Kinetochores In Mitosis Research Articles

ZW10: an emerging orchestrator of organelle dynamics during the cell division cycle.

Zeste white 10 (ZW10) was first identified as a centromere/kinetochore protein encoded by the ZW10 gene in Drosophila. ZW10 guides the spindle assembly checkpoint signaling during mitotic chromosome segregation in metazoans. Recent studies have shown that ZW10 is also involved in membranous organelle interactions during interphase and plays a vital role in membrane transport between the endoplasmic reticulum and Golgi apparatus. Despite these findings, the precise molecular mechanisms by which ZW10 regulates interactions between membranous organelles in interphase and the assembly of membraneless organelle kinetochore in mitosis remain elusive. Here, we highlight how ZW10 forms context-dependent protein complexes during the cell cycle. These complexes are essential for mediating membrane trafficking in interphase and ensuring the accurate segregation of chromosomes in mitosis.

Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint.

Cryptococcus neoformans is an opportunistic, human fungal pathogen which undergoes fascinating switches in cell cycle control and ploidy when it encounters stressful environments such as the human lung. Here we carry out a mechanistic analysis of the spindle checkpoint which regulates the metaphase to anaphase transition, focusing on Mps1 kinase and the downstream checkpoint components Mad1 and Mad2. We demonstrate that Cryptococcus mad1Δ or mad2Δ strains are unable to respond to microtubule perturbations, continuing to re-bud and divide, and die as a consequence. Fluorescent tagging of Chromosome 3, using a lacO array and mNeonGreen-lacI fusion protein, demonstrates that mad mutants are unable to maintain sister-chromatid cohesion in the absence of microtubule polymers. Thus, the classic checkpoint functions of the SAC are conserved in Cryptococcus. In interphase, GFP-Mad1 is enriched at the nuclear periphery, and it is recruited to unattached kinetochores in mitosis. Purification of GFP-Mad1 followed by mass spectrometric analysis of associated proteins show that it forms a complex with Mad2 and that it interacts with other checkpoint signalling components (Bub1) and effectors (Cdc20 and APC/C sub-units) in mitosis. We also demonstrate that overexpression of Mps1 kinase is sufficient to arrest Cryptococcus cells in mitosis, and show that this arrest is dependent on both Mad1 and Mad2. We find that a C-terminal fragment of Mad1 is an effective in vitro substrate for Mps1 kinase and map several Mad1 phosphorylation sites. Some sites are highly conserved within the C-terminal Mad1 structure and we demonstrate that mutation of threonine 667 (T667A) leads to loss of checkpoint signalling and abrogation of the GAL-MPS1 arrest. Thus Mps1-dependent phosphorylation of C-terminal Mad1 residues is a critical step in Cryptococcus spindle checkpoint signalling. We conclude that CnMps1 protein kinase, Mad1 and Mad2 proteins have all conserved their important, spindle checkpoint signalling roles helping ensure high fidelity chromosome segregation.

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.

Histone H3K79 methylation by DOT1L promotes Aurora B localization at centromeres in mitosis

Centromere localization of the chromosome passenger complex (CPC) is paramount for achieving accurate sister chromosome segregation in mitosis. Although it has been widely recognized that the recruitment of CPC is directly regulated by two histone codes, phosphorylation of histone H3 at threonine 3 (H3T3ph) and phosphorylation of histone H2A at threonine 120 (H2AT120ph), the regulation of CPC localization by other histone codes remains elusive. We show that dysfunction of disruptor of telomeric silencing 1 like (DOT1L) leads to mislocation of the CPC in prometaphase, caused by disturbing the level of H3T3ph and its reader Survivin. This cascade is initiated by over-dephosphorylation of H3T3ph mediated by the phosphatase RepoMan-PP1, whose scaffold RepoMan translocalizes to chromosomes, while the level of H3K79me2/3 is diminished. Together, our findings uncover a biological function of DOT1L and H3K79 methylation in mitosis and give insight into how genomic stability is coordinated by different histone codes.

The function, structure and dynamic organization of centromeres and kinetochores

It is a fundamental task to ensure the faithful transmission of genetic information across generations for eukaryote species. The centromere is a specialized chromosomal region that is essential for mediating sister chromatid alignment and separation during mitosis. Centromere identity is epigenetically determined by nucleosome-containing centromere protein A (CENP-A). The CENP-A nucleosome provides the foundation for the association of the inner kinetochore and the assembly of the outer kinetochore in mitosis. Here we review centromere identity determination, inner kinetochore function and assembly, and outer kinetochore function and assembly. In particular, we focus on the recent advances in the structure-activity relationship of the constitutive centromere-associated network (CCAN). CCAN structure information sheds new light on our understanding of centromere and kinetochore functions and dynamic organization.

CBIO-24. KINETOCHORE MISREGULATION IN GLIOBLASTOMA AND OTHER CANCERS

Abstract Aneuploidy is a hallmark of most late stage cancers and contributes to tumor cell evolution, invasiveness, therapy resistance, and recurrence. A major cause of aneuploidy is lagging anaphase chromosomes that have merotelic kinetochore (KT) attachments in mitosis. In this scenario, a KT (i.e., the proteinaceous structure assembled onto centromeres in mitosis) is bound to microtubules (MTs) emanating from both spindle poles, causing chromosome mis-segregation. “Background” chromosome missegregation likely contributes to the initiation of multiple cancers, including glioblastoma (GBM), where chr 7 gain and chr 10 loss are early events. However, we have found that in GBM and transformed cells activation of the RAS signaling pathway significantly increases lagging anaphase chromosomes, and, as a result, aneuploidy and tumor heterogeneity. However, the underlying mechanism of RAS-induced chromosome instability has remained elusive. Here we present one possible cause: MAPK-driven KT stress. The hallmarks of KT stress include chronic KT-MT attachment instability in mitosis, dramatic changes in KT morphology, and increases in merotelic KT attachments and chromosome segregation errors. KT stress is lethal to cancer cells. However, stressed cells survive by relying on BUB1B/BubR1 activity to strengthen KT-MT attachments. We first observed KT stress in GBM patient isolates, where ~60–70% display its hallmarks. We subsequently found that RAS or MEK activity is sufficient to induce KT stress in both CNS and non-CNS derived cell types. We propose that KT stress is caused by aberrant mitotic RAS/MAPK activity, which likely targets one or more KT proteins resulting in KT-MT attachment defects and chromosome instability. In addition, Dr. Jun Zhu’s lab has also created a computational classifier that can identify KT stressed cells and tumors based on the expression of 838 genes associated with KT stress, which can be also used to identify orthogonal therapeutic sensitivities. We will present these findings as well at this meeting.

Kinetochore protein Spindly controls microtubule polarity in Drosophila axons

Microtubule polarity in axons and dendrites defines the direction of intracellular transport in neurons. Axons contain arrays of uniformly polarized microtubules with plus-ends facing the tips of the processes (plus-end-out), while dendrites contain microtubules with a minus-end-out orientation. It has been shown that cytoplasmic dynein, targeted to cortical actin, removes minus-end-out microtubules from axons. Here we have identified Spindly, a protein known for recruitment of dynein to kinetochores in mitosis, as a key factor required for dynein-dependent microtubule sorting in axons of Drosophila neurons. Depletion of Spindly affects polarity of axonal microtubules in vivo and in primary neuronal cultures. In addition to these defects, depletion of Spindly in neurons causes major collapse of axonal patterning in the third-instar larval brain as well as severe coordination impairment in adult flies. These defects can be fully rescued by full-length Spindly, but not by variants with mutations in its dynein-binding site. Biochemical analysis demonstrated that Spindly binds F-actin, suggesting that Spindly serves as a link between dynein and cortical actin in axons. Therefore, Spindly plays a critical role during neurodevelopment by mediating dynein-driven sorting of axonal microtubules.

Molecular basis of MKLP2-dependent Aurora B transport from chromatin to the anaphase central spindle.

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.

Mps1-mediated release of Mad1 from nuclear pores ensures the fidelity of chromosome segregation.

The spindle assembly checkpoint (SAC) relies on the recruitment of Mad1-C-Mad2 to unattached kinetochores but also on its binding to Megator/Tpr at nuclear pore complexes (NPCs) during interphase. However, the molecular underpinnings controlling the spatiotemporal redistribution of Mad1-C-Mad2 as cells progress into mitosis remain elusive. Here, we show that activation of Mps1 during prophase triggers Mad1 release from NPCs and that this is required for kinetochore localization of Mad1-C-Mad2 and robust SAC signaling. We find that Mps1 phosphorylates Megator/Tpr to reduce its interaction with Mad1 in vitro and in Drosophila cells. Importantly, preventing Mad1 from binding to Megator/Tpr restores Mad1 accumulation at kinetochores, the fidelity of chromosome segregation, and genome stability in larval neuroblasts of mps1-null mutants. Our findings demonstrate that the subcellular localization of Mad1 is tightly coordinated with cell cycle progression by kinetochore-extrinsic activity of Mps1. This ensures that both NPCs in interphase and kinetochores in mitosis can generate anaphase inhibitors to efficiently preserve genomic stability.

A Kinesin-5, Cin8, Recruits Protein Phosphatase 1 to Kinetochores and Regulates Chromosome Segregation

Kinesin-5 is a highly conserved homo-tetrameric protein complex responsible for crosslinking microtubules and pushing spindle poles apart. The budding yeast Kinesin-5, Cin8, is highly concentrated at kinetochores in mitosis before anaphase, but its functions there are largely unsolved. Here, we show that Cin8 localizes to kinetochores in a cell-cycle-dependent manner and concentrates near the microtubule binding domains of Ndc80 at metaphase. Cin8's kinetochore localization depends on the Ndc80 complex, kinetochore microtubules, and the Dam1 complex. Consistent with its kinetochore localization, a Cin8 deletion induces a loss of tension at the Ndc80 microtubule binding domains and a major delay in mitotic progression. Cin8 associates with Protein Phosphatase 1 (PP1), and mutants that inhibit its PP1 binding also induce a loss of tension at the Ndc80 microtubule binding domains and delay mitotic progression. Taken together, our results suggest that Cin8-PP1 plays a critical role at kinetochores to promote accurate chromosome segregation by controlling Ndc80 attachment to microtubules.

Chromosomes trapped in micronuclei are liable to segregation errors.

ABSTRACTDNA in micronuclei is likely to get damaged. When shattered DNA from micronuclei gets reincorporated into the primary nucleus, aberrant rearrangements can take place, a phenomenon referred to as chromothripsis. Here, we investigated how chromatids from micronuclei act in subsequent divisions and how this affects their fate. We observed that the majority of chromatids derived from micronuclei fail to establish a proper kinetochore in mitosis, which is associated with problems in chromosome alignment, segregation and spindle assembly checkpoint activation. Remarkably, we found that, upon their formation, micronuclei already display decreased levels of important kinetochore assembly factors. Importantly, these defects favour the exclusion of the micronucleus over the reintegration into the primary nucleus over several divisions. Interestingly, the defects observed in micronuclei are likely overcome once micronuclei are reincorporated into the primary nuclei, as they further propagate normally. We conclude that the formation of a separate small nuclear entity represents a mechanism for the cell to delay the stable propagation of excess chromosome(s) and/or damaged DNA, by inducing kinetochore defects.

Seh1 targets GATOR2 and Nup153 to mitotic chromosomes

ABSTRACTIn metazoa, the Nup107 complex (also known as the nucleoporin Y-complex) plays a major role in formation of the nuclear pore complex in interphase and is localised to kinetochores in mitosis. The Nup107 complex shares a single highly conserved subunit, Seh1 (also known as SEH1L in mammals) with the GATOR2 complex, an essential activator of mTORC1 kinase. mTORC1/GATOR2 has a central role in the coordination of cell growth and proliferation. Here, we use chemical genetics and quantitative chromosome proteomics to study the role of the Seh1 protein in mitosis. Surprisingly, Seh1 is not required for the association of the Nup107 complex with mitotic chromosomes, but it is essential for the association of both the GATOR2 complex and nucleoporin Nup153 with mitotic chromosomes. Our analysis also reveals a role for Seh1 at human centromeres, where it is required for efficient localisation of the chromosomal passenger complex (CPC). Furthermore, this analysis detects a functional interaction between the Nup107 complex and the small kinetochore protein SKAP (also known as KNSTRN).

Disentangling the molecular determinants for Cenp-F localization to nuclear pores and kinetochores.

Cenp-F is a multifaceted protein implicated in cancer and developmental pathologies. The Cenp-F C-terminal region contains overlapping binding sites for numerous proteins that contribute to its functions throughout the cell cycle. Here, we focus on the nuclear pore protein Nup133 that interacts with Cenp-F both at nuclear pores in prophase and at kinetochores in mitosis, and on the kinase Bub1, known to contribute to Cenp-F targeting to kinetochores. By combining in silico structural modeling and yeast two-hybrid assays, we generate an interaction model between a conserved helix within the Nup133 β-propeller and a short leucine zipper-containing dimeric segment of Cenp-F. We thereby create mutants affecting the Nup133/Cenp-F interface and show that they prevent Cenp-F localization to the nuclear envelope, but not to kinetochores. Conversely, a point mutation within an adjacent leucine zipper affecting the kinetochore targeting of Cenp-F KT-core domain impairs its interaction with Bub1, but not with Nup133, identifying Bub1 as the direct KT-core binding partner of Cenp-F. Finally, we show that Cenp-E redundantly contributes together with Bub1 to the recruitment of Cenp-F to kinetochores.

Spindly is required for rapid migration of human cells.

ABSTRACTDynein is the sole processive minus-end-directed microtubule motor found in animals. It has roles in cell division, membrane trafficking, and cell migration. Together with dynactin, dynein regulates centrosomal orientation to establish and maintain cell polarity, controls focal adhesion turnover and anchors microtubules at the leading edge. In higher eukaryotes, dynein/dynactin requires additional components such as Bicaudal D to form an active motor complex and for regulating its cellular localization. Spindly is a protein that targets dynein/dynactin to kinetochores in mitosis and can activate its motility in vitro. However, no role for Spindly in interphase dynein/dynactin function has been found. We show that Spindly binds to the cell cortex and microtubule tips and colocalizes with dynein/dynactin at the leading edge of migrating U2OS cells and primary fibroblasts. U2OS cells that lack Spindly migrated slower in 2D than control cells, although centrosome polarization appeared to happen properly in the absence of Spindly. Re-expression of Spindly rescues migration, but the expression of a mutant, which is defective for dynactin binding, failed to rescue this defect. Taken together, these data demonstrate that Spindly plays an important role in mediating a subset of dynein/dynactin's function in cell migration.

Unexpected roles of a shugoshin protein at subtelomeres

A chromosome is composed of structurally and functionally distinct domains. Telomeres, which are located at the ends of linear chromosomes, play crucial roles in genome stability. Although substantial knowledge of telomeres has been accumulated, the regulation and function of subtelomeres, which are the domains adjacent to telomeres, remain largely unknown. In this review, I describe recent discoveries about the multiple roles of a shugoshin family protein, Sgo2, which is localized at centromeres in mitosis and contributes to precise chromosome segregation, in defining chromatin structure and functions of the subtelomeres in fission yeast. Sgo2 becomes enriched at the subtelomeres, particularly during G2 phase, and is essential for the formation of a highly condensed subtelomeric chromatin body called the knob. Furthermore, Sgo2 maintains the expression levels of subtelomeric genes and the timing of DNA replication at subtelomeric late origins.

A noncatalytic function of the topoisomerase II CTD in Aurora B recruitment to inner centromeres during mitosis.

Faithful chromosome segregation depends on the precise timing of chromatid separation, which is enforced by checkpoint signals generated at kinetochores. Here, we provide evidence that the C-terminal domain (CTD) of DNA topoisomerase IIα (Topo II) provides a novel function at inner centromeres of kinetochores in mitosis. We find that the yeast CTD is required for recruitment of the tension checkpoint kinase Ipl1/Aurora B to inner centromeres in metaphase but is not required in interphase. Conserved CTD SUMOylation sites are required for Ipl1 recruitment. This inner-centromere CTD function is distinct from the catalytic activity of Topo II. Genetic and biochemical evidence suggests that Topo II recruits Ipl1 via the Haspin-histone H3 threonine 3 phosphorylation pathway. Finally, Topo II and Sgo1 are equally important for Ipl1 recruitment to inner centromeres. This indicates H3 T3-Phos/H2A T120-Phos is a universal epigenetic signature that defines the eukaryotic inner centromere and provides the binding site for Ipl1/Aurora B.

SUMOylation of DNA topoisomerase IIα regulates histone H3 kinase Haspin and H3 phosphorylation in mitosis.

DNA topoisomerase II (TOP2) plays a pivotal role in faithful chromosome separation through its strand-passaging activity that resolves tangled genomic DNA during mitosis. Additionally, TOP2 controls progression of mitosis by activating cell cycle checkpoints. Recent work showed that the enzymatically inert C-terminal domain (CTD) of TOP2 and its posttranslational modification are critical to this checkpoint regulation. However, the molecular mechanism has not yet been determined. By using Xenopus laevis egg extract, we found that SUMOylation of DNA topoisomerase IIα (TOP2A) CTD regulates the localization of the histone H3 kinase Haspin and phosphorylation of histone H3 at threonine 3 at the centromere, two steps known to be involved in the recruitment of the chromosomal passenger complex (CPC) to kinetochores in mitosis. Robust centromeric Haspin localization requires SUMOylated TOP2A CTD binding activity through SUMO-interaction motifs and the phosphorylation of Haspin. We propose a novel mechanism through which the TOP2 CTD regulates the CPC via direct interaction with Haspin at mitotic centromeres.

ChromoShake: a chromosome dynamics simulator reveals that chromatin loops stiffen centromeric chromatin.

ChromoShake is a three-dimensional simulator designed to find the thermodynamically favored states for given chromosome geometries. The simulator has been applied to a geometric model based on experimentally determined positions and fluctuations of DNA and the distribution of cohesin and condensin in the budding yeast centromere. Simulations of chromatin in differing initial configurations reveal novel principles for understanding the structure and function of a eukaryotic centromere. The entropic position of DNA loops mirrors their experimental position, consistent with their radial displacement from the spindle axis. The barrel-like distribution of cohesin complexes surrounding the central spindle in metaphase is a consequence of the size of the DNA loops within the pericentromere to which cohesin is bound. Linkage between DNA loops of different centromeres is requisite to recapitulate experimentally determined correlations in DNA motion. The consequences of radial loops and cohesin and condensin binding are to stiffen the DNA along the spindle axis, imparting an active function to the centromere in mitosis.

High evolutionary turnover of satellite families in Caenorhabditis.

BackgroundThe high density of tandem repeat sequences (satellites) in nematode genomes and the availability of genome sequences from several species in the group offer a unique opportunity to better understand the evolutionary dynamics and the functional role of these sequences. We take advantage of the previously developed SATFIND program to study the satellites in four Caenorhabditis species and investigate these questions.MethodsThe identification and comparison of satellites is carried out in three steps. First we find all the satellites present in each species with the SATFIND program. Each satellite is defined by its length, number of repeats, and repeat sequence. Only satellites with at least ten repeats are considered. In the second step we build satellite families with a newly developed alignment program. Satellite families are defined by a consensus sequence and the number of satellites in the family. Finally we compare the consensus sequence of satellite families in different species.ResultsWe give a catalog of individual satellites in each species. We have also identified satellite families with a related sequence and compare them in different species. We analyze the turnover of satellites: they increased in size through duplications of fragments of 100-300 bases. It appears that in many cases they have undergone an explosive expansion. In C. elegans we have identified a subset of large satellites that have strong affinity for the centromere protein CENP-A. We have also compared our results with those obtained from other species, including one nematode and three mammals.ConclusionsMost satellite families found in Caenorhabditis are species-specific; in particular those with long repeats. A subset of these satellites may facilitate the formation of kinetochores in mitosis. Other satellite families in C. elegans are either related to Helitron transposons or to meiotic pairing centers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0495-x) contains supplementary material, which is available to authorized users.

DNA loops generate intracentromere tension in mitosis.

The centromere is the DNA locus that dictates kinetochore formation and is visibly apparent as heterochromatin that bridges sister kinetochores in metaphase. Sister centromeres are compacted and held together by cohesin, condensin, and topoisomerase-mediated entanglements until all sister chromosomes bi-orient along the spindle apparatus. The establishment of tension between sister chromatids is essential for quenching a checkpoint kinase signal generated from kinetochores lacking microtubule attachment or tension. How the centromere chromatin spring is organized and functions as a tensiometer is largely unexplored. We have discovered that centromere chromatin loops generate an extensional/poleward force sufficient to release nucleosomes proximal to the spindle axis. This study describes how the physical consequences of DNA looping directly underlie the biological mechanism for sister centromere separation and the spring-like properties of the centromere in mitosis.