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Microtubule (MT) filaments, composed of α/β-tubulin dimers, are fundamental to cellular architecture, function and organismal development. They are nucleated from MT organizing centers by the evolutionarily conserved γ-tubulin ring complex (γTuRC). However, the molecular mechanism of nucleation remains elusive. Here we used cryo-electron tomography to determine the structure of the native γTuRC capping the minus end of a MT in the context of enriched budding yeast spindles. In our structure, γTuRC presents a ring of γ-tubulin subunits to seed nucleation of exclusively 13-protofilament MTs, adopting an active closed conformation to function as a perfect geometric template for MT nucleation. Our cryo-electron tomography reconstruction revealed that a coiled-coil protein staples the first row of α/β-tubulin of the MT to alternating positions along the γ-tubulin ring of γTuRC. This positioning of α/β-tubulin onto γTuRC suggests a role for the coiled-coil protein in augmenting γTuRC-mediated MT nucleation. Based on our results, we describe a molecular model for budding yeast γTuRC activation and MT nucleation., (© 2024. The Author(s).)

During cell division, kinetochore microtubules (KMTs) provide a physical linkage between the chromosomes and the rest of the spindle. KMTs in mammalian cells are organized into bundles, so-called kinetochore-fibers (k-fibers), but the ultrastructure of these fibers is currently not well characterized. Here, we show by large-scale electron tomography that each k-fiber in HeLa cells in metaphase is composed of approximately nine KMTs, only half of which reach the spindle pole. Our comprehensive reconstructions allowed us to analyze the three-dimensional (3D) morphology of k-fibers and their surrounding MTs in detail. We found that k-fibers exhibit remarkable variation in circumference and KMT density along their length, with the pole-proximal side showing a broadening. Extending our structural analysis then to other MTs in the spindle, we further observed that the association of KMTs with non-KMTs predominantly occurs in the spindle pole regions. Our 3D reconstructions have implications for KMT growth and k-fiber self-organization models as covered in a parallel publication applying complementary live-cell imaging in combination with biophysical modeling (Conway et al., 2022). Finally, we also introduce a new visualization tool allowing an interactive display of our 3D spindle data that will serve as a resource for further structural studies on mitosis in human cells., Competing Interests: RK, GF, WC, DB, DN, TM No competing interests declared, (© 2022, Kiewisz et al.)

Sirtuins play an important role in female mammalian reproductive function, participating in folliculogenesis and oocyte maturation. Studies exploring the consequences of inhibition/deletion of a specific sirtuin (SIRT) have demonstrated a deleterious effect on follicular growth, oocyte maturation, fertilization rates and embryo development, suggesting that sirtuins must have a relevant role in these processes. However, the exact mechanisms behind sirtuin function are still unclear. Most of the knowledge currently available derives from mouse studies and the literature is scarce in other species. So far, there is insufficient information about the subcellular localization of sirtuins during bovine meiosis, which would contribute to understanding the role and participation of sirtuins in the process of oocyte maturation, due to the close relation between location and function. Using in vitro maturation (IVM) of bovine oocytes we comprehensively documented and illustrated the subcellular localization pattern and distribution of SIRT1, 2 and 3 during meiotic progression. Moreover, we also detailed and quantified the colocalization of those sirtuins with the meiotic spindle, from the germinal-vesicle (GV)-stage until the Metaphase-II (MII)-stage. Our study demonstrated an increase in the expression of SIRT1, 2 and 3 during in vitro oocyte maturation and, for the first time, colocalization of SIRT1, 2 and 3 with both metaphase-I and metaphase-II spindles. These findings suggest that all three sirtuins may have a role in meiotic spindle assembly and microtubule dynamics in the bovine model. In addition, we have demonstrated the nuclear presence of SIRT1 and SIRT2 in the GV-stage. The apparent perinucleolar location of SIRT2 suggests that SIRT2 may shuttle into the nucleus at the GV-stage to regulate heterochromatin. This study reinforces the value of sirtuins during in vitro bovine meiotic progression and indicates potential molecular targets to improve maturation rates and embryo development., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Published by Elsevier Inc.)

Human oocytes are prone to assembling meiotic spindles with unstable poles, which can favor aneuploidy in human eggs. The underlying causes of spindle instability are unknown. We found that NUMA (nuclear mitotic apparatus protein)-mediated clustering of microtubule minus ends focused the spindle poles in human, bovine, and porcine oocytes and in mouse oocytes depleted of acentriolar microtubule-organizing centers (aMTOCs). However, unlike human oocytes, bovine, porcine, and aMTOC-free mouse oocytes have stable spindles. We identified the molecular motor KIFC1 (kinesin superfamily protein C1) as a spindle-stabilizing protein that is deficient in human oocytes. Depletion of KIFC1 recapitulated spindle instability in bovine and aMTOC-free mouse oocytes, and the introduction of exogenous KIFC1 rescued spindle instability in human oocytes. Thus, the deficiency of KIFC1 contributes to spindle instability in human oocytes.

The centrosome of Dictyostelium amoebae contains no centrioles and consists of a cylindrical layered core structure surrounded by a corona harboring microtubule-nucleating γ-tubulin complexes. It is the major centrosomal model beyond animals and yeasts. Proteomics, protein interaction studies by BioID and superresolution microscopy methods led to considerable progress in our understanding of the composition, structure and function of this centrosome type. We discuss all currently known components of the Dictyostelium centrosome in comparison to other centrosomes of animals and yeasts.

Aurora A is a serine/threonine kinase essential for mitotic entry and spindle assembly. Recent molecular studies have revealed the existence of multiple, distinct mechanisms of Aurora A activation, each occurring at specific subcellular locations, optimized for cellular context, and primed by signaling events including phosphorylation and oxidation., (© 2021 Tavernier et al.)

DNA loop extrusion by condensins and decatenation by DNA topoisomerase II (topo II) are thought to drive mitotic chromosome compaction and individualization. Here, we reveal that the linker histone H1.8 antagonizes condensins and topo II to shape mitotic chromosome organization. In vitro chromatin reconstitution experiments demonstrate that H1.8 inhibits binding of condensins and topo II to nucleosome arrays. Accordingly, H1.8 depletion in Xenopus egg extracts increased condensins and topo II levels on mitotic chromatin. Chromosome morphology and Hi-C analyses suggest that H1.8 depletion makes chromosomes thinner and longer through shortening the average loop size and reducing the DNA amount in each layer of mitotic loops. Furthermore, excess loading of condensins and topo II to chromosomes by H1.8 depletion causes hyper-chromosome individualization and dispersion. We propose that condensins and topo II are essential for chromosome individualization, but their functions are tuned by the linker histone to keep chromosomes together until anaphase., Competing Interests: PC, BD, EC, AV, HF none, JD Reviewing editor, eLife, (© 2021, Choppakatla et al.)

This study investigated the effect of the antioxidant dieckol, a component of Ecklonia cava, on maturation and developmental competence of porcine oocytes exposed to oxidative stress in vitro. Oocytes were matured in in vitro maturation (IVM) medium containing various concentrations of dieckol. The blastocyst formation rate was highest in the 0.5 μM dieckol-treated (0.5 DEK) group. The reactive oxygen species level was decreased, and the level of glutathione and expression of antioxidant genes (NFE2L, SOD1, and SOD2) at metaphase II were increased in the 0.5 DEK group. Abnormal spindle organization and chromosome misalignment were prevented in the 0.5 DEK group. Expression of maternal markers (CCNB1 and MOS) and activity of p44/42 mitogen-activated protein kinase were increased in the 0.5 DEK group. After parthenogenetic activation, the total number of cells per blastocyst was increased and the percentage of apoptotic cells was decreased in the 0.5 DEK group. Expression of development-related genes (CX45, CDX2, POU5F1, and NANOG), antiapoptotic genes (BCL2L1 and BIRC5), and a proapoptotic gene (CASP3) were altered in the 0.5 DEK group. These results indicate that the antioxidant dieckol improves IVM and subsequent development of porcine oocytes and can be used to improve the quality of oocytes under peroxidation experimental conditions., (© 2021 Wiley Periodicals LLC.)

Dysfunction of splicing factors often result in abnormal cell differentiation and apoptosis, especially in neural tissues. Mutations in pre-mRNAs processing factor 31 (PRPF31) cause autosomal dominant retinitis pigmentosa, a progressive retinal degeneration disease. The transcriptome-wide splicing events specifically regulated by PRPF31 and their biological roles in the development and maintenance of retina are still unclear. Here, we showed that the differentiation and viability of retinal progenitor cells (RPCs) are severely perturbed in prpf31 knockout zebrafish when compared with other tissues at an early embryonic stage. At the cellular level, significant mitotic arrest and DNA damage were observed. These defects could be rescued by the wild-type human PRPF31 rather than the disease-associated mutants. Further bioinformatic analysis and experimental verification uncovered that Prpf31 deletion predominantly causes the skipping of exons with a weak 5' splicing site. Moreover, genes necessary for DNA repair and mitotic progression are most enriched among the differentially spliced events, which may explain the cellular and tissular defects in prpf31 mutant retinas. This is the first time that Prpf31 is demonstrated to be essential for the survival and differentiation of RPCs during retinal neurogenesis by specifically modulating the alternative splicing of genes involved in DNA repair and mitosis., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The effects of prostaglandin F2α on the cytoskeleton and membrane organelles of oocytes was investigated by culturing ovulated mouse oocytes in its presence (50 or 100 ng/ml) for 3 h. Tubulin, fibrillar actin, membranes and chromatin were visualized by specific antibodies, phalloidin, lipophilic dye DiOC6 and Hoechst 33342, respectively. Control oocytes were characterized by a meiotic spindle with chromosomes aligned at its equator, and a cortical layer of microfilaments with an actin cap. Intracellular membranes were localized mostly in the central region in metaphase I and in a broader volume, but still excluding the cell periphery, in metaphase II, and were slightly concentrated around the chromosomes. In oocytes treated with 50 ng/ml prostaglandin, cortical actin staining was diminished, the membrane distribution was clustered, and chromosomes showed signs of misalignment despite the apparently preserved spindle. In cells treated with 100 ng/ml prostaglandin, both the spindle and the actin cortex had degenerated or disappeared as microscopic objects. Metaphase plates were on average broader and more disorganized than in the 50 ng/ml group, and the distribution of membrane organelles had become uniform. These effects, to our knowledge observed for the first time, did not require presence of the cumulus during the incubation. They could be regarded as acceleration of the oocyte postovulatory aging, in which cytoskeletal deterioration seemed to have a leading role.

During mitosis microtubules self-organize to form a bipolar mitotic spindle structure, which positions the sister chromatids on the spindle mid-plane and separates them afterwards. Previous studies have identified many spindle associated proteins. Yet, we do not fully understand how these nanoscopic proteins lead to force generation through interactions of individual microtubules, motor proteins and chromosomes, and how a large number of these local interactions ultimately determine the structure and mechanics of the spindle in micron scale. Here we review the current understanding and open questions related to the structure and mechanics of the mitotic spindle. We then discuss how a combination of electron microscopy and computational modeling can be used to tackle some of these open questions., (Published by Elsevier Ltd.)

Cell division without growth results in progressive cell size reductions during early embryonic development. How do the sizes of intracellular structures and organelles scale with cell size and what are the functional implications of such scaling relationships? Model organisms, in particular Caenorhabditis elegans worms, Drosophila melanogaster flies, Xenopus laevis frogs, and Mus musculus mice, have provided insights into developmental size scaling of the nucleus, mitotic spindle, and chromosomes. Nuclear size is regulated by nucleocytoplasmic transport, nuclear envelope proteins, and the cytoskeleton. Regulators of microtubule dynamics and chromatin compaction modulate spindle and mitotic chromosome size scaling, respectively. Developmental scaling relationships for membrane-bound organelles, like the endoplasmic reticulum, Golgi, mitochondria, and lysosomes, have been less studied, although new imaging approaches promise to rectify this deficiency. While models that invoke limiting components and dynamic regulation of assembly and disassembly can account for some size scaling relationships in early embryos, it will be exciting to investigate the contribution of newer concepts in cell biology such as phase separation and interorganellar contacts. With a growing understanding of the underlying mechanisms of organelle size scaling, future studies promise to uncover the significance of proper scaling for cell function and embryonic development, as well as how aberrant scaling contributes to disease. This article is categorized under: Establishment of Spatial and Temporal Patterns > Regulation of Size, Proportion, and Timing Early Embryonic Development > Fertilization to Gastrulation Comparative Development and Evolution > Model Systems., (© 2020 Wiley Periodicals, Inc.)

The cAMP-dependent protein kinase Pka1 is known as a regulator of glycogenesis, transition into meiosis, proper chromosome segregation, and stress responses in Schizosaccharomyces pombe . We demonstrated that both the cAMP/PKA pathway and glucose limitation play roles in appropriate spindle formation. Overexpression of Mal3 (1-308), an EB1 family protein, caused growth defects, increased 4C DNA content, and induced monopolar spindle formation. Overproduction of a high-affinity microtubule binding mutant (Q89R) and a recombinant protein possessing the CH and EB1 domains (1-241) both resulted in more severe phenotypes than Mal3 (1-308). Loss of functional Pka1 and glucose limitation rescued the phenotypes of Mal3-overexpressing cells, whereas deletion of Tor1 or Ssp2 did not. Growth defects and monopolar spindle formation in a kinesin-5 mutant, cut7-446 , was partially rescued by pka1 deletion or glucose limitation. These findings suggest that Pka1 and glucose limitation regulate proper spindle formation in Mal3-overexpressing cells and the cut7-446 mutant.

Error-free cell division depends on the accurate assembly of the spindle midzone from dynamic spindle microtubules to ensure chromatid segregation during metaphase-anaphase transition. However, the mechanism underlying the key transition from the mitotic spindle to central spindle before anaphase onset remains elusive. Given the prevalence of chromosome instability phenotype in gastric tumorigenesis, we developed a strategy to model context-dependent cell division using a combination of light sheet microscope and 3D gastric organoids. Light sheet microscopic image analyses of 3D organoids showed that CENP-E inhibited cells undergoing aberrant metaphase-anaphase transition and exhibiting chromosome segregation errors during mitosis. High-resolution real-time imaging analyses of 2D cell culture revealed that CENP-E inhibited cells undergoing central spindle splitting and chromosome instability phenotype. Using biotinylated syntelin as an affinity matrix, we found that CENP-E forms a complex with PRC1 in mitotic cells. Chemical inhibition of CENP-E in metaphase by syntelin prevented accurate central spindle assembly by perturbing temporal assembly of PRC1 to the midzone. Thus, CENP-E-mediated PRC1 assembly to the central spindle constitutes a temporal switch to organize dynamic kinetochore microtubules into stable midzone arrays. These findings reveal a previously uncharacterized role of CENP-E in temporal control of central spindle assembly. Since CENP-E is absent from yeast, we reasoned that metazoans evolved an elaborate central spindle organization machinery to ensure accurate sister chromatid segregation during anaphase and cytokinesis., (© The Author(s) (2019). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.)

The Aurora B chromosomal passenger complex (CPC) is a conserved regulator of mitosis. Its functions require localization first to the chromosome arms and then centromeres in mitosis and subsequently the central spindle in anaphase. Here, we analyze the requirements for core CPC subunits, survivin and INCENP, and the mitotic kinesin-like protein 2 (MKLP2) in targeting to these distinct localizations. Centromere recruitment of the CPC requires interaction of survivin with histone H3 phosphorylated at threonine 3, and we provide a complete structure of this assembly. Furthermore, we show that the INCENP RRKKRR-motif is required for both centromeric localization of the CPC in metaphase and MKLP2-dependent transport in anaphase. MKLP2 and DNA bind competitively to this motif, and INCENP T59 phosphorylation acts as a switch preventing MKLP2 binding in metaphase. In anaphase, CPC binding promotes the microtubule-dependent ATPase activity of MKLP2. These results explain how centromere targeting of the CPC in mitosis is coupled to its movement to the central spindle in anaphase., (© 2020 Serena et al.)

CRISPR-Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences into human organoids is lacking robust knock-in approaches. Here, we describe CRISPR-Cas9-mediated homology-independent organoid transgenesis (CRISPR-HOT), which enables efficient generation of knock-in human organoids representing different tissues. CRISPR-HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences into desired loci, without the necessity to inactivate TP53 in untransformed cells, which was previously used to increase HDR-mediated knock-in. CRISPR-HOT was used to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter-in which the mitotic spindle was labelled by endogenously tagged tubulin and the cell membrane by endogenously tagged E-cadherin-uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed that TP53 is involved in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR-HOT simplifies genome editing in human organoids.

Objective: To evaluate oocyte meiotic spindle (OMS) morphology at intracytoplasmic sperm injection (ICSI) as a predictor of blastocyst ploidy and whether OMS morphology could aid standard morphology-based blastocyst selection., Design: Prospective cohort study., Setting: In vitro fertilization clinic., Patient(s): Patients undergoing ICSI cycles with an intention to perform preimplantation genetic testing for aneuploidy (PGT-A) from October 2014 to December 2017., Intervention(s): The OMS was visualized with the use of polarized light microscopy at the time of ICSI and the morphology classified as normal, dysmorphic, translucent, not visible, or in telophase. Blastocyst biopsy for PGT-A was performed on embryos with suitable development., Main Outcome Measure(s): The association of OMS morphology with the resulting blastocyst ploidy was evaluated on an "intention-to-treat" (ITT) and an "as-treated analysis" (ATA) basis., Result(s): The morphology of 2,056 OMSs were classified. A strong association of OMS morphology with fertilization, cleavage to at least 6 cells on day 3, and good/top-quality blastocyst formation was present. Normal OMS was positively associated with blastocyst euploidy compared with all other OMS types combined, per either ITT or ATA. Even after controlling for female age, blastocyst quality, and developmental stage, the presence of a normal OMS was strongly associated with the probability of blastocyst euploidy., Conclusion(s): OMS morphology is a predictive marker of blastocyst ploidy and can potentially aid standard morphology-based blastocyst selection., (Copyright © 2019 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

During organogenesis, precise control of spindle orientation balances proliferation and differentiation. In the developing murine epidermis, planar and perpendicular divisions yield symmetric and asymmetric fate outcomes, respectively. Classically, division axis specification involves centrosome migration and spindle rotation, events occurring early in mitosis. Here, we identify a novel orientation mechanism which corrects erroneous anaphase orientations during telophase. The directionality of reorientation correlates with the maintenance or loss of basal contact by the apical daughter. While the scaffolding protein LGN is known to determine initial spindle positioning, we show that LGN also functions during telophase to reorient oblique divisions toward perpendicular. The fidelity of telophase correction also relies on the tension-sensitive adherens junction proteins vinculin, α-E-catenin, and afadin. Failure of this corrective mechanism impacts tissue architecture, as persistent oblique divisions induce precocious, sustained differentiation. The division orientation plasticity provided by telophase correction may enable progenitors to adapt to local tissue needs., Competing Interests: KL, KB, CD, DS, AB, GB, LR, SW No competing interests declared, (© 2019, Lough et al.)

Predictive biomarkers for tumor response to neoadjuvant chemotherapy are needed in breast cancer. This study investigates the predictive value of 280 genes encoding proteins that regulate microtubule assembly and function. By analyzing 3 independent multicenter randomized cohorts of breast cancer patients, we identified 17 genes that are differentially regulated in tumors achieving pathological complete response (pCR) to neoadjuvant chemotherapy. We focused on the MTUS1 gene, whose major product, ATIP3, is a microtubule-associated protein down-regulated in aggressive breast tumors. We show here that low levels of ATIP3 are associated with an increased pCR rate, pointing to ATIP3 as a predictive biomarker of breast tumor chemosensitivity. Using preclinical models of patient-derived xenografts and 3-dimensional models of breast cancer cell lines, we show that low ATIP3 levels sensitize tumors to the effects of taxanes but not DNA-damaging agents. ATIP3 silencing improves the proapoptotic effects of paclitaxel and induces mitotic abnormalities, including centrosome amplification and multipolar spindle formation, which results in chromosome missegregation leading to aneuploidy. As shown by time-lapse video microscopy, ATIP3 depletion exacerbates cytokinesis failure and mitotic death induced by low doses of paclitaxel. Our results favor a mechanism by which the combination of ATIP3 deficiency and paclitaxel treatment induces excessive aneuploidy, which in turn results in elevated cell death. Together, these studies highlight ATIP3 as an important regulator of mitotic integrity and a useful predictive biomarker for a population of chemoresistant breast cancer patients., Competing Interests: The authors declare no competing interest.

Mammalian oocytes assemble a bipolar acentriolar microtubule spindle to segregate chromosomes during asymmetric division. There is increasing evidence that actin in the spindle interior not only participates in spindle migration and positioning but also protects oocytes from chromosome segregation errors leading to aneuploidy. Here we show that actin is an integral component of the meiotic machinery that closely interacts with microtubules during all major events of human oocyte maturation from the time point of spindle assembly till polar body extrusion and metaphase arrest. With the aid of drugs selectively affecting cytoskeleton dynamics and transiently disturbing the integrity of the two cytoskeleton systems, we identify interdependent structural rearrangements indicative of a close communication between actin and microtubules as fundamental feature of human oocytes. Our data support a model of actin-microtubule interplay that is essential for bipolar spindle assembly and correct partitioning of the nuclear genome in human oocyte meiosis.

The organization of microtubules in spindles is complex and not fully understood. Here we report on current advances in generating 3D reconstructions of staged spindles by serial-section electron tomography, exemplified by the first mitotic spindle in early Caenorhabditis elegans embryo. We then review how advances in correlative light microscopy and quantitative electron tomography enable the development of theory and stochastic simulations, which describe how the microtubule organization in spindles emerges from their dynamics. We show how theory and simulations can be used to address long-standing questions in cell division research, advancing the field beyond a pure structural description of microtubules in spindles., (Published by Elsevier Ltd.)

In mitosis, the spindle assembly checkpoint (SAC) monitors the formation of microtubule-kinetochore attachments during capture of chromosomes by the mitotic spindle. Spindle assembly is complete once there are no longer any unattached kinetochores. Here, we will discuss the mechanism and key components of spindle checkpoint signalling. Unattached kinetochores bind the principal spindle checkpoint kinase monopolar spindle 1 (MPS1). MPS1 triggers the recruitment of other spindle checkpoint proteins and the formation of a soluble inhibitor of anaphase, thus preventing exit from mitosis. On microtubule attachment, kinetochores become checkpoint silent due to the actions of PP2A-B56 and PP1. This SAC responsive period has to be coordinated with mitotic spindle formation to ensure timely mitotic exit and accurate chromosome segregation. We focus on the molecular mechanisms by which the SAC permissive state is created, describing a central role for CDK1-cyclin B1 and its counteracting phosphatase PP2A-B55. Furthermore, we discuss how CDK1-cyclin B1, through its interaction with MAD1, acts as an integral component of the SAC, and actively orchestrates checkpoint signalling and thus contributes to the faithful execution of mitosis., (© 2019 Federation of European Biochemical Societies.)

The accuracy of the two sequential meiotic divisions in oocytes is essential for creating a haploid gamete with a normal chromosomal content. Here, we have analysed the 3D dynamics of chromosomes during the second meiotic division in live mouse oocytes. We find that chromosomes form stable kinetochore-microtubule attachments at the end of prometaphase II stage that are retained until anaphase II onset. Remarkably, we observe that more than 20% of the kinetochore-microtubule attachments at the metaphase II stage are merotelic or lateral. However, < 1% of all chromosomes at onset of anaphase II are found to lag at the spindle equator and < 10% of the laggards missegregate and give rise to aneuploid gametes. Our results demonstrate that aberrant kinetochore-microtubule attachments are not corrected at the metaphase stage of the second meiotic division. Thus, the accuracy of the chromosome segregation process in mouse oocytes during meiosis II is ensured by an efficient correction process acting at the anaphase stage., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Sister centromere fusion is a process unique to meiosis that promotes co-orientation of the sister kinetochores, ensuring they attach to microtubules from the same pole during metaphase I. We have found that the kinetochore protein SPC105R/KNL1 and Protein Phosphatase 1 (PP1-87B) regulate sister centromere fusion in Drosophila oocytes. The analysis of these two proteins, however, has shown that two independent mechanisms maintain sister centromere fusion. Maintenance of sister centromere fusion by SPC105R depends on Separase, suggesting cohesin proteins must be maintained at the core centromeres. In contrast, maintenance of sister centromere fusion by PP1-87B does not depend on either Separase or WAPL. Instead, PP1-87B maintains sister centromeres fusion by regulating microtubule dynamics. We demonstrate that this regulation is through antagonizing Polo kinase and BubR1, two proteins known to promote stability of kinetochore-microtubule (KT-MT) attachments, suggesting that PP1-87B maintains sister centromere fusion by inhibiting stable KT-MT attachments. Surprisingly, C(3)G, the transverse element of the synaptonemal complex (SC), is also required for centromere separation in Pp1-87B RNAi oocytes. This is evidence for a functional role of centromeric SC in the meiotic divisions, that might involve regulating microtubule dynamics. Together, we propose two mechanisms maintain co-orientation in Drosophila oocytes: one involves SPC105R to protect cohesins at sister centromeres and another involves PP1-87B to regulate spindle forces at end-on attachments., Competing Interests: The authors have declared that no competing interests exist.

Cdk1 has been found to phosphorylate the majority of its substrates in disordered regions, but some substrates maintain precise phosphosite positions over billions of years. Here, we examined the phosphoregulation of the kinesin-5, Cin8, using synthetic Cdk1-sites. We first analyzed the three native Cdk1 sites within the catalytic motor domain. Any single site conferred regulation, but to different extents. Synthetic sites were then systematically generated by single amino-acid substitutions, starting from a phosphodeficient variant of Cin8. Out of 29 synthetic Cdk1 sites, 8 disrupted function; 19 were neutral, similar to the phospho-deficient variant; and only two gave rise to phosphorylation-dependent spindle phenotypes. Of these two, one was immediately adjacent to a native Cdk1 site. Only one novel site position resulted in phospho-regulation. This site was sampled elsewhere in evolution, but the synthetic version was inefficient in S. cerevisiae . This study shows that a single phosphorylation site can modulate complex spindle dynamics, but likely requires further evolution to optimally regulate the precise reaction cycle of a mitotic motor., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Zinc plays an essential role in mammalian oocyte maturation, fertilization, and early embryogenesis, and depletion of zinc impairs cell cycle control, asymmetric division, and cytokinesis in oocyte. We report that zinc, via the actin nucleator Spire, acts as an essential regulator of the actin cytoskeleton remodeling during mouse oocyte maturation and fertilization. Depletion of zinc in the mouse oocyte impaired cortical and cytoplasmic actin formation. Spire is colocalized with zinc-containing vesicles via its zinc finger-containing Fab1, YOTB, Vac 1, EEA1 (FYVE) domain. Improper localization of Spire by zinc depletion or mutations in the FYVE domain impair cytoplasmic actin mesh formations and asymmetric division and cytokinesis of oocyte. All 3 major domains of the Spire are required for its proper localization and activity. After fertilization or parthenogenetic activation, Spire localization was dramatically altered following zinc release from the oocyte. Collectively, our data reveal novel roles for zinc in the regulation of the actin nucleator Spire by controlling its localization in mammalian oocyte.-Jo, Y.-J., Lee, I.-W., Jung, S.-M., Kwon, J., Kim, N.-H., Namgoong, S. Spire localization via zinc finger-containing domain is crucial for the asymmetric division of mouse oocyte.

In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinetochores. While kinetochore subcomplexes have been studied extensively in vitro, little is known about their in vivo structure and interactions with microtubules or their response to spindle damage. Here we combine electron cryotomography of serial cryosections with genetic and pharmacological perturbation to study the yeast chromosome segregation machinery in vivo. Each kinetochore microtubule has one (rarely, two) Dam1C/DASH outer kinetochore assemblies. Dam1C/DASH contacts the microtubule walls and does so with its flexible "bridges"; there are no contacts with the protofilaments' curved tips. In metaphase, ∼40% of the Dam1C/DASH assemblies are complete rings; the rest are partial rings. Ring completeness and binding position along the microtubule are sensitive to kinetochore attachment and tension, respectively. Our study and those of others support a model in which each kinetochore must undergo cycles of conformational change to couple microtubule depolymerization to chromosome movement., (© 2019 Ng et al.)

During asymmetric cell division, the molecular motor dynein generates cortical pulling forces that position the spindle to reflect polarity and adequately distribute cell fate determinants. In Caenorhabditis elegans embryos, despite a measured anteroposterior force imbalance, antibody staining failed to reveal dynein enrichment at the posterior cortex, suggesting a transient localization there. Dynein accumulates at the microtubule plus ends, in an EBP-2 EB -dependent manner. This accumulation, although not transporting dynein, contributes modestly to cortical forces. Most dyneins may instead diffuse to the cortex. Tracking of cortical dynein revealed two motions: one directed and the other diffusive-like, corresponding to force-generating events. Surprisingly, while dynein is not polarized at the plus ends or in the cytoplasm, diffusive-like tracks were more frequently found at the embryo posterior tip, where the forces are higher. This asymmetry depends on GPR-1/2 LGN and LIN-5 NuMA , which are enriched there. In csnk-1(RNAi) embryos, the inverse distribution of these proteins coincides with an increased frequency of diffusive-like tracks anteriorly. Importantly, dynein cortical residence time is always symmetric. We propose that the dynein-binding rate at the posterior cortex is increased, causing the polarity-reflecting force imbalance. This mechanism of control supplements the regulation of mitotic progression through the nonpolarized dynein detachment rate.

Microtubule nucleation was uncovered as a key principle of spindle assembly. However, the mechanistic details about microtubule nucleation and the organization of spindle formation and symmetry are currently being revealed. Here we describe the function of coiled-coil domain containing 61 (Ccdc61), a so far uncharacterized centrosomal protein, in spindle assembly and symmetry. Our data describe that Ccdc61 is required for spindle assembly and precise chromosome alignments in mitosis. Microtubule tip-tracking experiments in the absence of Ccdc61 reveal a clear loss of the intrinsic symmetry of microtubule tracks within the spindle. Furthermore, we show that Ccdc61 controls the centrosomal localization of centrosomal protein of 170 kDa (Cep170), a protein that was shown previously to localize to centrosomes as well as spindle microtubules and promotes microtubule organization and microtubule assembly. Interestingly, selective disruption of Ccdc61 impairs the binding between Cep170 and TANK binding kinase 1, an interaction that is required for microtubule stability. In summary, we have discovered Ccdc61 as a centrosomal protein with an important function in mitotic microtubule organization.

Accurate chromosome segregation relies on bioriented amphitelic attachments of chromosomes to microtubules of the mitotic spindle, in which sister chromatids are connected to opposite spindle poles. BUB-1 is a protein of the Spindle Assembly Checkpoint (SAC) that coordinates chromosome attachment with anaphase onset. BUB-1 is also required for accurate sister chromatid segregation independently of its SAC function, but the underlying mechanism remains unclear. Here we show that, in Caenorhabditis elegans embryos, BUB-1 accelerates the establishment of non-merotelic end-on kinetochore-microtubule attachments by recruiting the RZZ complex and its downstream partner dynein-dynactin at the kinetochore. In parallel, BUB-1 limits attachment maturation by the SKA complex. This activity opposes kinetochore-microtubule attachment stabilisation promoted by CLS-2 CLASP -dependent kinetochore-microtubule assembly. BUB-1 is therefore a SAC component that coordinates the function of multiple downstream kinetochore-associated proteins to ensure accurate chromosome segregation., Competing Interests: FE, GM, NG, JC, JD No competing interests declared, (© 2018, Edwards et al.)

Centrosomes play various critical roles in animal cells such as microtubule nucleation and stabilization, mitotic spindle morphogenesis, and spindle orientation. Land plants have lost centrosomes and yet must execute many of these functions. Recent studies have revealed the crucial roles played by morphologically distinct cytoplasmic microtubule-organizing centers (MTOCs) in initiating spindle bipolarity and maintaining spindle orientation robustness. These MTOCs resemble centrosomes in many aspects, implying an evolutionary divergence of MT-organizing structures in plants. However, their functions rely on conserved nucleation and amplification mechanisms, indicating a similarity in MT network establishment between animals and plants. Moreover, recent characterization of a plant-specific MT minus-end tracking protein suggests that plants have developed functionally equivalent modules to stabilize and organize MTs at minus ends. These findings support the theory that plants overcome centrosome loss by utilizing modified but substantially conserved mechanisms to organize MT networks., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Several studies have shown that RNAi-mediated depletion of splicing factors (SFs) results in mitotic abnormalities. However, it is currently unclear whether these abnormalities reflect defective splicing of specific pre-mRNAs or a direct role of the SFs in mitosis. Here, we show that two highly conserved SFs, Sf3A2 and Prp31, are required for chromosome segregation in both Drosophila and human cells. Injections of anti-Sf3A2 and anti-Prp31 antibodies into Drosophila embryos disrupt mitotic division within 1 min, arguing strongly against a splicing-related mitotic function of these factors. We demonstrate that both SFs bind spindle microtubules (MTs) and the Ndc80 complex, which in Sf3A2- and Prp31-depleted cells is not tightly associated with the kinetochores; in HeLa cells the Ndc80/HEC1-SF interaction is restricted to the M phase. These results indicate that Sf3A2 and Prp31 directly regulate interactions among kinetochores, spindle microtubules and the Ndc80 complex in both Drosophila and human cells., Competing Interests: CP, EB, FR, DH, AP, JC, SB, JW, MG, MS No competing interests declared, (© 2018, Pellacani et al.)

Cell division is a fundamental biological process that has been extensively investigated in different systems. Similar to most eukaryotic cells, plant cells assemble a mitotic spindle to separate replicated chromosomes. In contrast, to complete cell division, plant cells assemble a phragmoplast, which is composed of aligned microtubules and actin filaments. This structure helps transport vesicles containing new cell wall material, which then fuse to form the cell plate; the cell plate will expand to create the new dividing cell wall. Because vesicles are known to be transported by myosin motors during interphase, we hypothesized this could also be the case during cell division and we investigated the localization of the plant homologue of myosin V - myosin XI, in cell division. In this work, we used the protonemal cells of the moss Physcomitrella patens as a model, because of its simple cellular morphology and ease to generate transgenic cell lines expressing fluorescent tagged proteins. Using a fluorescent protein fusion of myosin XI, we found that, during mitosis, this molecule appears to associate with the kinetochores immediately after nuclear envelope breakdown. Following metaphase, myosin XI stays associated with the spindle's midzone during the rest of mitosis, and when the phragmoplast is formed, it concentrates at the cell plate. Using an actin polymerization inhibitor, latrunculin B, we found that the association of myosin XI with the mitotic spindle and the phragmoplast are only partially dependent on the presence of filamentous actin. We also showed that myosin XI on the spindle partially overlaps with a v-SNARE vesicle marker but is not co-localized with the endoplasmic reticulum and a RabA vesicle marker. These observations suggest an actin-dependent and an actin-independent behavior of myosin XI during cell division, and provide novel insights to our understanding of the function of myosin XI during plant cell division., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In cytoplasm, protein γ-tubulin joins with various γ-tubulin complex proteins (GCPs) to form a heterotetramer γ-tubulin small complex (γ-TuSC) that can grow into a ring-shaped structure called the γ-tubulin ring complex (γ-TuRC). Both γ-TuSC and γ-TuRC are required for microtubule nucleation. Recent knowledge on γ-tubulin with regard to its cellular functions beyond participation in its creation of microtubules suggests that this protein forms a cellular meshwork. The present review summarizes the recognized functions of γ-tubulin and aims to unite the current views on this protein.

Nucleoporins (Nups) are a large and diverse family of proteins that mediate nucleocytoplasmic transport at interphase of vertebrate cells. Nups also function in mitosis progression. However, whether Nups are involved in oocyte meiosis progression is still rarely known. In this study, we delineated the roles and regulatory mechanisms of Nucleoporin35 (Nup35) during oocyte meiotic maturation. The immunofluorescent signal of Nup35 was localized in the nuclear membrane at germinal vesicle (GV) stage, the microtubules and spindle at pro-metaphase I (pro-MI), metaphase I (MI), and metaphase II (MII), but to the spindle poles at anaphase I (AI) and telophase I (TI). The dynamic localization pattern of Nup35 during oocyte meiotic maturation implied its specific roles. We also found that Nup35 existed as a putatively phosphorylated form after resumption of meiosis (GVBD), but not at GV stage, implying its functional switch from nuclear membrane to meiotic progression. Further study uncovered that knockdown of Nup35 by specific siRNA significantly compromised the extrusion of first polar body (PBE), but not GVBD, with defects of spindle assembly and chromosome alignment and dissociated some localization signal of p-ERK1/2 from spindle poles to cytoplasm. A defective kinetochore - microtubule attachment (K-MT) was also identified in oocytes after knockdown of Nup35, which activates spindle assembly checkpoint. In conclusion, our results suggest that Nup35 is putatively phosphorylated and released to the cytoplasm after resumption of meiosis, and regulates spindle assembly and chromosome alignment., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Kinetochore fibers (K-fibers) are microtubule bundles attached to chromosomes. Efficient K-fiber formation is required for chromosome congression, crucial for faithful chromosome segregation in cells. However, the mechanisms underlying K-fiber formation before chromosome biorientation remain unclear. Depletion of hepatoma up-regulated protein (HURP), a RanGTP-dependent microtubule-associated protein localized on K-fibers, has been shown to result in low-efficiency K-fiber formation. Therefore, here we sought to identify critical interaction partners of HURP that may modulate this function. Using co-immunoprecipitation and bimolecular fluorescence complementation assays, we determined that HURP interacts directly with the centrosomal protein transforming acidic coiled coil-containing protein 3 (TACC3), a centrosomal protein, both in vivo and in vitro through the HURP 1-625 region. We found that HURP is important for TACC3 function during kinetochore microtubule assembly at the chromosome region in prometaphase. Moreover, HURP regulates stable lateral kinetochore attachment and chromosome congression in early mitosis by modulation of TACC3. These findings provide new insight into the coordinated regulation of K-fiber formation and chromosome congression in prometaphase by microtubule-associated proteins., (© 2018 Zhang et al.)

Mitosis relies on forces generated in the spindle, a micro-machine composed of microtubules and associated proteins. Forces are required for the congression of chromosomes to the metaphase plate and their separation in anaphase. However, besides forces, torques may exist in the spindle, yet they have not been investigated. Here we show that the spindle is chiral. Chirality is evident from the finding that microtubule bundles in human spindles follow a left-handed helical path, which cannot be explained by forces but rather by torques. Kinesin-5 (Kif11/Eg5) inactivation abolishes spindle chirality. Our theoretical model predicts that bending and twisting moments may generate curved shapes of bundles. We found that bundles turn by about -2 deg µm -1 around the spindle axis, which we explain by a twisting moment of roughly -10 pNµm. We conclude that torques, in addition to forces, exist in the spindle and determine its chiral architecture.

The mitotic spindle orientation and position is crucial for the fidelity of chromosome segregation during asymmetric cell division to generate daughter cells with different sizes or fates. This mechanism is best understood in the budding yeast Saccharomyces cerevisiae, named the spindle position checkpoint (SPOC). The SPOC inhibits cells from exiting mitosis until the mitotic spindle is properly oriented along the mother-daughter polarity axis. Despite many experimental studies, the mechanisms underlying SPOC regulation remains elusive and unexplored theoretically. Here, a minimal mathematical is developed to describe SPOC activation and silencing having autocatalytic feedback-loop. Numerical simulations of the nonlinear ordinary differential equations (ODEs) model accurately reproduce the phenotype of SPOC mechanism. Bifurcation analysis of the nonlinear ODEs reveals the orientation dependency on spindle pole bodies, and how this dependence is altered by parameter values. Partial differential equation (PDEs) model as well as linear stability analysis indicate that diffusion play no major role using experimental high diffusion values. These results provide for systems understanding on the molecular organization of spindle orientation system via mathematical modeling. The presented mathematical model is easy to understand and, within the above mentioned context, can be used as a base for further development of quantitative models in asymmetric cell-division., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In plant cytokinesis, actin is thought to be crucial in cell plate guidance to the cortical division zone (CDZ), but its organization and function are not fully understood. To elucidate actin organization during cytokinesis, we employed an experimental system, in which the mitotic apparatus is displaced and separated from the CDZ by centrifugation and observed using a global-local live imaging microscope that enabled us to record behavior of actin filaments in the CDZ and the whole cell division process in parallel. In this system, returning movement of the cytokinetic apparatus in cultured-tobacco BY-2 cells occurs, and there is an advantage to observe actin organization clearly during the cytokinetic phase because more space was available between the CDZ and the distantly formed phragmoplast. Actin cables were clearly observed between the CDZ and the phragmoplast in BY-2 cells expressing GFP-fimbrin after centrifugation. Both the CDZ and the edge of the expanding phragmoplast had actin bulges. Using live-cell imaging including the global-local live imaging microscopy, we found actin filaments started to accumulate at the actin-depleted zone when cell plate expansion started even in the cell whose cell plate failed to reach the CDZ. These results suggest that specific accumulation of actin filaments at the CDZ and the appearance of actin cables between the CDZ and the phragmoplast during cell plate formation play important roles in the guidance of cell plate edges to the CDZ.

Enhanced microtubule acetylation has been identified as a negative prognostic indicator in breast cancer. We reported previously that primary cultured human mammary epithelial cells manifest breast cancer-related aneuploidization via the activation of severing protein katanin-like (KL)1 when tau is deficient. To address in this current study whether microtubule hyperacetylation is involved in breast carcinogenesis through mitosis, the effects of tubacin on human mammary epithelial cells were tested using immunofluorescence techniques. Tau-knockdown cells showed enhancement of KL1-dependent events, chromosome-bridging and micronucleation in response to tubacin. These enhancements were suppressed by further expression of an acetylation-deficient tubulin mutant. Consistently, using a rat fibroblast-based microtubule sensitivity test, it was confirmed that KL1 also shows enhanced activity in response to microtubule hyperacetylation as well as katanin. It was further observed in rat fibroblasts that exogenously expressed KL1 results in more micronucleation under microtubule hyperacetylation conditions. These data suggest that microtubule acetylation upregulates KL1 and induces more aneuploidy if tau is deficient. It is thus plausible that microtubule hyperacetylation promotes tumor progression by enhancing microtubule sensitivity to KL1, thereby disrupting spindle microtubules and this process could be reversed by the microtubule-binding and microtubule protective octapeptide NAPVSIPQ (NAP) which recruits tau to the microtubules.

Epithelial cells form tissues with many functions, including secretion and environmental separation and protection. Glandular epithelial tissues comprise cysts and tubules that are formed from a polarized, single-epithelial cell layer surrounding a central, fluid-filled lumen. The pathways regulating key processes in epithelial tissue morphogenesis such as mitotic spindle formation are incompletely understood, but are important to investigate, as their dysregulation is a signature of epithelial tumors. Here, we describe a signaling axis that manifests in a defect in mitotic spindle orientation during epithelial growth and cystogenesis. We found that activation of the small GTPase ADP-ribosylation factor 6 (ARF6) results in the sustained internalization of cell-surface components such as the cMet receptor and the cell-adhesion molecule E-cadherin. The spindle orientation defect arising from elevated levels of ARF6-GTP required an increase in cMet endocytosis, but was independent of E-cadherin internalization or elevated extracellular signal-regulated kinase (ERK) activity resulting from internalized receptor signaling on endosomes. Misorientation of the mitotic spindle resulted in the development of epithelial cysts with structural abnormalities, the most conspicuous of which was the presence of multiple intercellular lumens. Abnormal mitotic spindle orientation was necessary but insufficient to disrupt glandular development, as blocking the strong prosurvival signal resulting from ERK hyperactivation yielded structurally normal cysts despite continued manifestation of spindle orientation defects. Our findings highlight a previously unknown link between ARF6 activation, cMet receptor internalization, and mitotic spindle orientation during epithelial glandular morphogenesis., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

The chromokinesin Kif4A controls proper chromosome condensation, congression/alignment, and cytokinesis to ensure faithful genetic inheritance. Here, we report that Cdk phosphorylation of human Kif4A at T1161 licenses Kif4A chromosomal localization, which, in turn, controls Kif4A early mitotic function. Phosphorylated Kif4A (Kif4AWT) or Cdk phospho-mimetic Kif4A mutant (Kif4ATE) associated with chromosomes and condensin I (non-SMC subunit CAP-G and core subunit SMC2) to regulate chromosome condensation, spindle morphology, and chromosome congression/alignment in early mitosis. In contrast, Cdk non-phosphorylatable Kif4A mutant (Kif4ATA) could neither localize on chromosomes nor associate with CAP-G and SMC2. Furthermore, Kif4ATA could not rescue defective chromosome condensation, spindle morphology, or chromosome congression/alignment in cells depleted of endogenous Kif4A, which activated a mitotic checkpoint and delayed early mitotic progression. However, targeting Kif4ATA to chromosomes by fusion of Kif4ATA with Histone H1 resulted in restoration of chromosome and spindle functions of Kif4A, similar to Kif4AWT and Kif4ATE, in cells depleted of endogenous Kif4A. Thus, our results demonstrate that Cdk phosphorylation-licensed chromosomal localization of Kif4A plays a critical role in regulating early mitotic functions of Kif4A that are important for early mitotic progression.

An increasing number of observations suggest an evolutionary switch of centromere position on monocentric eukaryotic chromosomes which otherwise display a conserved sequence of genes and markers. Such observations are particularly frequent for primates and equidae (for review see Heredity 108:59-67, 2012) but occur also in marsupials (J Hered 96:217-224, 2005) and in plants (Chromosome Res 25:299-311, 2017 and references therein). The actual mechanism(s) behind remained unclear in many cases (Proc Natl Acad Sci USA 101:6542-6547, 2004; Trends Genet 30:66-74, 2014). The same is true for de novo centromere formation on chromosomes lacking an active centromere. This article focuses on recent reports on centromere repositioning and possible mechanisms behind and addresses open questions.

Cell division is fundamental to life and its perturbation can disrupt organismal development, alter tissue homeostasis, and cause disease. Analysis of mitotic abnormalities provides insight into how certain perturbations affect the fidelity of cell division and how specific cellular structures, molecules, and enzymatic activities contribute to the accuracy of this process. However, accurate classification of mitotic defects is instrumental for correct interpretation of data and formulation of new hypotheses. In this article, we provide guidelines for identifying specific mitotic stages and for classifying normal and deviant mitotic phenotypes. We hope this will clarify confusion about how certain defects are classified and help investigators avoid misnomers, misclassification, and/or misinterpretation, thus leading to a unified and standardized system to classify mitotic defects.

It is widely accepted in eukaryotes that the cleavage furrow only initiates after mitosis completion. In fission yeast, cytokinesis requires the synthesis of a septum tightly coupled to cleavage furrow ingression. The current cytokinesis model establishes that simultaneous septation and furrow ingression only initiate after spindle breakage and mitosis exit. Thus, this model considers that although Cdk1 is inactivated at early-anaphase, septation onset requires the long elapsed time until mitosis completion and full activation of the Hippo-like SIN pathway. Here, we studied the precise timing of septation onset regarding mitosis by exploiting both the septum-specific detection with the fluorochrome calcofluor and the high-resolution electron microscopy during anaphase and telophase. Contrarily to the existing model, we found that both septum and cleavage furrow start to ingress at early anaphase B, long before spindle breakage, with a slow ingression rate during anaphase B, and greatly increasing after telophase onset. This shows that mitosis and cleavage furrow ingression are not concatenated but simultaneous events in fission yeast. We found that the timing of septation during early anaphase correlates with the cell size and is regulated by the corresponding levels of SIN Etd1 and Rho1. Cdk1 inactivation was directly required for timely septation in early anaphase. Strikingly the reduced SIN activity present after Cdk1 loss was enough to trigger septation by immediately inducing the medial recruitment of the SIN kinase complex Sid2-Mob1. On the other hand, septation onset did not depend on the SIN asymmetry establishment, which is considered a hallmark for SIN activation. These results recalibrate the timing of key cytokinetic events in fission yeast; and unveil a size-dependent control mechanism that synchronizes simultaneous nuclei separation with septum and cleavage furrow ingression to safeguard the proper chromosome segregation during cell division., Competing Interests: The authors have declared that no competing interests exist.

Sustained spindle tension applied to sister centromeres during mitosis eventually leads to uncoordinated loss of sister chromatid cohesion, a phenomenon known as "cohesion fatigue." We report that Aurora A-dependent phosphorylation of serine 7 of the centromere histone variant CENP-A (p-CENP-AS7) protects bioriented chromosomes against cohesion fatigue. Expression of a non-phosphorylatable version of CENP-A (CENP-AS7A) weakens sister chromatid cohesion only when sister centromeres are under tension, providing the first evidence of a regulated mechanism involved in protection against passive cohesion loss. Consistent with this observation, p-CENP-AS7 is detected at the inner centromere where it forms a discrete domain. The depletion or inhibition of Aurora A phenocopies the expression of CENP-AS7A and we show that Aurora A is recruited to centromeres in a Bub1-dependent manner. We propose that Aurora A-dependent phosphorylation of CENP-A at the inner centromere protects chromosomes against tension-induced cohesion fatigue until the last kinetochore is attached to spindle microtubules.

In budding yeast, the microtubule plus-end tracking proteins Bik1 (CLIP-170) and Bim1 (EB1) form a complex that interacts with partners involved in spindle positioning, including Stu2 and Kar9. Here, we show that the CAP-Gly and coiled-coil domains of Bik1 interact with the C-terminal ETF peptide of Bim1 and the C-terminal tail region of Stu2, respectively. The crystal structures of the CAP-Gly domain of Bik1 (Bik1CG) alone and in complex with an ETF peptide revealed unique, functionally relevant CAP-Gly elements, establishing Bik1CG as a specific C-terminal phenylalanine recognition domain. Unlike the mammalian CLIP-170-EB1 complex, Bik1-Bim1 forms ternary complexes with the EB1-binding motifs SxIP and LxxPTPh, which are present in diverse proteins, including Kar9. Perturbation of the Bik1-Bim1 interaction in vivo affected Bik1 localization and astral microtubule length. Our results provide insight into the role of the Bik1-Bim1 interaction for cell division, and demonstrate that the CLIP-170-EB1 module is evolutionarily flexible., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Before fertilization, mammalian oocyte undergoes an asymmetric division which depends on eccentric positioning of the spindle at the oocyte cortex to form a polar body and an egg. Since the centriole is absent and, as a result, the polar array microtubules are not fully developed in oocytes, microtubules have seldom been considered as required for eccentric positioning of the spindle, while actin-related forces have instead been proposed to be primarily responsible for this process. However, the existing models are largely conflicting and the underlying mechanism of asymmetric division is still elusive. Here we show that poly(ADP-ribose) (PAR) is enriched at mouse oocyte cortical area throughout meiosis. Specific removal of cortical PAR results in an ectopic spindle and a failure of asymmetric division. During spindle migration, when the spindle deviates from the center of oocyte by a pushing force of cytoplasmic actin, the short polar array microtubules emanating from the juxtacortical spindle pole extend to the cortex and penetrate into cortical PAR, docking and stabilizing the spindle at the cortex which facilitates the asymmetric division. This process depends on the affinity between PAR and microtubule-associated proteins such as Spindly, which contributes to a physical link for cortical PAR and the spindle. Notably, fusing a PAR-binding domain to end-binding protein 3, a plus-end tracking protein at the polar array microtubules, restores the asymmetric division of oocytes with Spindly knockdown. Thus, our work demonstrates a comprehensive mechanism for oocyte spindle positioning and asymmetric division.