Cyclin B3 activates the Anaphase-Promoting Complex/Cyclosome in meiosis and mitosis

In mitosis and meiosis, chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit ubiquitin ligase that targets proteins for degradation, leading to the separation of chromatids. APC/C activation requires phosphorylation of its APC3 and APC1 subunits, which allows the APC/C to bind its co-activator Cdc20. The identity of the kinase(s) responsible for APC/C activation in vivo is unclear. Cyclin B3 (CycB3) is an activator of the Cyclin-Dependent Kinase 1 (Cdk1) that is required for meiotic anaphase in flies, worms and vertebrates. It has been hypothesized that CycB3-Cdk1 may be responsible for APC/C activation in meiosis but this remains to be determined. Using Drosophila, we found that mutations in CycB3 genetically enhance mutations in tws, which encodes the B55 regulatory subunit of Protein Phosphatase 2A (PP2A) known to promote mitotic exit. Females heterozygous for CycB3 and tws loss-of-function alleles lay embryos that arrest in mitotic metaphase in a maternal effect, indicating that CycB3 promotes anaphase in mitosis in addition to meiosis. This metaphase arrest is not due to the Spindle Assembly Checkpoint (SAC) because mutation of mad2 that inactivates the SAC does not rescue the development of embryos from CycB3-/+, tws-/+ females. Moreover, we found that CycB3 promotes APC/C activity and anaphase in cells in culture. We show that CycB3 physically associates with the APC/C, is required for phosphorylation of APC3, and promotes APC/C association with its Cdc20 co-activators Fizzy and Cortex. Our results strongly suggest that CycB3-Cdk1 directly activates the APC/C to promote anaphase in both meiosis and mitosis.

PP2A-Cdc55 is responsible for mitotic arrest in DNA re-replicating cells in S. cerevisiae

The cell cycle is an ordered process in which cells replicate their DNA in S-phase and divide them into two identical daughter cells in mitosis. DNA replication takes place only once per cell cycle to preserve genome integrity, which is tightly regulated by Cyclin Dependent Kinase (CDK). Formation of the pre-replicative complex, a platform for origin licensing, is inhibited through CDK-dependent phosphorylation. Failure of this control leads to re-licensing, re-replication and DNA damage. Eukaryotic cells have evolved surveillance mechanisms to maintain genome integrity, termed cell cycle checkpoints. It has been shown that the DNA damage checkpoint is activated upon the induction of DNA re-replication and arrests cell cycle in mitosis in S. cerevisiae. In this study, we show that PP2A-Cdc55 is responsible for the metaphase arrest induced by DNA re-replication, leading to dephosphorylation of APC component, Exclusion of Cdc55 from the nucleus bypassed the mitotic arrest and resulted in enhanced cell lethality in re-replicating cells. The metaphase arrest in re-replication cells was retained in the absence of Mad2, a key component of the spindle assembly checkpoint. Moreover, re-replicating cells showed the same rate of DNA damage induction in the presence or absence of Cdc55. These results indicate that PP2A-Cdc55 maintains metaphase arrest upon DNA re-replication and DNA damage through APC inhibition.

Actin-microtubule interplay coordinates spindle assembly in human oocytes

Mammalian oocytes assemble a bipolar acentriolar microtubule spindle to segregate chromosomes during asymmetric division. There is increasing evidence that actin in the spindle interior not only participates in spindle migration and positioning but also protects oocytes from chromosome segregation errors leading to aneuploidy. Here we show that actin is an integral component of the meiotic machinery that closely interacts with microtubules during all major events of human oocyte maturation from the time point of spindle assembly till polar body extrusion and metaphase arrest. With the aid of drugs selectively affecting cytoskeleton dynamics and transiently disturbing the integrity of the two cytoskeleton systems, we identify interdependent structural rearrangements indicative of a close communication between actin and microtubules as fundamental feature of human oocytes. Our data support a model of actin-microtubule interplay that is essential for bipolar spindle assembly and correct partitioning of the nuclear genome in human oocyte meiosis.

The molecular architecture of the meiotic spindle is remodeled during metaphase arrest in oocytes

Before fertilization, oocytes of most species undergo a long, natural arrest in metaphase. Before this, prometaphase I is also prolonged, due to late stable kinetochore–microtubule attachment. How oocytes stably maintain the dynamic spindle for hours during these periods is poorly understood. Here we report that the bipolar spindle changes its molecular architecture during the long prometaphase/metaphase I in Drosophila melanogaster oocytes. By generating transgenic flies expressing GFP-tagged spindle proteins, we found that 14 of 25 spindle proteins change their distribution in the bipolar spindle. Among them, microtubule cross-linking kinesins, MKlp1/Pavarotti and kinesin-5/Klp61F, accumulate to the spindle equator in late metaphase. We found that the late equator accumulation of MKlp1/Pavarotti is regulated by a mechanism distinct from that in mitosis. While MKlp1/Pavarotti contributes to the control of spindle length, kinesin-5/Klp61F is crucial for maintaining a bipolar spindle during metaphase I arrest. Our study provides novel insight into how oocytes maintain a bipolar spindle during metaphase arrest.

A kinetochore-based ATM/ATR-independent DNA damage checkpoint maintains genomic integrity in trypanosomes

DNA damage-induced cell cycle checkpoints serve as surveillance mechanisms to maintain genomic stability, and are regulated by ATM/ATR-mediated signaling pathways that are conserved from yeast to humans. Trypanosoma brucei, an early divergent microbial eukaryote, lacks key components of the conventional DNA damage-induced G2/M cell cycle checkpoint and the spindle assembly checkpoint, and nothing is known about how T. brucei controls its cell cycle checkpoints. Here we discover a kinetochore-based, DNA damage-induced metaphase checkpoint in T. brucei. MMS-induced DNA damage triggers a metaphase arrest by modulating the abundance of the outer kinetochore protein KKIP5 in an Aurora B kinase- and kinetochore-dependent, but ATM/ATR-independent manner. Overexpression of KKIP5 arrests cells at metaphase through stabilizing the mitotic cyclin CYC6 and the cohesin subunit SCC1, mimicking DNA damage-induced metaphase arrest, whereas depletion of KKIP5 alleviates the DNA damage-induced metaphase arrest and causes chromosome mis-segregation and aneuploidy. These findings suggest that trypanosomes employ a novel DNA damage-induced metaphase checkpoint to maintain genomic integrity.

A Cdk1 phosphomimic mutant of MCAK impairs microtubule end recognition

The microtubule depolymerising kinesin-13, MCAK, is phosphorylated at residue T537 by Cdk1. This is the only known phosphorylation site within MCAK’s motor domain. To understand the impact of phosphorylation by Cdk1 on microtubule depolymerisation activity, we have investigated the molecular mechanism of the phosphomimic mutant T537E. This mutant significantly impairs microtubule depolymerisation activity and when transfected into cells causes metaphase arrest and misaligned chromosomes. We show that the molecular mechanism underlying the reduced depolymerisation activity of this phosphomimic mutant is an inability to recognise the microtubule end. The microtubule-end residence time is reduced relative to wild-type MCAK, whereas the lattice residence time is unchanged by the phosphomimic mutation. Further, the microtubule-end specific stimulation of ADP dissociation, characteristic of MCAK, is abolished by this mutation. Our data shows that T537E is unable to distinguish between the microtubule end and the microtubule lattice.

A Cdk1 phosphomimic mutant of MCAK impairs microtubule end recognition

The microtubule depolymerising kinesin-13, MCAK, is phosphorylated at residue T537 by Cdk1. This is the only known phosphorylation site within MCAK’s motor domain. To understand the impact of phosphorylation by Cdk1 microtubule depolymerisation activity, we have investigated the molecular mechanism of the phosphomimic mutant T537E. This mutant significantly impairs microtubule depolymerisation activity and when transfected into cells causes metaphase arrest and misaligned chromosomes. We show that the molecular mechanism underlying the reduced depolymerisation activity of this phosphomimic mutant is an inability to recognise the microtubule end. The microtubule-end residence time is reduced relative to wild-type MCAK, whereas the lattice residence time is unchanged by the phosphomimic mutation. Further, the microtubule-end specific stimulation of ADP dissociation, characteristic of MCAK, is abolished by this mutation. Our data shows that T537E is unable to distinguish between the microtubule end and the microtubule lattice.

The BTG4 and CAF1 complex prevents the spontaneous activation of eggs by deadenylating maternal mRNAs

Once every menstrual cycle, eggs are ovulated into the oviduct where they await fertilization. The ovulated eggs are arrested in metaphase of the second meiotic division, and only complete meiosis upon fertilization. It is crucial that the maintenance of metaphase arrest is tightly controlled, because the spontaneous activation of the egg would preclude the development of a viable embryo (Zhang et al. 2015 J. Genet. Genomics 42 , 477–485. ( doi:10.1016/j.jgg.2015.07.004 ); Combelles et al. 2011 Hum. Reprod. 26 , 545–552. ( doi:10.1093/humrep/deq363 ); Escrich et al. 2011 J. Assist. Reprod. Genet. 28 , 111–117. ( doi:10.1007/s10815-010-9493-5 )). However, the mechanisms that control the meiotic arrest in mammalian eggs are only poorly understood. Here, we report that a complex of BTG4 and CAF1 safeguards metaphase II arrest in mammalian eggs by deadenylating maternal mRNAs. As a follow-up of our recent high content RNAi screen for meiotic genes (Pfender et al. 2015 Nature 524 , 239–242. ( doi:10.1038/nature14568 )), we identified Btg4 as an essential regulator of metaphase II arrest. Btg4- depleted eggs progress into anaphase II spontaneously before fertilization. BTG4 prevents the progression into anaphase by ensuring that the anaphase-promoting complex/cyclosome (APC/C) is completely inhibited during the arrest. The inhibition of the APC/C relies on EMI2 (Tang et al. 2010 Mol. Biol. Cell 21 , 2589–2597. ( doi:10.1091/mbc.E09-08-0708 ); Ohe et al. 2010 Mol. Biol. Cell 21 , 905–913. ( doi:10.1091/mbc.E09-11-0974 )), whose expression is perturbed in the absence of BTG4. BTG4 controls protein expression during metaphase II arrest by forming a complex with the CAF1 deadenylase and we hypothesize that this complex is recruited to the mRNA via interactions between BTG4 and poly(A)-binding proteins. The BTG4–CAF1 complex drives the shortening of the poly(A) tails of a large number of transcripts at the MI–MII transition, and this wave of deadenylation is essential for the arrest in metaphase II. These findings establish a BTG4-dependent pathway for controlling poly(A) tail length during meiosis and identify an unexpected role for mRNA deadenylation in preventing the spontaneous activation of eggs.