near-normal proficiency. Our findings reveal substantial divergence in mitotic regulation between yeast and mammalian cells, as the latter possess efficient mechanisms for completing late M-phase events in the absence of a nucleolar Cdc14-related phosphatase.