A theoretical model of anaphase.

This paper develops a theory for anaphase in cells. After a brief description of microtubules, the mitotic spindle and the centrosome, a mathematical model for anaphase is introduced and developed in the context of the cell cytoplasm and liquid crystalline structures. Prophase, prometaphase and metaphase are then briefly described in order to focus on anaphase, which is the main study of this paper. The entities involved are modelled in terms of liquid crystal defects and microtubules are represented as defect flux lines. The mathematical techniques employed make extensive use of energy considerations based on the work that was developed by Dafermos (1970) from the classical Frank-Oseen nematic liquid crystal energy (Frank, 1958; Oseen, 1933). With regard to liquid crystal theory we introduce the concept of regions of influence for defects which it is believed have important implications beyond the subject of this paper. The results of this paper align with observed biochemical phenomena and are explored in application to HeLa cells and Caenorhabditis elegans. This unified approach offers the possibility of gaining insight into various consequences of mitotic abnormalities which may result in Down syndrome, Hodgkin lymphoma, breast, prostate and various other types of cancer.

Kinetochore life histories reveal an Aurora-B-dependent error correction mechanism in anaphase.

Chromosome mis-segregation during mitosis leads to aneuploidy, which is a hallmark of cancer and linked to cancer genome evolution. Errors can manifest as "lagging chromosomes" in anaphase, although their mechanistic origins and likelihood of correction are incompletely understood. Here, we combine lattice light-sheet microscopy, endogenous protein labeling, and computational analysis to define the life history of >104 kinetochores. By defining the "laziness" of kinetochores in anaphase, we reveal that chromosomes are at a considerable risk of mis-segregation. We show that the majority of lazy kinetochores are corrected rapidly in anaphase by Aurora B; if uncorrected, they result in a higher rate of micronuclei formation. Quantitative analyses of the kinetochore life histories reveal a dynamic signature of metaphase kinetochore oscillations that forecasts their anaphase fate. We propose that in diploid human cells chromosome segregation is fundamentally error prone, with an additional layer of anaphase error correction required for stable karyotype propagation.

Quantitative and Site-Specific Chemoproteomic Profiling of Protein O-GlcNAcylation in the Cell Cycle.

Mammalian cell cycle is a central process for tissue growth and maintenance. Protein O-linked ß-N-acetylglucosamine (O-GlcNAc) modification has been found to occur on several important cell cycle regulators. However, the O-GlcNAcylated proteome has not been extensively profiled during cell cycle progression. Herein, we report a quantitative profiling of protein O-GlcNAcylation sites in cell proliferation, by using an O-GlcNAc chemoproteomic strategy. In HeLa cells, a total of 902, 439, and 872 high-confidence O-GlcNAcylation sites distributed on 414, 265, and 425 proteins are identified in the interphase, early mitosis, and mitotic exit stages, respectively. The identified O-GlcNAcylation events occur on a variety of important regulators, which are involved in the processes of cell division, DNA repair, and cell death. Furthermore, we show that O-GlcNAcylation is dynamically regulated in a cell cycle stage-dependent manner. Our results provide a valuable resource for investigating the functional roles of O-GlcNAc in the mammalian cell cycle.

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.

Coupling of Cdc20 inhibition and activation by BubR1.

Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by cyclin B1-Cdk1 independently inhibits APC/C-Cdc20 activation. This creates a conundrum for how Cdc20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively, our work reveals how Cdc20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset.

Cyclin B3 activates the Anaphase-Promoting Complex/Cyclosome in meiosis and mitosis.

In mitosis and meiosis, chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit ubiquitin ligase that targets proteins for degradation, leading to the separation of chromatids. APC/C activation requires phosphorylation of its APC3 and APC1 subunits, which allows the APC/C to bind its co-activator Cdc20. The identity of the kinase(s) responsible for APC/C activation in vivo is unclear. Cyclin B3 (CycB3) is an activator of the Cyclin-Dependent Kinase 1 (Cdk1) that is required for meiotic anaphase in flies, worms and vertebrates. It has been hypothesized that CycB3-Cdk1 may be responsible for APC/C activation in meiosis but this remains to be determined. Using Drosophila, we found that mutations in CycB3 genetically enhance mutations in tws, which encodes the B55 regulatory subunit of Protein Phosphatase 2A (PP2A) known to promote mitotic exit. Females heterozygous for CycB3 and tws loss-of-function alleles lay embryos that arrest in mitotic metaphase in a maternal effect, indicating that CycB3 promotes anaphase in mitosis in addition to meiosis. This metaphase arrest is not due to the Spindle Assembly Checkpoint (SAC) because mutation of mad2 that inactivates the SAC does not rescue the development of embryos from CycB3-/+, tws-/+ females. Moreover, we found that CycB3 promotes APC/C activity and anaphase in cells in culture. We show that CycB3 physically associates with the APC/C, is required for phosphorylation of APC3, and promotes APC/C association with its Cdc20 co-activators Fizzy and Cortex. Our results strongly suggest that CycB3-Cdk1 directly activates the APC/C to promote anaphase in both meiosis and mitosis.

Evidence for anaphase pulling forces during C. elegans meiosis.

Anaphase chromosome movement is thought to be mediated by pulling forces generated by end-on attachment of microtubules to the outer face of kinetochores. However, it has been suggested that during C. elegans female meiosis, anaphase is mediated by a kinetochore-independent pushing mechanism with microtubules only attached to the inner face of segregating chromosomes. We found that the kinetochore proteins KNL-1 and KNL-3 are required for preanaphase chromosome stretching, suggesting a role in pulling forces. In the absence of KNL-1,3, pairs of homologous chromosomes did not separate and did not move toward a spindle pole. Instead, each homolog pair moved together with the same spindle pole during anaphase B spindle elongation. Two masses of chromatin thus ended up at opposite spindle poles, giving the appearance of successful anaphase.

Ase1 domains dynamically slow anaphase spindle elongation and recruit Bim1 to the midzone.

How cells regulate microtubule cross-linking activity to control the rate and duration of spindle elongation during anaphase is poorly understood. In this study, we test the hypothesis that PRC1/Ase1 proteins use distinct microtubule-binding domains to control the spindle elongation rate. Using the budding yeast Ase1, we identify unique contributions for the spectrin and carboxy-terminal domains during different phases of spindle elongation. We show that the spectrin domain uses conserved basic residues to promote the recruitment of Ase1 to the midzone before anaphase onset and slow spindle elongation during early anaphase. In contrast, a partial Ase1 carboxy-terminal truncation fails to form a stable midzone in late anaphase, produces higher elongation rates after early anaphase, and exhibits frequent spindle collapses. We find that the carboxy-terminal domain interacts with the plus-end tracking protein EB1/Bim1 and recruits Bim1 to the midzone to maintain midzone length. Overall, our results suggest that the Ase1 domains provide cells with a modular system to tune midzone activity and control elongation rates.

MKLP2 Is a Motile Kinesin that Transports the Chromosomal Passenger Complex during Anaphase.

During cytokinesis, signals from the anaphase spindle direct the formation and position of a contractile ring at the cell cortex [1]. The chromosomal passenger complex (CPC) participates in cytokinesis initiation by signaling from the spindle midzone and equatorial cortex [2], but the mechanisms underlying the anaphase-specific CPC localization are currently unresolved. Accumulation of the CPC at these sites requires the presence of microtubules and the mitotic kinesin-like protein 2, MKLP2 (KIF20A), a member of the kinesin-6 family [2-7], and this has led to the hypothesis that the CPC is transported along microtubules by MKLP2 [3-5, 7]. However, the structure of the MKLP2 motor domain with its extended neck-linker region suggests that this kinesin might not be able to drive processive transport [8, 9]. Furthermore, experiments in Xenopus egg extracts indicated that the CPC might be transported by kinesin-4, KIF4A [10]. Finally, CPC-MKLP2 complexes might be directly recruited to the equatorial cortex via association with actin and myosin II, independent of kinesin activity [4, 8]. Using microscopy-based assays with purified proteins, we demonstrate that MKLP2 is a processive plus-end directed motor that can transport the CPC along microtubules in vitro. In cells, strong suppression of MKLP2-dependent CPC motility by expression of an MKLP2 P-loop mutant perturbs CPC accumulation at both the spindle midzone and equatorial cortex, whereas a weaker inhibition of MKLP2 motor using Paprotrain mainly affects CPC localization to the equatorial cortex. Our data indicate that control of cytokinesis initiation by the CPC requires its directional MKLP2-dependent transport.

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

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

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

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

Aurora A promotes chromosome congression by activating the condensin-dependent pool of KIF4A.

Aurora kinases create phosphorylation gradients within the spindle during prometaphase and anaphase, thereby locally regulating factors that promote spindle organization, chromosome condensation and movement, and cytokinesis. We show that one such factor is the kinesin KIF4A, which is present along the chromosome axes throughout mitosis and the central spindle in anaphase. These two pools of KIF4A depend on condensin I and PRC1, respectively. Previous work has shown KIF4A is activated by Aurora B at the anaphase central spindle. However, whether or not chromosome-associated KIF4A bound to condensin I is regulated by Aurora kinases remain unclear. To determine the roles of the two different pools of KIF4A, we generated specific point mutants that are unable to interact with either condensin I or PRC1 or are deficient for Aurora kinase regulation. By analyzing these mutants, we show that Aurora A phosphorylates the condensin I-dependent pool of KIF4A and thus actively promotes chromosome congression from the spindle poles to the metaphase plate.

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.

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

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

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

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

Direct observation of branching MT nucleation in living animal cells.

Centrosome-mediated microtubule (MT) nucleation has been well characterized; however, numerous noncentrosomal MT nucleation mechanisms exist. The branching MT nucleation pathway envisages that the γ-tubulin ring complex (γ-TuRC) is recruited to MTs by the augmin complex to initiate nucleation of new MTs. While the pathway is well conserved at a molecular and functional level, branching MT nucleation by core constituents has never been directly observed in animal cells. Here, multicolor TIRF microscopy was applied to visualize and quantitatively define the entire process of branching MT nucleation in dividing Drosophila cells during anaphase. The steps of a stereotypical branching nucleation event entailed augmin binding to a mother MT and recruitment of γ-TuRC after 15 s, followed by nucleation 16 s later of a daughter MT at a 36° branch angle. Daughters typically remained attached throughout their ∼40-s lifetime unless the mother depolymerized past the branch point. Assembly of branched MT arrays, which did not require Drosophila TPX2, enhanced localized RhoA activation during cytokinesis.

Self-Organization of Minimal Anaphase Spindle Midzone Bundles.

In anaphase spindles, antiparallel microtubules associate to form tight midzone bundles, as required for functional spindle architecture and correct chromosome segregation. Several proteins selectively bind to these overlaps to control cytokinesis. How midzone bundles assemble is poorly understood. Here, using an in vitro reconstitution approach, we demonstrate that minimal midzone bundles can reliably self-organize in solution from dynamic microtubules, the microtubule crosslinker PRC1, and the motor protein KIF4A. The length of the central antiparallel overlaps in these microtubule bundles is similar to that observed in cells and is controlled by the PRC1/KIF4A ratio. Experiments and computer simulations demonstrate that minimal midzone bundle formation results from promoting antiparallel microtubule crosslinking, stopping microtubule plus-end dynamicity, and motor-driven midzone compaction and alignment. The robustness of this process suggests that a similar self-organization mechanism may contribute to the reorganization of the spindle architecture during the metaphase to anaphase transition in cells.

Role for polo-like kinase 4 in mediation of cytokinesis.

The mitotic protein polo-like kinase 4 (PLK4) plays a critical role in centrosome duplication for cell division. By using immunofluorescence, we confirm that PLK4 is localized to centrosomes. In addition, we find that phospho-PLK4 (pPLK4) is cleaved and distributed to kinetochores (metaphase and anaphase), spindle midzone/cleavage furrow (anaphase and telophase), and midbody (cytokinesis) during cell division in immortalized epithelial cells as well as breast, ovarian, and colorectal cancer cells. The distribution of pPLK4 midzone/cleavage furrow and midbody positions pPLK4 to play a functional role in cytokinesis. Indeed, we found that inhibition of PLK4 kinase activity with a small-molecule inhibitor, CFI-400945, prevents translocation to the spindle midzone/cleavage furrow and prevents cellular abscission, leading to the generation of cells with polyploidy, increased numbers of duplicated centrosomes, and vulnerability to anaphase or mitotic catastrophe. The regulatory role of PLK4 in cytokinesis makes it a potential target for therapeutic intervention in appropriately selected cancers.

Shugoshin protects centromere pairing and promotes segregation of nonexchange partner chromosomes in meiosis.

Faithful chromosome segregation during meiosis I depends upon the formation of connections between homologous chromosomes. Crossovers between homologs connect the partners, allowing them to attach to the meiotic spindle as a unit, such that they migrate away from one another at anaphase I. Homologous partners also become connected by pairing of their centromeres in meiotic prophase. This centromere pairing can promote proper segregation at anaphase I of partners that have failed to become joined by a crossover. Centromere pairing is mediated by synaptonemal complex (SC) proteins that persist at the centromere when the SC disassembles. Here, using mouse spermatocyte and yeast model systems, we tested the role of shugoshin in promoting meiotic centromere pairing by protecting centromeric synaptonemal components from disassembly. The results show that shugoshin protects the centromeric SC in meiotic prophase and, in anaphase, promotes the proper segregation of partner chromosomes that are not linked by a crossover.