Profilin 1 deficiency drives mitotic defects and reduces genome stability.

Profilin 1-encoded by PFN1-is a small actin-binding protein with a tumour suppressive role in various adenocarcinomas and pagetic osteosarcomas. However, its contribution to tumour development is not fully understood. Using fix and live cell imaging, we report that Profilin 1 inactivation results in multiple mitotic defects, manifested prominently by anaphase bridges, multipolar spindles, misaligned and lagging chromosomes, and cytokinesis failures. Accordingly, next-generation sequencing technologies highlighted that Profilin 1 knock-out cells display extensive copy-number alterations, which are associated with complex genome rearrangements and chromothripsis events in primary pagetic osteosarcomas with Profilin 1 inactivation. Mechanistically, we show that Profilin 1 is recruited to the spindle midzone at anaphase, and its deficiency reduces the supply of actin filaments to the cleavage furrow during cytokinesis. The mitotic defects are also observed in mouse embryonic fibroblasts and mesenchymal cells deriving from a newly generated knock-in mouse model harbouring a Pfn1 loss-of-function mutation. Furthermore, nuclear atypia is also detected in histological sections of mutant femurs. Thus, our results indicate that Profilin 1 has a role in regulating cell division, and its inactivation triggers mitotic defects, one of the major mechanisms through which tumour cells acquire chromosomal instability.

Depletion or cleavage of cohesin during anaphase differentially affects chromatin structure and segregation.

Chromosome segregation requires both the separation of sister chromatids and the sustained condensation of chromatids during anaphase. In yeast cells, cohesin is not only required for sister chromatid cohesion but also plays a major role determining the structure of individual chromatids in metaphase. Separase cleavage is thought to remove all cohesin complexes from chromosomes to initiate anaphase. It is thus not clear how the length and organisation of segregating chromatids is maintained during anaphase in the absence of cohesin. Here, we show that degradation of cohesin at the anaphase onset causes aberrant chromatid segregation. Hi-C analysis on segregating chromatids demonstrates that cohesin depletion causes loss of intrachromatid organisation. Surprisingly, tobacco etch virus (TEV)-mediated cleavage of cohesin does not dramatically disrupt chromatid organisation in anaphase, explaining why bulk segregation is achieved. In addition, we identified a small pool of cohesin complexes bound to telophase chromosomes in wild-type cells and show that they play a role in the organisation of centromeric regions. Our data demonstrates that in yeast cells cohesin function is not over in metaphase, but extends to the anaphase period when chromatids are segregating.

Control of division in Chlamydomonas by cyclin B/CDKB1 and the anaphase-promoting complex.

In yeast and animals, cyclin B binds and activates the cyclin-dependent kinase ('CDK') CDK1 to drive entry into mitosis. We show that CYCB1, the sole cyclin B in Chlamydomonas, activates the plant-specific CDKB1 rather than the CDK1 ortholog CDKA1, confirming and extending previous results. Time-lapse microscopy shows that CYCB1 is synthesized before each division in the multiple fission cycle, then is rapidly degraded 3-5 minutes before division occurs. CYCB1 degradation is dependent on the anaphase-promoting complex (APC). Like CYCB1, CDKB1 is not synthesized until late G1; however, CDKB1 is not degraded with each division within the multiple fission cycle, but is degraded after all divisions have ceased. The microtubule plus-end-binding protein EB1 labeled with mNeonGreen allowed detection of mitotic events in live cells. The earliest detectable step in mitosis, splitting of polar EB1 signal into two foci, likely associated with future spindle poles, was dependent on CYCB1. CYCB1-GFP localized close to these foci immediately before spindle formation. Spindle breakdown, cleavage furrow formation and accumulation of EB1 in the furrow were dependent on the APC. In interphase, rapidly growing microtubules are marked by 'comets' of EB1; comets are absent in the absence of APC function. Thus CYCB1/CDKB1 and the APC modulate microtubule function and assembly while regulating mitotic progression. Genetic results suggest an independent additional role for the APC in regulating sister chromatid cohesion; this role is likely conserved across eukaryotes.

Protein phosphatase 2A (PP2A) promotes anaphase entry after DNA replication stress in budding yeast.

DNA replication stress activates the S-phase checkpoint that arrests the cell cycle, but it is poorly understood how cells recover from this arrest. Cyclin-dependent kinase (CDK) and protein phosphatase 2A (PP2A) are key cell cycle regulators, and Cdc55 is a regulatory subunit of PP2A in budding yeast. We found that yeast cells lacking functional PP2ACdc55 showed slow growth in the presence of hydroxyurea (HU), a DNA synthesis inhibitor, without obvious viability loss. Moreover, PP2A mutants exhibited delayed anaphase entry and sustained levels of anaphase inhibitor Pds1 after HU treatment. A DNA damage checkpoint Chk1 phosphorylates and stabilizes Pds1. We show that chk1Δ and mutation of the Chk1 phosphorylation sites in Pds1 largely restored efficient anaphase entry in PP2A mutants after HU treatment. In addition, deletion of SWE1, which encodes the inhibitory kinase for CDK or mutation of the Swe1 phosphorylation site in CDK (cdc28F19), also suppressed the anaphase entry delay in PP2A mutants after HU treatment. Our genetic data suggest that Swe1/CDK acts upstream of Pds1. Surprisingly, cdc55Δ showed significant suppression to the viability loss of S-phase checkpoint mutants during DNA synthesis block. Together, our results uncover a PP2A-Swe1-CDK-Chk1-Pds1 axis that promotes recovery from DNA replication stress.

The RAD51 recombinase protects mitotic chromatin in human cells.

The RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. While interphase functions of RAD51 in maintaining genome stability are well-characterised, its role in mitosis remains contentious. In this study, we show that RAD51 protects under-replicated DNA in mitotic human cells and, in this way, promotes mitotic DNA synthesis (MiDAS) and successful chromosome segregation. In cells experiencing mild replication stress, MiDAS was detected irrespective of mitotically generated DNA damage. MiDAS broadly required de novo RAD51 recruitment to single-stranded DNA, which was supported by the phosphorylation of RAD51 by the key mitotic regulator Polo-like kinase 1. Importantly, acute inhibition of MiDAS delayed anaphase onset and induced centromere fragility, suggesting a mechanism that prevents the satisfaction of the spindle assembly checkpoint while chromosomal replication remains incomplete. This study hence identifies an unexpected function of RAD51 in promoting genomic stability in mitosis.

PRC1 is a critical regulator for anaphase spindle midzone assembly and cytokinesis in mouse oocyte meiosis.

Protein regulator of cytokinesis 1 (PRC1) is a microtubule bundling protein that is involved in the regulation of the central spindle bundle and spindle orientation during mitosis. However, the functions of PRC1 during meiosis have rarely been studied. In this study, we explored the roles of PRC1 during meiosis using an oocyte model. Our results found that PRC1 was expressed at all stages of mouse oocyte meiosis, and PRC1 accumulated in the midzone/midbody during anaphase/telophase I. Moreover, depleting PRC1 caused defects in polar body extrusion during mouse oocyte maturation. Further analysis found that PRC1 knockdown did not affect meiotic spindle formation or chromosome segregation; however, deleting PRC1 prevented formation of the midzone and midbody at the anaphase/telophase stage of meiosis I, which caused cytokinesis defects and further induced the formation of two spindles in the oocytes. PRC1 knockdown increased the level of tubulin acetylation, indicating that microtubule stability was affected. Furthermore, KIF4A and PRC1 showed similar localization in the midzone/midbody of oocytes at anaphase/telophase I, while the depletion of KIF4A affected the expression and localization of PRC1. The PRC1 mRNA injection rescued the defects caused by PRC1 knockdown in oocytes. In summary, our results suggest that PRC1 is critical for midzone/midbody formation and cytokinesis under regulation of KIF4A in mouse oocytes.

The 3D Topography of Mitotic Chromosomes.

A long-standing conundrum is how mitotic chromosomes can compact, as required for clean separation to daughter cells, while maintaining close parallel alignment of sister chromatids. Pursuit of this question, by high resolution 3D fluorescence imaging of living and fixed mammalian cells, has led to three discoveries. First, we show that the structural axes of separated sister chromatids are linked by evenly spaced "mini-axis" bridges. Second, when chromosomes first emerge as discrete units, at prophase, they are organized as co-oriented sister linear loop arrays emanating from a conjoined axis. We show that this same basic organization persists throughout mitosis, without helical coiling. Third, from prophase onward, chromosomes are deformed into sequential arrays of half-helical segments of alternating handedness (perversions), accompanied by correlated kinks. These arrays fluctuate dynamically over <15 s timescales. Together these discoveries redefine the foundation for thinking about the evolution of mitotic chromosomes as they prepare for anaphase segregation.

Polo kinase recruitment via the constitutive centromere-associated network at the kinetochore elevates centromeric RNA.

The kinetochore, a multi-protein complex assembled on centromeres, is essential to segregate chromosomes during cell division. Deficiencies in kinetochore function can lead to chromosomal instability and aneuploidy-a hallmark of cancer cells. Kinetochore function is controlled by recruitment of regulatory proteins, many of which have been documented, however their function often remains uncharacterized and many are yet to be identified. To identify candidates of kinetochore regulation we used a proteome-wide protein association strategy in budding yeast and detected many proteins that are involved in post-translational modifications such as kinases, phosphatases and histone modifiers. We focused on the Polo-like kinase, Cdc5, and interrogated which cellular components were sensitive to constitutive Cdc5 localization. The kinetochore is particularly sensitive to constitutive Cdc5 kinase activity. Targeting Cdc5 to different kinetochore subcomplexes produced diverse phenotypes, consistent with multiple distinct functions at the kinetochore. We show that targeting Cdc5 to the inner kinetochore, the constitutive centromere-associated network (CCAN), increases the levels of centromeric RNA via an SPT4 dependent mechanism.

A prognostic model based on cell-cycle control predicts outcome of breast cancer patients.

BACKGROUND: A prognostic model combining biomarkers of metaphase-anaphase transition of the cell cycle was developed for invasive breast cancer. The prognostic value and clinical applicability of the model was evaluated in comparison with the routine prognosticators of invasive breast carcinoma. METHODS: The study comprised 1135 breast cancer patients with complete clinical data and up to 22-year follow-up. Regulators of metaphase-anaphase transition were detected immunohistochemically and the biomarkers with the strongest prognostic impacts were combined into a prognostic model. The prognostic value of the model was tested and evaluated in separate patient materials originating from two Finnish breast cancer centers. RESULTS: The designed model comprising immunoexpressions of Securin, Separase and Cdk1 identified 8.4-fold increased risk of breast cancer mortality (p < 0.0001). A survival difference exceeding 15 years was observed between the majority (> 75%) of patients resulting with favorable as opposed to unfavorable outcome of the model. Along with nodal status, the model showed independent prognostic impact for all breast carcinomas and for subgroups of luminal, N+ and N- disease. CONCLUSIONS: The impact of the proposed prognostic model in predicting breast cancer survival was comparable to nodal status. However, the model provided additional information in N- breast carcinoma in identifying patients with aggressive course of disease, potentially in need of adjuvant treatments. Concerning N+, in turn, the model could provide evidence for withholding chemotherapy from patients with favorable outcome.

Budding yeast complete DNA synthesis after chromosome segregation begins.

To faithfully transmit genetic information, cells must replicate their entire genome before division. This is thought to be ensured by the temporal separation of replication and chromosome segregation. Here we show that in 20-40% of unperturbed yeast cells, DNA synthesis continues during anaphase, late in mitosis. High cyclin-Cdk activity inhibits DNA synthesis in metaphase, and the decrease in cyclin-Cdk activity during mitotic exit allows DNA synthesis to finish at subtelomeric and some difficult-to-replicate regions. DNA synthesis during late mitosis correlates with elevated mutation rates at subtelomeric regions, including copy number variation. Thus, yeast cells temporally overlap DNA synthesis and chromosome segregation during normal growth, possibly allowing cells to maximize population-level growth rate while simultaneously exploring greater genetic space.

The Aurora B specificity switch is required to protect from non-disjunction at the metaphase/anaphase transition.

The Aurora B abscission checkpoint delays cytokinesis until resolution of DNA trapped in the cleavage furrow. This process involves PKCε phosphorylation of Aurora B S227. Assessing if this PKCε-Aurora B module provides a more widely exploited genome-protective control for the cell cycle, we show Aurora B phosphorylation at S227 by PKCε also occurs during mitosis. Expression of Aurora B S227A phenocopies inhibition of PKCε in by-passing the delay and resolution at anaphase entry that is associated with non-disjunction and catenation of sister chromatids. Implementation of this anaphase delay is reflected in PKCε activation following cell cycle dependent cleavage by caspase 7; knock-down of caspase 7 phenocopies PKCε loss, in a manner rescued by ectopically expressing/generating a free PKCε catalytic domain. Molecular dynamics indicates that Aurora B S227 phosphorylation induces conformational changes and this manifests in a profound switch in specificity towards S29 TopoIIα phosphorylation, a response necessary for catenation resolution during mitosis.

Cohesin Removal Reprograms Gene Expression upon Mitotic Entry.

As cells enter mitosis, the genome is restructured to facilitate chromosome segregation, accompanied by dramatic changes in gene expression. However, the mechanisms that underlie mitotic transcriptional regulation are unclear. In contrast to transcribed genes, centromere regions retain transcriptionally active RNA polymerase II (Pol II) in mitosis. Here, we demonstrate that chromatin-bound cohesin is necessary to retain elongating Pol II at centromeres. We find that WAPL-mediated removal of cohesin from chromosome arms during prophase is required for the dissociation of Pol II and nascent transcripts, and failure of this process dramatically alters mitotic gene expression. Removal of cohesin/Pol II from chromosome arms in prophase is important for accurate chromosome segregation and normal activation of gene expression in G1. We propose that prophase cohesin removal is a key step in reprogramming gene expression as cells transition from G2 through mitosis to G1.

CDC6 regulates both G2/M transition and metaphase-to-anaphase transition during the first meiosis of mouse oocytes.

Cell division cycle protein, CDC6, is essential for the initiation of DNA replication. CDC6 was recently shown to inhibit the microtubule-organizing activity of the centrosome. Here, we show that CDC6 is localized to the spindle from pro-metaphase I (MI) to MII stages of oocytes, and it plays important roles at two critical steps of oocyte meiotic maturation. CDC6 depletion facilitated the G2/M transition (germinal vesicle breakdown [GVBD]) through regulation of Cdh1 and cyclin B1 expression and CDK1 (CDC2) phosphorylation in a GVBD-inhibiting culture system containing milrinone. Furthermore, GVBD was significantly decreased after knockdown of cyclin B1 in CDC6-depleted oocytes, indicating that the effect of CDC6 loss on GVBD stimulation was mediated, at least in part, by raising cyclin B1. Knockdown of CDC6 also caused abnormal localization of γ-tubulin, resulting in defective spindles, misaligned chromosomes, cyclin B1 accumulation, and spindle assembly checkpoint (SAC) activation, leading to significant pro-MI/MI arrest and PB1 extrusion failure. These phenotypes were also confirmed by time-lapse live cell imaging analysis. The results indicate that CDC6 is indispensable for maintaining G2 arrest of meiosis and functions in G2/M checkpoint regulation in mouse oocytes. Moreover, CDC6 is also a key player regulating meiotic spindle assembly and metaphase-to-anaphase transition in meiotic oocytes.

Autophagy of an Amyloid-like Translational Repressor Regulates Meiotic Exit.

We explored the potential for autophagy to regulate budding yeast meiosis. Following pre-meiotic DNA replication, we blocked autophagy by chemical inhibition of Atg1 kinase or engineered degradation of Atg14 and observed homologous chromosome segregation followed by sister chromatid separation; cells then underwent additional rounds of spindle formation and disassembly without DNA re-replication, leading to aberrant chromosome segregation. Analysis of cell-cycle regulators revealed that autophagy inhibition prevents meiosis II-specific expression of Clb3 and leads to the aberrant persistence of Clb1 and Cdc5, two substrates of a meiotic ubiquitin ligase activated by Ama1. Lastly, we found that during meiosis II, autophagy degrades Rim4, an amyloid-like translational repressor whose timed clearance regulates protein production from its mRNA targets, which include CLB3 and AMA1. Strikingly, engineered Clb3 or Ama1 production restored meiotic termination in the absence of autophagy. Thus, autophagy destroys a master regulator of meiotic gene expression to enable irreversible meiotic exit.

The role of phosphorylation in the elasticity of the tethers that connect telomeres of separating anaphase chromosomes.

Elastic tethers, connecting telomeres of all separating anaphase chromosome pairs, lose elasticity when they lengthen during anaphase. Treatment with phosphatase inhibitor CalyculinA causes anaphase chromosomes to move backwards after they reach the poles, suggesting that dephosphorylation causes loss of tether elasticity. We added 50nM CalyculinA to living anaphase crane-fly spermatocytes with different length tethers. When tethers were short, almost all partner chromosomes moved backwards after nearing the poles. When tethers were longer, fewer chromosomes moved backwards. With yet longer tethers none moved backward. This is consistent with tether elasticity being lost by dephosphorylation. 50nM CalyculinA blocks both PP1 and PP2A. To distinguish between PP1 and PP2A we treated cells with short tethers with 50nM okadaic acid which blocks solely PP2A, or with 1µM okadaic acid which blocks both PP1 and PP2A. Only 1µM okadaic acid caused chromosomes to move backward. Thus, tether elasticity is lost because of dephosphorylation by PP1.

The anaphase-promoting complex: A key mitotic regulator associated with somatic mutations occurring in cancer.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that helps control chromosome separation and exit from mitosis in many different kinds of organisms, including yeast, flies, worms, and humans. This review represents a new perspective on the connection between APC/C subunit mutations and cancer. The complex nature of APC/C and limited mutation analysis of its subunits has made it difficult to determine the relationship of each subunit to cancer. In this work, cancer genomic data were examined to identify APC/C subunits with a greater than 5% alteration frequency in 11 representative cancers using the cBioPortal database. Using the Genetic Determinants of Cancer Patient Survival database, APC/C subunits were also studied and found to be significantly associated with poor patient prognosis in several cases. In comparing these two kinds of cancer genomics data to published large-scale genomic analyses looking for cancer driver genes, ANAPC1 and ANAPC3/CDC27 stood out as being represented in all three types of analyses. Seven other subunits were found to be associated both with >5% alteration frequency in certain cancers and being associated with an effect on cancer patient prognosis. The aim of this review is to provide new approaches for investigators conducting in vivo studies of APC/C subunits and cancer progression. In turn, a better understanding of these APC/C subunits and their role in different cancers will help scientists design drugs that are more precisely targeted to certain cancers, using APC/C mutation status as a biomarker.

First meiotic anaphase requires Cep55-dependent inhibitory cyclin-dependent kinase 1 phosphorylation.

During mitosis, anaphase is triggered by anaphase-promoting complex (APC)-mediated destruction of securin and cyclin B1, which leads to inactivation of cyclin-dependent kinase 1 (Cdk1). By regulating APC activity, the mitotic spindle assembly checkpoint (SAC) therefore has robust control over anaphase timing to prevent chromosome mis-segregation. Mammalian oocytes are prone to aneuploidy, the reasons for which remain obscure. In mitosis, Cep55 is required post-anaphase for the final steps of cytokinesis. We found that Cep55-depleted mouse oocytes progress normally through early meiosis I, but that anaphase I fails as a result of persistent Cdk1 activity. Unexpectedly, Cdk1 inactivation was compromised following Cep55 depletion, despite on-time SAC silencing and intact APC-mediated proteolysis. We found that impaired Cdk1 inactivation was caused by inadequate inhibitory Cdk1 phosphorylation consequent upon failure to suppress Cdc25 phosphatase, identifying a proteolysis-independent step necessary for anaphase I. Thus, the SAC in oocytes does not exert exclusive control over anaphase I initiation, providing new insight into vulnerability to error.

Kinesin-8 and Dis1/TOG collaborate to limit spindle elongation from prophase to anaphase A for proper chromosome segregation in fission yeast.

High-fidelity chromosome segregation relies on proper microtubule regulation. Kinesin-8 has been shown to destabilise microtubules to reduce metaphase spindle length and chromosome movements in multiple species. XMAP215/chTOG polymerases catalyse microtubule growth for spindle assembly, elongation and kinetochore-microtubule attachment. Understanding of their biochemical activity has advanced, but little work directly addresses the functionality and interplay of these conserved factors. We utilised the synthetic lethality of fission yeast kinesin-8 (Klp5-Klp6) and XMAP215/chTOG (Dis1) to study their individual and overlapping roles. We found that the non-motor kinesin-8 tailbox is essential for mitotic function; mutation compromises plus-end-directed processivity. Klp5-Klp6 induces catastrophes to control microtubule length and, surprisingly, Dis1 collaborates with kinesin-8 to slow spindle elongation. Together, they enforce a maximum spindle length for a viable metaphase-anaphase transition and limit elongation during anaphase A to prevent lagging chromatids. Our work provides mechanistic insight into how kinesin-8 negatively regulates microtubules and how this functionally overlaps with Dis1 and highlights the importance of spindle length control in mitosis.

Central-spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B.

Spindle microtubules, whose dynamics vary over time and at different locations, cooperatively drive chromosome segregation. Measurements of microtubule dynamics and spindle ultrastructure can provide insight into the behaviors of microtubules, helping elucidate the mechanism of chromosome segregation. Much work has focused on the dynamics and organization of kinetochore microtubules, that is, on the region between chromosomes and poles. In comparison, microtubules in the central-spindle region, between segregating chromosomes, have been less thoroughly characterized. Here, we report measurements of the movement of central-spindle microtubules during chromosome segregation in human mitotic spindles and Caenorhabditis elegans mitotic and female meiotic spindles. We found that these central-spindle microtubules slide apart at the same speed as chromosomes, even as chromosomes move toward spindle poles. In these systems, damaging central-spindle microtubules by laser ablation caused an immediate and complete cessation of chromosome motion, suggesting a strong coupling between central-spindle microtubules and chromosomes. Electron tomographic reconstruction revealed that the analyzed anaphase spindles all contain microtubules with both ends between segregating chromosomes. Our results provide new dynamical, functional, and ultrastructural characterizations of central-spindle microtubules during chromosome segregation in diverse spindles and suggest that central-spindle microtubules and chromosomes are strongly coupled in anaphase.

The stoichiometry of the outer kinetochore is modulated by microtubule-proximal regulatory factors.

The kinetochore is a large molecular machine that attaches chromosomes to microtubules and facilitates chromosome segregation. The kinetochore includes submodules that associate with the centromeric DNA and submodules that attach to microtubules. Additional copies of several submodules of the kinetochore are added during anaphase, including the microtubule binding module Ndc80. While the factors governing plasticity are not known, they could include regulation based on microtubule-kinetochore interactions. We report that Fin1 localizes to the microtubule-proximal edge of the kinetochore cluster during anaphase based on single-particle averaging of super-resolution images. Fin1 is required for the assembly of normal levels of Dam1 and Ndc80 submodules. Levels of Ndc80 further depend on the Dam1 microtubule binding complex. Our results suggest the stoichiometry of outer kinetochore submodules is strongly influenced by factors at the kinetochore-microtubule interface such as Fin1 and Dam1, and phosphorylation by cyclin-dependent kinase. Outer kinetochore stoichiometry is remarkably plastic and responsive to microtubule-proximal regulation.