Silke Hauf

The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore–microtubule attachment and in maintaining the spindle assembly checkpoint

The proper segregation of sister chromatids in mitosis depends on bipolar attachment of all chromosomes to the mitotic spindle. We have identified the small molecule Hesperadin as an inhibitor of chromosome alignment and segregation. Our data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B. Mammalian cells treated with Hesperadin enter anaphase in the presence of numerous monooriented chromosomes, many of which may have both sister kinetochores attached to one spindle pole (syntelic attachment). Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3–5 h. Together, our data suggest that Aurora B is required to generate unattached kinetochores on monooriented chromosomes, which in turn could promote bipolar attachment as well as maintain checkpoint signaling.

Dissociation of cohesin from chromosome arms and loss of arm cohesion during early mitosis depends on phosphorylation of SA2.

Cohesin is a protein complex that is required to hold sister chromatids together. Cleavage of the Scc1 subunit of cohesin by the protease separase releases the complex from chromosomes and thereby enables the separation of sister chromatids in anaphase. In vertebrate cells, the bulk of cohesin dissociates from chromosome arms already during prophase and prometaphase without cleavage of Scc1. Polo-like kinase 1 (Plk1) and Aurora-B are required for this dissociation process, and Plk1 can phosphorylate the cohesin subunits Scc1 and SA2 in vitro, consistent with the possibility that cohesin phosphorylation by Plk1 triggers the dissociation of cohesin from chromosome arms. However, this hypothesis has not been tested yet, and in budding yeast it has been found that phosphorylation of Scc1 by the Polo-like kinase Cdc5 enhances the cleavability of cohesin, but does not lead to separase-independent dissociation of cohesin from chromosomes. To address the functional significance of cohesin phosphorylation in human cells, we have searched for phosphorylation sites on all four subunits of cohesin by mass spectrometry. We have identified numerous mitosis-specific sites on Scc1 and SA2, mutated them, and expressed nonphosphorylatable forms of both proteins stably at physiological levels in human cells. The analysis of these cells lines, in conjunction with biochemical experiments in vitro, indicate that Scc1 phosphorylation is dispensable for cohesin dissociation from chromosomes in early mitosis but enhances the cleavability of Scc1 by separase. In contrast, our data reveal that phosphorylation of SA2 is essential for cohesin dissociation during prophase and prometaphase, but is not required for cohesin cleavage by separase. The similarity of the phenotype obtained after expression of nonphosphorylatable SA2 in human cells to that seen after the depletion of Plk1 suggests that SA2 is the critical target of Plk1 in the cohesin dissociation pathway.

Human Bub1 defines the persistent cohesion site along the mitotic chromosome by affecting Shugoshin localization.

Shugoshin (Sgo) proteins constitute a conserved protein family defined as centromeric protectors of Rec8-containing cohesin complexes in meiosis . In vertebrate mitosis, Scc1/Rad21-containing cohesin complexes are also protected at centromeres because arm cohesin, but not centromeric cohesin, is largely dissociated in pro- and prometaphase . The dissociation process is dependent on the activity of polo-like kinase (Plk1) and partly dependent on Aurora B . Recently, it has been demonstrated that vertebrate shugoshin is required for preserving centromeric cohesion during mitosis ; however, it was not addressed whether human shugoshin protects cohesin itself. Here, we show that the persistence of human Scc1 at centromeres in mitosis is indeed dependent on human Sgo1. In fission yeast, Sgo localization depends on Bub1, a conserved spindle checkpoint protein, which is enigmatically also required for chromosome congression during prometaphase in vertebrate cells. We demonstrate that human Sgo1 fails to localize at centromeres in Bub1-repressed cells, and centromeric cohesion is significantly loosened. Remarkably, in these cells, Sgo1 relocates to chromosomes all along their length and provokes ectopic protection from dissociation of Scc1 on chromosome arms. These results reveal a hitherto concealed role for human Bub1 in defining the persistent cohesion site of mitotic chromosomes.

Regulation of Sister Chromatid Cohesion between Chromosome Arms

Sister chromatid separation in anaphase depends on the removal of cohesin complexes from chromosomes. In vertebrates, the bulk of cohesin is already removed from chromosome arms during prophase and prometaphase, whereas cohesin remains at centromeres until metaphase, when cohesin is cleaved by the protease separase. In unperturbed mitoses, arm cohesion nevertheless persists throughout metaphase and is principally sufficient to maintain sister chromatid cohesion. How arm cohesion is maintained until metaphase is unknown. Here we show that small amounts of cohesin can be detected in the interchromatid region of metaphase chromosome arms. If prometaphase is prolonged by treatment of cells with microtubule poisons, these cohesin complexes dissociate from chromosome arms, and arm cohesion is dissolved. If cohesin dissociation in prometaphase-arrested cells is prevented by depletion of Plk1 or inhibition of Aurora B, arm cohesion is maintained. These observations imply that, in unperturbed mitoses, small amounts of cohesin maintain arm cohesion until metaphase. When cells lacking Plk1 and Aurora B activity enter anaphase, chromatids lose cohesin. This loss is prevented by proteasome inhibitors, implying that it depends on separase activation. Separase may therefore be able to cleave cohesin at centromeres and on chromosome arms.

Kinetochore Orientation in Mitosis and Meiosis

Kinetochores are the major point of contact between spindle microtubules and chromosomes. They are assemblies of more than 50 different proteins and take part in regulating and controlling their own interaction with the spindle. We review recent advance in understanding how kinetochores are properly placed onto the chromosome, and how their interaction with the microtubules of the spindle is regulated. Kinetochore orientation in meiosis I shows some particular features, and we also discuss similarities and differences between mitosis and meiosis I.

Aurora controls sister kinetochore mono-orientation and homolog bi-orientation in meiosis-I

Aurora‐B kinases are important regulators of mitotic chromosome segregation, where they are required for the faithful bi‐orientation of sister chromatids. In contrast to mitosis, sister chromatids have to be oriented toward the same spindle pole in meiosis‐I, while homologous chromosomes are bi‐oriented. We find that the fission yeast Aurora kinase Ark1 is required for the faithful bi‐orientation of sister chromatids in mitosis and of homologous chromosomes in meiosis‐I. Unexpectedly, Ark1 is also necessary for the faithful mono‐orientation of sister chromatids in meiosis‐I, even though the canonical mono‐orientation pathway, which depends on Moa1 and Rec8, seems intact. Our data suggest that Ark1 prevents unified sister kinetochores during metaphase‐I from merotelic attachment to both spindle poles and thus from being torn apart during anaphase‐I, revealing a novel mechanism promoting monopolar attachment. Furthermore, our results provide an explanation for the previously enigmatic observation that fission yeast Shugoshin Sgo2, which assists in loading Aurora to centromeres, and its regulator Bub1 are required for the mono‐orientation of sister chromatids in meiosis‐I.

Mitotic Substrates of the Kinase Aurora with Roles in Chromatin Regulation Identified Through Quantitative Phosphoproteomics of Fission Yeast

System-wide analysis of proteins phosphorylated by the mitotic kinase Aurora in fission yeast suggests widespread functions for this kinase in regulating chromatin dynamics. Expanding Aurora’s World Yeast are an attractive system for investigating complex biological processes, such as mitosis, because they frequently have a smaller set of regulatory genes than do mammals and they are genetically tractable. For instance, the mitotic kinase Aurora is encoded by a single gene in yeast, in contrast to metazoans, which have two or more Aurora-encoding genes. Koch et al. used fission yeast engineered to express an inhibitor-sensitive form of Aurora and then performed phosphoproteomic analysis to identify previously unknown targets of this key regulator of mitosis. Although Aurora has a well-established function in chromosome attachment to mitotic spindles, this work suggests that Aurora has a broader role in regulation of chromatin structure and may protect cells from DNA damage. These additional functions have implications for the investigation of Aurora inhibitors as cancer therapeutics. Kinases of the Aurora family are essential for the proper execution of mitosis in eukaryotes, and Aurora inhibitors are in clinical trials as anticancer drugs. We applied site-specific quantitative phosphoproteomics in conjunction with chemical inhibition of Aurora to identify mitotic Aurora substrates in fission yeast on a proteome-wide scale. We detected 8000 phosphorylation events, of which we assigned almost 6000 to a specific residue; 220 were reduced in cells exposed to the Aurora inhibitor. After controlling for unspecific effects of the inhibitor, we classified 70 sites (on 42 proteins) as probable targets of Aurora, which enabled refinement of the consensus sequence for phosphorylation by Aurora. Several of the substrate candidates were known targets of Aurora, validating the approach, but most represented newly detected Aurora substrates. The involvement of these Aurora substrates in diverse aspects of chromatin dynamics suggests that in addition to its established role in controlling chromosome compaction and attachment to the mitotic spindle, Aurora influences other aspects of chromatin architecture and function during mitosis.

Absolute Proteome and Phosphoproteome Dynamics during the Cell Cycle of Schizosaccharomyces pombe (Fission Yeast)

To quantify cell cycle-dependent fluctuations on a proteome-wide scale, we performed integrative analysis of the proteome and phosphoproteome during the four major phases of the cell cycle in Schizosaccharomyces pombe. In highly synchronized cells, we identified 3753 proteins and 3682 phosphorylation events and relatively quantified 65% of the data across all phases. Quantitative changes during the cell cycle were infrequent and weak in the proteome but prominent in the phosphoproteome. Protein phosphorylation peaked in mitosis, where the median phosphorylation site occupancy was 44%, about 2-fold higher than in other phases. We measured copy numbers of 3178 proteins, which together with phosphorylation site stoichiometry enabled us to estimate the absolute amount of protein-bound phosphate, as well as its change across the cell cycle. Our results indicate that 23% of the average intracellular ATP is utilized by protein kinases to phosphorylate their substrates to drive regulatory processes during cell division. Accordingly, we observe that phosphate transporters and phosphate-metabolizing enzymes are phosphorylated and therefore likely to be regulated in mitosis.

Determinants of robustness in spindle assembly checkpoint signalling

The spindle assembly checkpoint is a conserved signalling pathway that protects genome integrity. Given its central importance, this checkpoint should withstand stochastic fluctuations and environmental perturbations, but the extent of and mechanisms underlying its robustness remain unknown. We probed spindle assembly checkpoint signalling by modulating checkpoint protein abundance and nutrient conditions in fission yeast. For core checkpoint proteins, a mere 20% reduction can suffice to impair signalling, revealing a surprising fragility. Quantification of protein abundance in single cells showed little variability (noise) of critical proteins, explaining why the checkpoint normally functions reliably. Checkpoint-mediated stoichiometric inhibition of the anaphase activator Cdc20 (Slp1 in Schizosaccharomyces pombe) can account for the tolerance towards small fluctuations in protein abundance and explains our observation that some perturbations lead to non-genetic variation in the checkpoint response. Our work highlights low gene expression noise as an important determinant of reliable checkpoint signalling.

Repositioning of Aurora B Promoted by Chiasmata Ensures Sister Chromatid Mono-Orientation in Meiosis I

During meiosis I, kinetochores of sister chromatids are juxtaposed or fused and mono-orient, while homologous chromosomes that are paired by chiasmata (bivalents) have to biorient. In the absence of chiasmata, biorientation of sister chromatids (univalents), which carries a risk of aneuploidy, has been occasionally detected in several species, including humans. We show in fission yeast that biorientation of fused sister kinetochores predominates during early prometaphase I. Without chiasmata, this undesirable biorientation of univalents persists and eventually evades the spindle assembly checkpoint, provoking abnormal anaphase. When univalents are connected by chiasmata or by an artificial tether, this erroneous attachment is converted to monopolar attachment and stabilized. This stabilization is apparently achieved by a chromosome configuration that brings kinetochores to the outer edge of the bivalent, while bringing Aurora B, a destabilizer of kinetochore-microtubule attachment, inward. Our results elucidate how chiasmata favor biorientation of bivalents over that of univalents at meiosis I.