Haploidy-linked cell proliferation defects limit larval growth in zebrafish.

Haploid larvae in non-mammalian vertebrates are lethal, with characteristic organ growth retardation collectively called 'haploid syndrome'. In contrast to mammals, whose haploid intolerance is attributed to imprinting misregulation, the cellular principle of haploidy-linked defects in non-mammalian vertebrates remains unknown. Here, we investigated cellular defects that disrupt the ontogeny of gynogenetic haploid zebrafish larvae. Unlike diploid control larvae, haploid larvae manifested unscheduled cell death at the organogenesis stage, attributed to haploidy-linked p53 upregulation. Moreover, we found that haploid larvae specifically suffered the gradual aggravation of mitotic spindle monopolarization during 1-3 days post-fertilization, causing spindle assembly checkpoint-mediated mitotic arrest throughout the entire body. High-resolution imaging revealed that this mitotic defect accompanied the haploidy-linked centrosome loss occurring concomitantly with the gradual decrease in larval cell size. Either resolution of mitotic arrest or depletion of p53 partially improved organ growth in haploid larvae. Based on these results, we propose that haploidy-linked mitotic defects and cell death are parts of critical cellular causes shared among vertebrates that limit the larval growth in the haploid state, contributing to an evolutionary constraint on allowable ploidy status in the vertebrate life cycle.

Artificial kinetochore beads establish a biorientation-like state in the spindle.

Faithful chromosome segregation requires biorientation, where the pair of kinetochores on the chromosome establish bipolar microtubule attachment. The integrity of the kinetochore, a macromolecular complex built on centromeric DNA, is required for biorientation, but components sufficient for biorientation remain unknown. Here, we show that tethering the outer kinetochore heterodimer NDC80-NUF2 to the surface of apolar microbeads establishes their biorientation-like state in mouse cells. NDC80-NUF2 microbeads align at the spindle equator and self-correct alignment errors. The alignment is associated with stable bipolar microtubule attachment and is independent of the outer kinetochore proteins SPC24-SPC25, KNL1, the Mis12 complex, inner kinetochore proteins, and Aurora. Larger microbeads align more rapidly, suggesting a size-dependent biorientation mechanism. This study demonstrates a biohybrid kinetochore design for synthetic biorientation of microscale particles in cells.

The doublecortin-family kinase ZYG-8DCLK1 regulates microtubule dynamics and motor-driven forces to promote the stability of C. elegans acentrosomal spindles.

Although centrosomes help organize spindles in most cell types, oocytes of most species lack these structures. During acentrosomal spindle assembly in C. elegans oocytes, microtubule minus ends are sorted outwards away from the chromosomes where they form poles, but then these outward forces must be balanced to form a stable bipolar structure. Simultaneously, microtubule dynamics must be precisely controlled to maintain spindle length and organization. How forces and dynamics are tuned to create a stable bipolar structure is poorly understood. Here, we have gained insight into this question through studies of ZYG-8, a conserved doublecortin-family kinase; the mammalian homolog of this microtubule-associated protein is upregulated in many cancers and has been implicated in cell division, but the mechanisms by which it functions are poorly understood. We found that ZYG-8 depletion from oocytes resulted in overelongated spindles with pole and midspindle defects. Importantly, experiments with monopolar spindles revealed that ZYG-8 depletion led to excess outward forces within the spindle and suggested a potential role for this protein in regulating the force-generating motor BMK-1/kinesin-5. Further, we found that ZYG-8 is also required for proper microtubule dynamics within the oocyte spindle and that kinase activity is required for its function during both meiosis and mitosis. Altogether, our findings reveal new roles for ZYG-8 in oocytes and provide insights into how acentrosomal spindles are stabilized to promote faithful meiosis.

Unveiling inter-embryo variability in spindle length over time: Towards quantitative phenotype analysis.

How can inter-individual variability be quantified? Measuring many features per experiment raises the question of choosing them to recapitulate high-dimensional data. Tackling this challenge on spindle elongation phenotypes, we showed that only three typical elongation patterns describe spindle elongation in C. elegans one-cell embryo. These archetypes, automatically extracted from the experimental data using principal component analysis (PCA), accounted for more than 95% of inter-individual variability of more than 1600 experiments across more than 100 different conditions. The two first archetypes were related to spindle average length and anaphasic elongation rate. The third archetype, accounting for 6% of the variability, was novel and corresponded to a transient spindle shortening in late metaphase, reminiscent of kinetochore function-defect phenotypes. Importantly, these three archetypes were robust to the choice of the dataset and were found even considering only non-treated conditions. Thus, the inter-individual differences between genetically perturbed embryos have the same underlying nature as natural inter-individual differences between wild-type embryos, independently of the temperatures. We thus propose that beyond the apparent complexity of the spindle, only three independent mechanisms account for spindle elongation, weighted differently in the various conditions. Interestingly, the spindle-length archetypes covered both metaphase and anaphase, suggesting that spindle elongation in late metaphase is sufficient to predict the late anaphase length. We validated this idea using a machine-learning approach. Finally, given amounts of these three archetypes could represent a quantitative phenotype. To take advantage of this, we set out to predict interacting genes from a seed based on the PCA coefficients. We exemplified this firstly on the role of tpxl-1 whose homolog tpx2 is involved in spindle microtubule branching, secondly the mechanism regulating metaphase length, and thirdly the central spindle players which set the length at anaphase. We found novel interactors not in public databases but supported by recent experimental publications.

The Mitotic Checkpoint Complex controls the association of Cdc20 regulatory protein with the ubiquitin ligase APC/C in mitosis.

The ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) and its regulatory protein Cdc20 play important roles in the control of different stages of mitosis. APC/C associated with Cdc20 is active and promotes metaphase-anaphase transition by targeting for degradation inhibitors of anaphase initiation. Earlier in mitosis, premature action of APC/C is prevented by the mitotic checkpoint (or spindle assembly checkpoint) system, which ensures that anaphase is not initiated until all chromosomes are properly attached to the mitotic spindle. The active mitotic checkpoint system promotes the assembly of a Mitotic Checkpoint Complex (MCC), which binds to APC/C and inhibits its activity. The interaction of MCC with APC/C is strongly enhanced by Cdc20 bound to APC/C. While the association of Cdc20 with APC/C was known to be essential for both these stages of mitosis, it was not known how Cdc20 remains bound in spite of ongoing processes, phosphorylation and ubiquitylation, that stimulate its release from APC/C. We find that MCC strongly inhibits the release of Cdc20 from APC/C by the action of mitotic protein kinase Cdk1-cyclin B. This is not due to protection from phosphorylation of specific sites in Cdc20 that affect its interaction with APC/C. Rather, MCC stabilizes the binding to APC/C of partially phosphorylated forms of Cdc20. MCC also inhibits the autoubiquitylation of APC/C-bound Cdc20 and its ubiquitylation-promoted release from APC/C. We propose that these actions of MCC to maintain Cdc20 bound to APC/C in mitosis are essential for the control of mitosis during active mitotic checkpoint and in subsequent anaphase initiation.

miR-31-mediated local translation at the mitotic spindle is important for early development.

miR-31 is a highly conserved microRNA that plays crucial roles in cell proliferation, migration and differentiation. We discovered that miR-31 and some of its validated targets are enriched on the mitotic spindle of the dividing sea urchin embryo and mammalian cells. Using the sea urchin embryo, we found that miR-31 inhibition led to developmental delay correlated with increased cytoskeletal and chromosomal defects. We identified miR-31 to directly suppress several actin remodeling transcripts, including ß-actin, Gelsolin, Rab35 and Fascin. De novo translation of Fascin occurs at the mitotic spindle of sea urchin embryos and mammalian cells. Importantly, miR-31 inhibition leads to a significant a increase of newly translated Fascin at the spindle of dividing sea urchin embryos. Forced ectopic localization of Fascin transcripts to the cell membrane and translation led to significant developmental and chromosomal segregation defects, highlighting the importance of the regulation of local translation by miR-31 at the mitotic spindle to ensure proper cell division. Furthermore, miR-31-mediated post-transcriptional regulation at the mitotic spindle may be an evolutionarily conserved regulatory paradigm of mitosis.

Long-range repulsion between chromosomes in mammalian oocyte spindles.

During eukaryotic cell division, a microtubule-based structure called the spindle exerts forces on chromosomes. The best-studied spindle forces, including those responsible for the separation of sister chromatids, are directed parallel to the spindle's long axis. By contrast, little is known about forces perpendicular to the spindle axis, which determine the metaphase plate configuration and thus the location of chromosomes in the subsequent nucleus. Using live-cell microscopy, we find that metaphase chromosomes are spatially anti-correlated in mouse oocyte spindles, evidence of previously unknown long-range forces acting perpendicular to the spindle axis. We explain this observation by showing that the spindle's microtubule network behaves as a nematic liquid crystal and that deformation of the nematic field around embedded chromosomes causes long-range repulsion between them.

Thiostrepton induces spindle abnormalities and enhances Taxol cytotoxicity in MDA-MB-231 cells.

BACKGROUND: Thiostrepton (TST) is a known inhibitor of the transcription factor Forkhead box M1 (FoxM1) and inducer of heat shock response (HSR) and autophagy. TST thus may be one potential candidate of anticancer drugs for combination chemotherapy. METHODS AND RESULTS: Immunofluorescence staining of mitotic spindles and flow cytometry analysis revealed that TST induces mitotic spindle abnormalities, mitotic arrest, and apoptotic cell death in the MDA-MB-231 triple-negative breast cancer cell line. Interestingly, overexpression or depletion of FoxM1 in MDA-MB-231 cells did not affect TST induction of spindle abnormalities; however, TST-induced spindle defects were enhanced by inhibition of HSP70 or autophagy. Moreover, TST exhibited low affinity for tubulin and only slightly inhibited in vitro tubulin polymerization, but it severely impeded tubulin polymerization and destabilized microtubules in arrested mitotic MDA-MB-231 cells. Additionally, TST significantly enhanced Taxol cytotoxicity. TST also caused cytotoxicity and spindle abnormalities in a Taxol-resistant cell line, MDA-MB-231-T4R. CONCLUSIONS: These results suggest that, in addition to inhibiting FoxM1, TST may induce proteotoxicity and autophagy to disrupt cellular tubulin polymerization, and this mechanism might account for its antimitotic effects, enhancement of Taxol anticancer effects, and ability to overcome Taxol resistance in MDA-MB-231 cells. These data further imply that TST may be useful to improve the therapeutic efficacy of Taxol.

Centrosomal Protein 55 Regulates Chromosomal Instability in Cancer Cells by Controlling Microtubule Dynamics.

Centrosomal Protein 55 (CEP55) exhibits various oncogenic activities; it regulates the PI3K-Akt-pathway, midbody abscission, and chromosomal instability (CIN) in cancer cells. Here, we analyzed the mechanism of how CEP55 controls CIN in ovarian and breast cancer (OvCa) cells. Down-regulation of CEP55 reduced CIN in all cell lines analyzed, and CEP55 depletion decreased spindle microtubule (MT)-stability in OvCa cells. Moreover, recombinant CEP55 accelerated MT-polymerization and attenuated cold-induced MT-depolymerization. To analyze a potential relationship between CEP55-controlled CIN and its impact on MT-stability, we identified the CEP55 MT-binding peptides inside the CEP55 protein. Thereafter, a mutant with deficient MT-binding activity was re-expressed in CEP55-depleted OvCa cells and we could show that this mutant did not restore reduced CIN in CEP55-depleted cells. This finding strongly indicates that CEP55 regulates CIN by controlling MT dynamics.

Sumo-regulatory SENP2 controls the homeostatic squamous mitosis-differentiation checkpoint.

Squamous or epidermoid cancer arises in stratified epithelia but also is frequent in the non-epidermoid epithelium of the lung by unclear mechanisms. A poorly studied mitotic checkpoint drives epithelial cells bearing irreparable genetic damage into epidermoid differentiation. We performed an RNA-sequencing gene search to target unknown regulators of this response and selected the SUMO regulatory protein SENP2. Alterations of SENP2 expression have been associated with some types of cancer. We found the protein to be strongly localised to mitotic spindles of freshly isolated human epidermal cells. Primary cells rapidly differentiated after silencing SENP2 with specific shRNAs. Loss of SENP2 produced in synchronised epithelial cells delays in mitotic entry and exit and defects in chromosomal alignment. The results altogether strongly argue for an essential role of SENP2 in the mitotic spindle and hence in controlling differentiation. In addition, the expression of SENP2 displayed an inverse correlation with the immuno-checkpoint biomarker PD-L1 in a pilot collection of aggressive lung carcinomas. Consistently, metastatic head and neck cancer cells that do not respond to the mitosis-differentiation checkpoint were resistant to depletion of SENP2. Our results identify SENP2 as a novel regulator of the epithelial mitosis-differentiation checkpoint and a potential biomarker in epithelial cancer.

Spindle assembly checkpoint-dependent mitotic delay is required for cell division in absence of centrosomes.

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.

Kinesin-14 HSET and KlpA are non-processive microtubule motors with load-dependent power strokes.

Accurate chromosome segregation during cell division relies on coordinated actions of microtubule (MT)-based motor proteins in the mitotic spindle. Kinesin-14 motors play vital roles in spindle assembly and maintenance by crosslinking antiparallel MTs at the spindle midzone and anchoring spindle MTs' minus ends at the poles. In this study, we investigate the force generation and motility of the Kinesin-14 motors HSET and KlpA. Our findings reveal that both motors are non-processive, producing single load-dependent power strokes per MT encounter, with estimated load-free power strokes of ~30 and ~35 nm, respectively. Each homodimeric motor generates forces of ~0.5 pN, but when assembled in teams, they cooperate to generate forces of 1 pN or more. Notably, the cooperative activity among multiple motors leads to increased MT-sliding velocities. These results quantitatively elucidate the structure-function relationship of Kinesin-14 motors and underscore the significance of cooperative behavior in their cellular functions.

Ase1 selectively increases the lifetime of antiparallel microtubule overlaps.

Microtubules (MTs) are dynamically unstable polar biopolymers switching between periods of polymerization and depolymerization, with the switch from the polymerization to the depolymerization phase termed catastrophe and the reverse transition termed rescue.1 In presence of MT-crosslinking proteins, MTs form parallel or anti-parallel overlaps and self-assemble reversibly into complex networks, such as the mitotic spindle. Differential regulation of MT dynamics in parallel and anti-parallel overlaps is critical for the self-assembly of these networks.2,3 Diffusible MT crosslinkers of the Ase1/MAP65/PRC1 family associate with different affinities to parallel and antiparallel MT overlaps, providing a basis for this differential regulation.4,5,6,7,8,9,10,11 Ase1/MAP65/PRC1 family proteins directly affect MT dynamics12 and recruit other proteins that locally alter MT dynamics, such as CLASP or kinesin-4.7,13,14,15,16 However, how Ase1 differentially regulates MT stability in parallel and antiparallel bundles is unknown. Here, we show that Ase1 selectively promotes antiparallel MT overlap longevity by slowing down the depolymerization velocity and by increasing the rescue frequency, specifically in antiparallelly crosslinked MTs. At the retracting ends of depolymerizing MTs, concomitant with slower depolymerization, we observe retention and accumulation of Ase1 between crosslinked MTs and on isolated MTs. We hypothesize that the ability of Ase1 to reduce the dissociation of tubulin subunits is sufficient to promote its enrichment at MT ends. A mathematical model built on this idea shows good agreement with the experiments. We propose that differential regulation of MT dynamics by Ase1 contributes to mitotic spindle assembly by specifically stabilizing antiparallel overlaps, compared to parallel overlaps or isolated MTs.

An Arabidopsis Kinesin-14D motor is associated with midzone microtubules for spindle morphogenesis.

The acentrosomal spindle apparatus has kinetochore fibers organized and converged toward opposite poles; however, mechanisms underlying the organization of these microtubule fibers into an orchestrated bipolar array were largely unknown. Kinesin-14D is one of the four classes of Kinesin-14 motors that are conserved from green algae to flowering plants. In Arabidopsis thaliana, three Kinesin-14D members displayed distinct cell cycle-dependent localization patterns on spindle microtubules in mitosis. Notably, Kinesin-14D1 was enriched on the midzone microtubules of prophase and mitotic spindles and later persisted in the spindle and phragmoplast midzones. The kinesin-14d1 mutant had kinetochore fibers disengaged from each other during mitosis and exhibited hypersensitivity to the microtubule-depolymerizing herbicide oryzalin. Oryzalin-treated kinesin-14d1 mutant cells had kinetochore fibers tangled together in collapsed spindle microtubule arrays. Kinesin-14D1, unlike other Kinesin-14 motors, showed slow microtubule plus end-directed motility, and its localization and function were dependent on its motor activity and the novel malectin-like domain. Our findings revealed a Kinesin-14D1-dependent mechanism that employs interpolar microtubules to regulate the organization of kinetochore fibers for acentrosomal spindle morphogenesis.

Balancing Plk1 activity levels: The secret of synchrony between the cell and the centrosome cycle.

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.

Spindle architecture constrains karyotype evolution.

The eukaryotic cell division machinery must rapidly and reproducibly duplicate and partition the cell's chromosomes in a carefully coordinated process. However, chromosome numbers vary dramatically between genomes, even on short evolutionary timescales. We sought to understand how the mitotic machinery senses and responds to karyotypic changes by using a series of budding yeast strains in which the native chromosomes have been successively fused. Using a combination of cell biological profiling, genetic engineering and experimental evolution, we show that chromosome fusions are well tolerated up until a critical point. Cells with fewer than five centromeres lack the necessary number of kinetochore-microtubule attachments needed to counter outward forces in the metaphase spindle, triggering the spindle assembly checkpoint and prolonging metaphase. Our findings demonstrate that spindle architecture is a constraining factor for karyotype evolution.

Di(2-ethylexyl) phthalate and chromosomal damage: Insight on aneugenicity from the cytochalasin-block micronucleus assay.

Bis(2-ethylhexyl) phthalate is the most abundant phthalate used as plasticizer to soften plastics and polymers included in medical devices. Human and environmental exposure may occur because DEHP is not chemically bound to plastics and can easily leach out of the materials. This phthalate is classified as reproductive toxicant and possible carcinogen to humans. The genotoxic potential has still to be clarified, but there are indications suggesting that DEHP may have aneugenic effects. To further investigate DEHP genotoxicity, the cytochalasin-block micronucleus assay was applied and combined with the CREST staining to characterise micronucleus content and gain insights on its genotoxic mode of action. Chromosomal damage was also analysed in metaphase and ana-telophase cells and the morphology of the mitotic spindle was investigated to evaluate the possible involvement of this cellular apparatus as a target of DEHP. Our findings indicated that DEHP induced a statistically significant increase in the frequency of micronuclei as well as in the frequency of CREST-positive micronuclei. Consistently, disturbance of chromosome segregation and induction of numerical chromosome changes were observed together with changes in spindle morphology, formation of multipolar spindles and alteration of the microtubule network. Experiments performed without metabolic activation demonstrated a direct action of DEHP on chromosome segregation not mediated by its metabolites. In conclusion, there is consistent evidence for an aneugenic activity of DEHP. A thresholded genotoxic activity was identified for DEHP, disclosing possible implications for risk assessment.

Mitosis: An expanded view of mitotic mechanisms that arose in evolution.

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.

Chromosome biology: Too big to fail.

Spindles are microtubule-based machines that segregate chromosomes during cell division. Spindle morphology and dynamics are malleable based on forces within the spindle, and a new study reveals the extreme plasticity of the Saccharomyces cerevisiae spindle to adapt and segregate engineered mega-chromosomes.