The proper control of mitosis depends on the ubiquitin-mediated degradation of the right mitotic regulator at the right time. This is effected by the Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase that is regulated by the Spindle Assembly Checkpoint (SAC). The SAC prevents the APC/C from recognising Cyclin B1, the essential anaphase and cytokinesis inhibitor, until all chromosomes are attached to the spindle. Once chromosomes are attached, Cyclin B1 is rapidly degraded to enable chromosome segregation and cytokinesis. We have a good understanding of how the SAC inhibits the APC/C, but relatively little is known about how the APC/C recognises Cyclin B1 as soon as the SAC is turned off. Here, by combining live-cell imaging, in vitro reconstitution biochemistry, and structural analysis by cryo-electron microscopy, we provide evidence that the rapid recognition of Cyclin B1 in metaphase requires spatial regulation of the APC/C. Using fluorescence cross-correlation spectroscopy, we find that Cyclin B1 and the APC/C primarily interact at the mitotic apparatus. We show that this is because Cyclin B1, like the APC/C, binds to nucleosomes, and identify an \'arginine-anchor\' in the N-terminus as necessary and sufficient for binding to the nucleosome. Mutating the arginine anchor on Cyclin B1 reduces its interaction with the APC/C and delays its degradation: cells with the mutant, non-nucleosome-binding Cyclin B1 become aneuploid, demonstrating the physiological relevance of our findings. Together, our data demonstrate that mitotic chromosomes promote the efficient interaction between Cyclin B1 and the APC/C to ensure the timely degradation of Cyclin B1 and genomic stability.

The accuracy of cell division requires precise regulation of the cellular machinery governing DNA/genome duplication, ensuring its equal distribution among the daughter cells. The control of the centrosome cycle is crucial for the formation of a bipolar spindle, ensuring error-free segregation of the genome. The cell and centrosome cycles operate in close synchrony along similar principles. Both require a single duplication round in every cell cycle, and both are controlled by the activity of key protein kinases. Nevertheless, our comprehension of the precise cellular mechanisms and critical regulators synchronizing these two cycles remains poorly defined. Here, we present our hypothesis that the spatiotemporal regulation of a dynamic equilibrium of mitotic kinases activities forms a molecular clock that governs the synchronous progression of both the cell and the centrosome cycles.

Eukaryotic chromosome segregation requires kinetochores, multi-megadalton protein machines that assemble on the centromeres of chromosomes and mediate attachments to dynamic spindle microtubules. Kinetochores are built from numerous complexes, and there has been progress in structural studies on recombinant subassemblies. However, there is limited structural information on native kinetochore architecture. To address this, we purified functional, native kinetochores from the thermophilic yeast Kluyveromyces marxianus and examined them by electron microscopy (EM), cryoelectron tomography (cryo-ET), and atomic force microscopy (AFM). The kinetochores are extremely large, flexible assemblies that exhibit features consistent with prior models. We assigned kinetochore polarity by visualizing their interactions with microtubules and locating the microtubule binder, Ndc80c. This work shows that isolated kinetochores are more dynamic and complex than what might be anticipated based on the known structures of recombinant subassemblies and provides the foundation to study the global architecture and functions of kinetochores at a structural level.

The ring-shaped Cohesin complex, consisting of core subunits Smc1, Smc3, Scc1, and SA2 (or its paralog SA1), topologically entraps two duplicated sister DNA molecules to establish sister chromatid cohesion in S-phase. It remains largely elusive how the Cohesin release factor Wapl binds the Cohesin complex, thereby inducing Cohesin disassociation from mitotic chromosomes to allow proper resolution and separation of sister chromatids. Here, we show that Wapl uses two structural modules containing the FGF motif and the YNARHWN motif, respectively, to simultaneously bind distinct pockets in the extensive composite interface between Scc1 and SA2. Strikingly, only when both docking modules are mutated, Wapl completely loses the ability to bind the Scc1-SA2 interface and release Cohesin, leading to erroneous chromosome segregation in mitosis. Surprisingly, Sororin, which contains a conserved FGF motif and functions as a master antagonist of Wapl in S-phase and G2-phase, does not bind the Scc1-SA2 interface. Moreover, Sgo1, the major protector of Cohesin at mitotic centromeres, can only compete with the FGF motif but not the YNARHWN motif of Wapl for binding Scc1-SA2 interface. Our data uncover the molecular mechanism by which Wapl binds Cohesin to ensure precise chromosome segregation.

Mitosis exhibits astonishing evolutionary plasticity, with dividing eukaryotic cells differing in the organization of the mitotic spindle and the extent of nuclear envelope breakdown. A new study suggests that a multinucleated lifestyle may favor the evolution of closed nuclear division.

Mitosis in early embryos often proceeds at a rapid pace, but how this pace is achieved is not understood. Here, we show that cyclin B3 is the dominant driver of rapid embryonic mitoses in the C. elegans embryo. Cyclins B1 and B2 support slow mitosis (NEBD to anaphase ∼600 s), but the presence of cyclin B3 dominantly drives the approximately threefold faster mitosis observed in wildtype. Multiple mitotic events are slowed down in cyclin B1 and B2-driven mitosis, and cyclin B3-associated Cdk1 H1 kinase activity is ∼25-fold more active than cyclin B1-associated Cdk1. Addition of cyclin B1 to fast cyclin B3-only mitosis introduces an ∼60-s delay between completion of chromosome alignment and anaphase onset; this delay, which is important for segregation fidelity, is dependent on inhibitory phosphorylation of the anaphase activator Cdc20. Thus, cyclin B3 dominance, coupled to a cyclin B1-dependent delay that acts via Cdc20 phosphorylation, sets the rapid pace and ensures mitotic fidelity in the early C. elegans embryo.

Loss of Heterozygosity (LOH) due to mitotic recombination is frequently associated with the development of various cancers (e.g. retinoblastoma). LOH is also an important source of genetic diversity, especially in organisms where meiosis is infrequent. Irc20 is a putative helicase, and E3 ubiquitin ligase involved in DNA double-strand break repair pathway. We analyzed genome-wide LOH events, gross chromosomal changes, small insertion-deletions and single nucleotide mutations in eleven S. cerevisiae mutation accumulation lines of irc20∆, which underwent 50 mitotic bottlenecks. LOH enhancement in irc20∆ was small (1.6 fold), but statistically significant as compared to the wild type. Short (≤ 1 kb) and long (> 10 kb) LOH tracts were significantly enhanced in irc20∆. Both interstitial and terminal LOH events were also significantly enhanced in irc20∆ compared to the wild type. LOH events in irc20∆ were more telomere proximal and away from centromeres compared to the wild type. Gross chromosomal changes, single nucleotide mutations and in-dels were comparable between irc20∆ and wild type. Locus based and genome-wide analysis of meiotic recombination showed that meiotic crossover frequencies are not altered in irc20∆. These results suggest Irc20 primarily regulates mitotic recombination and does not affect meiotic crossovers. Our results suggest that the IRC20 gene is important for regulating LOH frequency and distribution.

During cell division, the mitotic spindle moves dynamically through the cell to position the chromosomes and determine the ultimate spatial position of the two daughter cells. These movements have been attributed to the action of cortical force generators which pull on the astral microtubules to position the spindle, as well as pushing events by these same microtubules against the cell cortex and plasma membrane. Attachment and detachment of cortical force generators working antagonistically against centring forces of microtubules have been modelled previously (Grill et al. in Phys Rev Lett 94:108104, 2005) via stochastic simulations and mean-field Fokker-Planck equations (describing random motion of force generators) to predict oscillations of a spindle pole in one spatial dimension. Using systematic asymptotic methods, we reduce the Fokker-Planck system to a set of ordinary differential equations (ODEs), consistent with a set proposed by Grill et al., which can provide accurate predictions of the conditions for the Fokker-Planck system to exhibit oscillations. In the limit of small restoring forces, we derive an algebraic prediction of the amplitude of spindle-pole oscillations and demonstrate the relaxation structure of nonlinear oscillations. We also show how noise-induced oscillations can arise in stochastic simulations for conditions in which the mean-field Fokker-Planck system predicts stability, but for which the period can be estimated directly by the ODE model and the amplitude by a related stochastic differential equation that incorporates random binding kinetics.

The spindle assembly checkpoint (SAC) temporally regulates mitosis by preventing progression from metaphase to anaphase until all chromosomes are correctly attached to the mitotic spindle. Centrosomes refine the spatial organization of the mitotic spindle at the spindle poles. However, centrosome loss leads to elongated mitosis, suggesting that centrosomes also inform the temporal organization of mitosis in mammalian cells. Here, we find that the mitotic delay in acentrosomal cells is enforced by the SAC in a MPS1-dependent manner, and that a SAC-dependent mitotic delay is required for bipolar cell division to occur in acentrosomal cells. Although acentrosomal cells become polyploid, polyploidy is not sufficient to cause dependency on a SAC-mediated delay to complete cell division. Rather, the division failure in absence of MPS1 activity results from mitotic exit occurring before acentrosomal spindles can become bipolar. Furthermore, prevention of centrosome separation suffices to make cell division reliant on a SAC-dependent mitotic delay. Thus, centrosomes and their definition of two spindle poles early in mitosis provide a \'timely two-ness\' that allows cell division to occur in absence of a SAC-dependent mitotic delay.

Temperature can impact every reaction essential to a cell. For organisms that cannot regulate their own temperature, adapting to temperatures that fluctuate unpredictably and on variable timescales is a major challenge. Extremes in the magnitude and frequency of temperature changes are increasing across the planet, raising questions as to how the biosphere will respond. To examine mechanisms of adaptation to temperature, we collected wild isolates from different climates of the fungus Ashbya gossypii, which has a compact genome of only ∼4,600 genes. We found control of the nuclear division cycle and polarized morphogenesis, both critical processes for fungal growth, were temperature sensitive and varied among the isolates. The phenotypes were associated with naturally varying sequences within the glutamine-rich region (QRR) IDR of an RNA-binding protein called Whi3. This protein regulates both nuclear division and polarized growth via its ability to form biomolecular condensates. In cells and in cell-free reconstitution assays, we found that temperature tunes the properties of Whi3-based condensates. Exchanging Whi3 sequences between isolates was sufficient to rescue temperature-sensitive phenotypes, and specifically, a heptad repeat sequence within the QRR confers temperature-sensitive behavior. Together, these data demonstrate that sequence variation in the size and composition of an IDR can promote cell adaptation to growth at specific temperature ranges. These data demonstrate the power of IDRs as tuning knobs for rapid adaptation to environmental fluctuations.