An anaphase surveillance mechanism prevents micronuclei formation from frequent chromosome segregation errors.

Micronuclei are a hallmark of cancer and several other human disorders. Recently, micronuclei were implicated in chromothripsis, a series of massive genomic rearrangements that may drive tumor evolution and progression. Here, we show that Aurora B kinase mediates a surveillance mechanism that integrates error correction during anaphase with spatial control of nuclear envelope reassembly to prevent micronuclei formation. Using high-resolution live-cell imaging of human cancer and non-cancer cells, we uncover that anaphase lagging chromosomes are more frequent than previously anticipated, yet they rarely form micronuclei. Micronuclei formation from anaphase lagging chromosomes is prevented by a midzone-based Aurora B phosphorylation gradient that stabilizes kinetochore-microtubule attachments and assists spindle forces required for anaphase error correction while delaying nuclear envelope reassembly on lagging chromosomes, independently of microtubule density. We propose that a midzone-based Aurora B phosphorylation gradient actively monitors and corrects frequent chromosome segregation errors to prevent micronuclei formation during human cell division.

Spatiotemporal control of mitotic exit during anaphase by an aurora B-Cdk1 crosstalk.

According to the prevailing 'clock' model, chromosome decondensation and nuclear envelope reformation when cells exit mitosis are byproducts of Cdk1 inactivation at the metaphase-anaphase transition, controlled by the spindle assembly checkpoint. However, mitotic exit was recently shown to be a function of chromosome separation during anaphase, assisted by a midzone Aurora B phosphorylation gradient - the 'ruler' model. Here we found that Cdk1 remains active during anaphase due to ongoing APC/CCdc20- and APC/CCdh1-mediated degradation of B-type Cyclins in Drosophila and human cells. Failure to degrade B-type Cyclins during anaphase prevented mitotic exit in a Cdk1-dependent manner. Cyclin B1-Cdk1 localized at the spindle midzone in an Aurora B-dependent manner, with incompletely separated chromosomes showing the highest Cdk1 activity. Slowing down anaphase chromosome motion delayed Cyclin B1 degradation and mitotic exit in an Aurora B-dependent manner. Thus, a crosstalk between molecular 'rulers' and 'clocks' licenses mitotic exit only after proper chromosome separation.

Late mitotic functions of Aurora kinases.

The coordination between late mitotic events such as poleward chromosome motion, spindle elongation, DNA decondensation, and nuclear envelope reformation (NER) is crucial for the completion of chromosome segregation at the anaphase-telophase transition. Mitotic exit is driven by a decrease of Cdk1 kinase activity and an increase of PP1/PP2A phosphatase activities. More recently, Aurora kinases have also emerged as master regulators of late mitotic events and cytokinesis. Aurora A is mainly associated with spindle poles throughout mitosis and midbody during telophase, whereas Aurora B re-localizes from centromeres in early mitosis to the spindle midzone and midbody as cells progress from anaphase to the completion of cytokinesis. Functional studies, together with the identification of a phosphorylation gradient during anaphase, established Aurora B as a major player in the organization of the spindle midzone and in the spatiotemporal coordination between chromosome segregation and NER. Aurora A has been less explored, but a cooperative role in spindle midzone stability has also been proposed, implying that both Aurora A and B contribute to accurate chromosome segregation during mitotic exit. Here, we review the roles of the Aurora kinases in the regulation of late mitotic events and discuss how they work together with other mitotic players to ensure an error-free mitosis.

A chromosome separation checkpoint: A midzone Aurora B gradient mediates a chromosome separation checkpoint that regulates the anaphase-telophase transition.

Here we discuss a "chromosome separation checkpoint" that might regulate the anaphase-telophase transition. The concept of cell cycle checkpoints was originally proposed to account for extrinsic control mechanisms that ensure the order of cell cycle events. Several checkpoints have been shown to regulate major cell cycle transitions, namely at G1-S and G2-M. At the onset of mitosis, the prophase-prometaphase transition is controlled by several potential checkpoints, including the antephase checkpoint, while the spindle assembly checkpoint guards the metaphase-anaphase transition. Our hypothesis is based on the recently uncovered feedback control mechanism that delays chromosome decondensation and nuclear envelope reassembly until effective separation of sister chromatids during anaphase is achieved. A central player in this potential checkpoint is the establishment of a constitutive, midzone-based Aurora B phosphorylation gradient that monitors the position of chromosomes along the spindle axis. We propose that this surveillance mechanism represents an additional step towards ensuring mitotic fidelity.

Feedback control of chromosome separation by a midzone Aurora B gradient.

Accurate chromosome segregation during mitosis requires the physical separation of sister chromatids before nuclear envelope reassembly (NER). However, how these two processes are coordinated remains unknown. Here, we identified a conserved feedback control mechanism that delays chromosome decondensation and NER in response to incomplete chromosome separation during anaphase. A midzone-associated Aurora B gradient was found to monitor chromosome position along the division axis and to prevent premature chromosome decondensation by retaining Condensin I. PP1/PP2A phosphatases counteracted this gradient and promoted chromosome decondensation and NER. Thus, an Aurora B gradient appears to mediate a surveillance mechanism that prevents chromosome decondensation and NER until effective separation of sister chromatids is achieved. This allows the correction and reintegration of lagging chromosomes in the main nuclei before completion of NER.

Genes involved in centrosome-independent mitotic spindle assembly in Drosophila S2 cells.

Animal mitotic spindle assembly relies on centrosome-dependent and centrosome-independent mechanisms, but their relative contributions remain unknown. Here, we investigated the molecular basis of the centrosome-independent spindle assembly pathway by performing a whole-genome RNAi screen in Drosophila S2 cells lacking functional centrosomes. This screen identified 197 genes involved in acentrosomal spindle assembly, eight of which had no previously described mitotic phenotypes and produced defective and/or short spindles. All 197 genes also produced RNAi phenotypes when centrosomes were present, indicating that none were entirely selective for the acentrosomal pathway. However, a subset of genes produced a selective defect in pole focusing when centrosomes were absent, suggesting that centrosomes compensate for this shape defect. Another subset of genes was specifically associated with the formation of multipolar spindles only when centrosomes were present. We further show that the chromosomal passenger complex orchestrates multiple centrosome-independent processes required for mitotic spindle assembly/maintenance. On the other hand, despite the formation of a chromosome-enriched RanGTP gradient, S2 cells depleted of RCC1, the guanine-nucleotide exchange factor for Ran on chromosomes, established functional bipolar spindles. Finally, we show that cells without functional centrosomes have a delay in chromosome congression and anaphase onset, which can be explained by the lack of polar ejection forces. Overall, these findings establish the constitutive nature of a centrosome-independent spindle assembly program and how this program is adapted to the presence/absence of centrosomes in animal somatic cells.

Stability of kinetochore-microtubule attachment and the role of different KMN network components in Drosophila.

Kinetochores bind spindle microtubules and also act as signaling centers that monitor this interaction. Defects in kinetochore assembly lead to chromosome missegregation and aneuploidy. The interaction between microtubules and chromosomes involves a conserved super-complex of proteins, known as the KNL1Mis12Ndc80 (KMN) network, composed by the KNL1 (Spc105), Mis12, and Ndc80 complexes. Previous studies indicate that all components of the network are required for kinetochore-microtubule attachment and all play relevant functions in chromosome congression, biorientation, and segregation. Here, we report a comparative study addressing the role of the different KMN components using dsRNA and in vivo fluorescence microscopy in Drosophila S2 cells allowing us to suggest that different KMN network components might perform different roles in chromosome segregation and the mitotic checkpoint signaling. Depletion of different components results in mostly lateral kinetochore-microtubule attachments that are relatively stable on depletion of Mis12 or Ndc80 but very unstable after Spc105 depletion. In vivo analysis on depletion of Mis12, Ndc80, and to some extent Spc105, shows that lateral kinetochore-microtubule interactions are still functional allowing poleward kinetochore movement. We also find that different KMN network components affect differently the localization of spindle assembly checkpoint (SAC) proteins at kinetochores. Depletion of Ndc80 and Spc105 abolishes the mitotic checkpoint, whereas depletion of Mis12 causes a delay in mitotic progression. Taken together, our results suggest that Mis12 and Ndc80 complexes help to properly orient microtubule attachment, whereas Spc105 plays a predominant role in the kinetochore-microtubule attachment as well as in the poleward movement of chromosomes, SAC response, and cell viability.

Driving chromosome segregation: lessons from the human and Drosophila centromere-kinetochore machinery.

The kinetochore is a complex molecular machine that serves as the interface between sister chromatids and the mitotic spindle. The kinetochore assembles at a particular chromosomal locus, the centromere, which is essential to maintain genomic stability during cell division. The kinetochore is a macromolecular puzzle of subcomplexes assembled in a hierarchical manner and fulfils three main functions: microtubule attachment, chromosome and sister chromatid movement, and regulation of mitotic progression though the spindle assembly checkpoint. In the present paper we compare recent results on the assembly, organization and function of the kinetochore in human and Drosophila cells and conclude that, although essential functions are highly conserved, there are important differences that might help define what is a minimal chromosome segregation machinery.

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