Vertebrate centromeres in mitosis are functionally bipartite structures stabilized by cohesin.

Centromeres are scaffolds for the assembly of kinetochores that ensure chromosome segregation during cell division. How vertebrate centromeres obtain a three-dimensional structure to accomplish their primary function is unclear. Using super-resolution imaging, capture-C, and polymer modeling, we show that vertebrate centromeres are partitioned by condensins into two subdomains during mitosis. The bipartite structure is found in human, mouse, and chicken cells and is therefore a fundamental feature of vertebrate centromeres. Super-resolution imaging and electron tomography reveal that bipartite centromeres assemble bipartite kinetochores, with each subdomain binding a distinct microtubule bundle. Cohesin links the centromere subdomains, limiting their separation in response to spindle forces and avoiding merotelic kinetochore-spindle attachments. Lagging chromosomes during cancer cell divisions frequently have merotelic attachments in which the centromere subdomains are separated and bioriented. Our work reveals a fundamental aspect of vertebrate centromere biology with implications for understanding the mechanisms that guarantee faithful chromosome segregation.

Life-cycle-coupled evolution of mitosis in close relatives of animals.

Eukaryotes have evolved towards one of two extremes along a spectrum of strategies for remodelling the nuclear envelope during cell division: disassembling the nuclear envelope in an open mitosis or constructing an intranuclear spindle in a closed mitosis1,2. Both classes of mitotic remodelling involve key differences in the core division machinery but the evolutionary reasons for adopting a specific mechanism are unclear. Here we use an integrated comparative genomics and ultrastructural imaging approach to investigate mitotic strategies in Ichthyosporea, close relatives of animals and fungi. We show that species in this clade have diverged towards either a fungal-like closed mitosis or an animal-like open mitosis, probably to support distinct multinucleated or uninucleated states. Our results indicate that multinucleated life cycles favour the evolution of closed mitosis.

Nuclear chromosome locations dictate segregation error frequencies.

Chromosome segregation errors during cell divisions generate aneuploidies and micronuclei, which can undergo extensive chromosomal rearrangements such as chromothripsis1-5. Selective pressures then shape distinct aneuploidy and rearrangement patterns-for example, in cancer6,7-but it is unknown whether initial biases in segregation errors and micronucleation exist for particular chromosomes. Using single-cell DNA sequencing8 after an error-prone mitosis in untransformed, diploid cell lines and organoids, we show that chromosomes have different segregation error frequencies that result in non-random aneuploidy landscapes. Isolation and sequencing of single micronuclei from these cells showed that mis-segregating chromosomes frequently also preferentially become entrapped in micronuclei. A similar bias was found in naturally occurring micronuclei of two cancer cell lines. We find that segregation error frequencies of individual chromosomes correlate with their location in the interphase nucleus, and show that this is highest for peripheral chromosomes behind spindle poles. Randomization of chromosome positions, Cas9-mediated live tracking and forced repositioning of individual chromosomes showed that a greater distance from the nuclear centre directly increases the propensity to mis-segregate. Accordingly, chromothripsis in cancer genomes9 and aneuploidies in early development10 occur more frequently for larger chromosomes, which are preferentially located near the nuclear periphery. Our findings reveal a direct link between nuclear chromosome positions, segregation error frequencies and micronucleus content, with implications for our understanding of tumour genome evolution and the origins of specific aneuploidies during development.

The TRIM69-MST2 signaling axis regulates centrosome dynamics and chromosome segregation.

Stringent control of centrosome duplication and separation is important for preventing chromosome instability. Structural and numerical alterations in centrosomes are hallmarks of neoplastic cells and contribute to tumorigenesis. We show that a Centrosome Amplification 20 (CA20) gene signature is associated with high expression of the Tripartite Motif (TRIM) family member E3 ubiquitin ligase, TRIM69. TRIM69-ablation in cancer cells leads to centrosome scattering and chromosome segregation defects. We identify Serine/threonine-protein kinase 3 (MST2) as a new direct binding partner of TRIM69. TRIM69 redistributes MST2 to the perinuclear cytoskeleton, promotes its association with Polo-like kinase 1 (PLK1) and stimulates MST2 phosphorylation at S15 (a known PLK1 phosphorylation site that is critical for centrosome disjunction). TRIM69 also promotes microtubule bundling and centrosome segregation that requires PRC1 and DYNEIN. Taken together, we identify TRIM69 as a new proximal regulator of distinct signaling pathways that regulate centrosome dynamics and promote bipolar mitosis.

Proliferative advantage of specific aneuploid cells drives evolution of tumor karyotypes.

Most tumors have abnormal karyotypes, which arise from mistakes during mitotic division of healthy euploid cells and evolve through numerous complex mechanisms. In a recent mouse model with increased chromosome missegregation, chromosome gains dominate over losses both in pretumor and tumor tissues, whereas T-cell lymphomas are characterized by gains of chromosomes 14 and 15. However, the quantitative understanding of clonal selection leading to tumor karyotype evolution remains unknown. Here we show, by introducing a mathematical model based on a concept of a macro-karyotype, that tumor karyotypes can be explained by proliferation-driven evolution of aneuploid cells. In pretumor cells, increased apoptosis and slower proliferation of cells with monosomies lead to predominant chromosome gains over losses. Tumor karyotypes with gain of one chromosome can be explained by karyotype-dependent proliferation, whereas, for those with two chromosomes, an interplay with karyotype-dependent apoptosis is an additional possible pathway. Thus, evolution of tumor-specific karyotypes requires proliferative advantage of specific aneuploid karyotypes.

Anaphase B: Long-standing models meet new concepts.

Mitotic cell divisions ensure stable transmission of genetic information from a mother to daughter cells in a series of generations. To ensure this crucial task is accomplished, the cell forms a bipolar structure called the mitotic spindle that divides sister chromatids to the opposite sides of the dividing mother cell. After successful establishment of stable attachments of microtubules to chromosomes and inspection of connections between them, at the heart of mitosis, the cell starts the process of segregation. This spectacular moment in the life of a cell is termed anaphase, and it involves two distinct processes: depolymerization of microtubules bound to chromosomes, which is also known as anaphase A, and elongation of the spindle or anaphase B. Both processes ensure physical separation of disjointed sister chromatids. In this chapter, we review the mechanisms of anaphase B spindle elongation primarily in mammalian systems, combining different pioneering ideas and concepts with more recent findings that shed new light on the force generation and regulation of biochemical modules operating during spindle elongation. Finally, we present a comprehensive model of spindle elongation that includes structural, biophysical, and molecular aspects of anaphase B.

Oblique circle method for measuring the curvature and twist of mitotic spindle microtubule bundles.

The highly ordered spatial organization of microtubule bundles in the mitotic spindle is crucial for its proper functioning. The recent discovery of twisted shapes of microtubule bundles and spindle chirality suggests that the bundles extend along curved paths in three dimensions, rather than being confined to a plane. This, in turn, implies that rotational forces, i.e., torques, exist in the spindle in addition to the widely studied linear forces. However, studies of spindle architecture and forces are impeded by a lack of a robust method for the geometric quantification of microtubule bundles in the spindle. In this work, we describe a simple method for measuring and evaluating the shapes of microtubule bundles by characterizing them in terms of their curvature and twist. By using confocal microscopy, we obtain three-dimensional images of spindles, which allows us to trace the entire microtubule bundle. For each traced bundle, we first fit a plane and then fit a circle lying in that plane. With this robust method, we extract the curvature and twist, which represent the geometric information characteristic for each bundle. As the bundle shapes reflect the forces within them, this method is valuable for the understanding of forces that act on chromosomes during mitosis.

Force-generating mechanisms of anaphase in human cells.

What forces drive chromosome segregation remains one of the most challenging questions in cell division. Even though the duration of anaphase is short, it is of utmost importance for genome fidelity that no mistakes are made. Seminal studies in model organisms have revealed different mechanisms operating during chromosome segregation in anaphase, but the translation of these mechanisms to human cells is not straightforward. Recent work has shown that kinetochore fiber depolymerization during anaphase A is largely motor independent, whereas spindle elongation during anaphase B is coupled to sliding of interpolar microtubules in human cells. In this Review, we discuss the current knowledge on the mechanisms of force generation by kinetochore, interpolar and astral microtubules. By combining results from numerous studies, we propose a comprehensive picture of the role of individual force-producing and -regulating proteins. Finally, by linking key concepts of anaphase to most recent data, we summarize the contribution of all proposed mechanisms to chromosome segregation and argue that sliding of interpolar microtubules and depolymerization at the kinetochore are the main drivers of chromosome segregation during early anaphase in human cells.

Mitotic Spindle Assembly: Building the Bridge between Sister K-Fibers.

The mitotic spindle performs the task of physically dividing the genetic material between the newly formed daughter cells. To achieve this, bundles of microtubules and associated proteins orchestrate forces that spatially organize and then separate the chromosomes. In the classic view of the spindle, the kinetochore microtubules (k-fibers) are tensed and, thus, straight, whereas interpolar bundles are curved and do not interact with k-fibers close to the spindle equator. The updated view of the spindle depicts k-fibers as curved and interacting with newly identified interpolar bundles, called bridging fibers, along their length. In this Opinion, we propose and discuss scenarios for the origin of this structure in the context of known spindle assembly mechanisms.

Pivoting of microtubules driven by minus-end-directed motors leads to spindle assembly.

BACKGROUND: At the beginning of mitosis, the cell forms a spindle made of microtubules and associated proteins to segregate chromosomes. An important part of spindle architecture is a set of antiparallel microtubule bundles connecting the spindle poles. A key question is how microtubules extending at arbitrary angles form an antiparallel interpolar bundle. RESULTS: Here, we show in fission yeast that microtubules meet at an oblique angle and subsequently rotate into antiparallel alignment. Our live-cell imaging approach provides a direct observation of interpolar bundle formation. By combining experiments with theory, we show that microtubules from each pole search for those from the opposite pole by performing random angular movement. Upon contact, two microtubules slide sideways along each other in a directed manner towards the antiparallel configuration. We introduce the contour length of microtubules as a measure of activity of motors that drive microtubule sliding, which we used together with observation of Cut7/kinesin-5 motors and our theory to reveal the minus-end-directed motility of this motor in vivo. CONCLUSION: Random rotational motion helps microtubules from the opposite poles to find each other and subsequent accumulation of motors allows them to generate forces that drive interpolar bundle formation.

PRC1-labeled microtubule bundles and kinetochore pairs show one-to-one association in metaphase.

In the mitotic spindle, kinetochore microtubules form k-fibers, whereas overlap or interpolar microtubules form antiparallel arrays containing the cross-linker protein regulator of cytokinesis 1 (PRC1). We have recently shown that an overlap bundle, termed bridging fiber, links outermost sister k-fibers. However, the relationship between overlap bundles and k-fibers throughout the spindle remained unknown. Here, we show that in a metaphase spindle more than 90% of overlap bundles act as a bridge between sister k-fibers. We found that the number of PRC1-GFP-labeled bundles per spindle is nearly the same as the number of kinetochore pairs. Live-cell imaging revealed that kinetochore movement in the equatorial plane of the spindle is highly correlated with the movement of the coupled PRC1-GFP-labeled fiber, whereas the correlation with other fibers decreases with increasing distance. Analysis of endogenous PRC1 localization confirmed the results obtained with PRC1-GFP PRC1 knockdown reduced the bridging fiber thickness and interkinetochore distance throughout the spindle, suggesting a function of PRC1 in bridging microtubule organization and force balance in the metaphase spindle.

Mitotic spindle: kinetochore fibers hold on tight to interpolar bundles.

When a cell starts to divide, it forms a spindle, a micro-machine made of microtubules, which separates the duplicated chromosomes. The attachment of microtubules to chromosomes is mediated by kinetochores, protein complexes on the chromosome. Spindle microtubules can be divided into three major classes: kinetochore microtubules, which form k-fibers ending at the kinetochore; interpolar microtubules, which extend from the opposite sides of the spindle and interact in the middle; and astral microtubules, which extend towards the cell cortex. Recent work in human cells has shown a close relationship between interpolar and kinetochore microtubules, where interpolar bundles are attached laterally to kinetochore fibers almost all along their length, acting as a bridge between sister k-fibers. Most of the interpolar bundles are attached to a pair of sister kinetochore fibers and vice versa. Thus, the spindle is made of modules consisting of a pair of sister kinetochore fibers and a bundle of interpolar microtubules that connects them. These interpolar bundles, termed bridging fibers, balance the forces acting at kinetochores and support the rounded shape of the spindle during metaphase. This review discusses the structure, function, and formation of kinetochore fibers and interpolar bundles, with an emphasis on how they interact. Their connections have an impact on the force balance in the spindle and on chromosome movement during mitosis because the forces in interpolar bundles are transmitted to kinetochore fibers and hence to kinetochores through these connections.

Fusion of protein aggregates facilitates asymmetric damage segregation.

Asymmetric segregation of damaged proteins at cell division generates a cell that retains damage and a clean cell that supports population survival. In cells that divide asymmetrically, such as Saccharomyces cerevisiae, segregation of damaged proteins is achieved by retention and active transport. We have previously shown that in the symmetrically dividing Schizosaccharomyces pombe there is a transition between symmetric and asymmetric segregation of damaged proteins. Yet how this transition and generation of damage-free cells are achieved remained unknown. Here, by combining in vivo imaging of Hsp104-associated aggregates, a form of damage, with mathematical modeling, we find that fusion of protein aggregates facilitates asymmetric segregation. Our model predicts that, after stress, the increased number of aggregates fuse into a single large unit, which is inherited asymmetrically by one daughter cell, whereas the other one is born clean. We experimentally confirmed that fusion increases segregation asymmetry, for a range of stresses, and identified Hsp16 as a fusion factor. Our work shows that fusion of protein aggregates promotes the formation of damage-free cells. Fusion of cellular factors may represent a general mechanism for their asymmetric segregation at division.

Asymmetric damage segregation at cell division via protein aggregate fusion and attachment to organelles.

The segregation of damaged components at cell division determines the survival and aging of cells. In cells that divide asymmetrically, such as Saccharomyces cerevisiae, aggregated proteins are retained by the mother cell. Yet, where and how aggregation occurs is not known. Recent work by Zhou and collaborators shows that the birth of protein aggregates, under specific stress conditions, requires active translation, and occurs mainly at the endoplasmic reticulum. Later, aggregates move to the mitochondrial surface through fis1-dependent association. During replicative aging, aggregate association with the mother-cell mitochondria contributes to the asymmetric segregation of aggregates, because mitochondria in the daughter cell do not carry aggregates. With increasing age of mother cells, aggregates lose their connection to the mitochondria, and segregation is less asymmetric. Relating these findings to other mechanisms of aggregate segregation in different organisms, we postulate that fusion between aggregates and their tethering to organelles such as the vacuole, nucleus, ER, or mitochondria are common principles that establish asymmetric segregation during stress resistance and aging.

Pulled Polymer Loops as a Model for the Alignment of Meiotic Chromosomes.

During recombination, the DNA of parents exchange their genetic information to give rise to a genetically unique offspring. For recombination to occur, homologous chromosomes need to find each other and align with high precision. Fission yeast solves this problem by folding chromosomes in loops and pulling them through the viscous nucleoplasm. We propose a theory of pulled polymer loops to quantify the effect of drag forces on the alignment of chromosomes. We introduce an external force field to the concept of a Brownian bridge and thus solve for the statistics of loop configurations in space.

Kinesin-8 motors improve nuclear centering by promoting microtubule catastrophe.

In fission yeast, microtubules push against the cell edge, thereby positioning the nucleus in the cell center. Kinesin-8 motors regulate microtubule catastrophe; however, their role in nuclear positioning is not known. Here we develop a physical model that describes how kinesin-8 motors affect nuclear centering by promoting a microtubule catastrophe. Our model predicts the improved centering of the nucleus in the presence of motors, which we confirmed experimentally in living cells. The model also predicts a characteristic time for the recentering of a displaced nucleus, which is supported by our experiments where we displaced the nucleus using optical tweezers.

Fusion leads to effective segregation of damage during cell division: An analytical treatment.

High levels of cellular damage are associated with impairment of cellular function and cell death. Partitioning the damage into a fraction of cells in the population improves population fitness and survival. We have previously shown that protein aggregates, resulting from misfolded, damaged proteins, fuse with each other leading to damage partitioning during cell division. Here, using an analytical treatment of aggregate fusion in dividing cells we present analytical expressions for two measures of damage partition: aggregate mass partition asymmetry between two dividing cells and standard deviation of total aggregate mass across the population. The scaling laws obtained demonstrate how damage partition may generally depend on characteristics of the cellular processes, facilitating better understanding of damage segregation in biological cells.

Maternal genetic variants in kinesin motor domains prematurely increase egg aneuploidy.

The female reproductive lifespan depends on egg quality, particularly euploidy. Mistakes in meiosis leading to egg aneuploidy are common, but the genetic landscape causing this is not well understood due to limited phenotypic data. We identify genetic determinants of reproductive aging via egg aneuploidy using a biobank of maternal exomes linked with maternal age and embryonic aneuploidy data. We found 404 genes with variants enriched in individuals with high egg aneuploidy rates and implicate kinesin protein family genes in aneuploidy risk. Experimental perturbations showed that motor domain variants in these genes increase aneuploidy in mouse oocytes. A knock-in mouse model validated that a specific variant in kinesin KIF18A accelerates reproductive aging and diminishes fertility. These findings suggest potential non-invasive biomarkers for egg quality, aiding personalized fertility medicine. One sentence summary: The study identifies novel genetic determinants of reproductive aging linked to egg aneuploidy by analyzing maternal exomes and demonstrates that variants in kinesin genes, specifically KIF18A , contribute to increased aneuploidy and accelerated reproductive aging, offering potential for personalized fertility medicine.

Centrosomal microtubule nucleation regulates radial migration of projection neurons independently of polarization in the developing brain.

Cortical projection neurons polarize and form an axon while migrating radially. Even though these dynamic processes are closely interwoven, they are regulated separately-the neurons terminate their migration when reaching their destination, the cortical plate, but continue to grow their axons. Here, we show that in rodents, the centrosome distinguishes these processes. Newly developed molecular tools modulating centrosomal microtubule nucleation combined with in vivo imaging uncovered that dysregulation of centrosomal microtubule nucleation abrogated radial migration without affecting axon formation. Tightly regulated centrosomal microtubule nucleation was required for periodic formation of the cytoplasmic dilation at the leading process, which is essential for radial migration. The microtubule nucleating factor γ-tubulin decreased at neuronal centrosomes during the migratory phase. As distinct microtubule networks drive neuronal polarization and radial migration, this provides insight into how neuronal migratory defects occur without largely affecting axonal tracts in human developmental cortical dysgeneses, caused by mutations in γ-tubulin.

Plasmodium ARK2-EB1 axis drives the unconventional spindle dynamics, scaffold formation and chromosome segregation of sexual transmission stages.

Mechanisms of cell division are remarkably diverse, suggesting the underlying molecular networks among eukaryotes differ extensively. The Aurora family of kinases orchestrates the process of chromosome segregation and cytokinesis during cell division through precise spatiotemporal regulation of their catalytic activities by distinct scaffolds. Plasmodium spp., the causative agents of malaria, are unicellular eukaryotes that have three divergent aurora-related kinases (ARKs) and lack most canonical scaffolds/activators. The parasite uses unconventional modes of chromosome segregation during endomitosis and meiosis in sexual transmission stages within mosquito host. This includes a rapid threefold genome replication from 1N to 8N with successive cycles of closed mitosis, spindle formation and chromosome segregation within eight minutes (termed male gametogony). Kinome studies had previously suggested likely essential functions for all three Plasmodium ARKs during asexual mitotic cycles; however, little is known about their location, function, or their scaffolding molecules during unconventional sexual proliferative stages. Using a combination of super-resolution microscopy, mass spectrometry, and live-cell fluorescence imaging, we set out to investigate the role of the atypical Aurora paralog ARK2 to proliferative sexual stages using rodent malaria model Plasmodium berghei . We find that ARK2 primarily localises to the spindle apparatus in the vicinity of kinetochores during both mitosis and meiosis. Interactomics and co-localisation studies reveal a unique ARK2 scaffold at the spindle including the microtubule plus end-binding protein EB1, lacking conserved Aurora scaffold proteins. Gene function studies indicate complementary functions of ARK2 and EB1 in driving endomitotic divisions and thereby parasite transmission. Our discovery of a novel Aurora kinase spindle scaffold underlines the emerging flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite Plasmodium .