Age-dependent loss of cohesion protection in human oocytes.

Aneuploid human eggs (oocytes) are a major cause of infertility, miscarriage, and chromosomal disorders. Such aneuploidies increase greatly as women age, with defective linkages between sister chromatids (cohesion) in meiosis as a common cause. We found that loss of a specific pool of the cohesin protector protein, shugoshin 2 (SGO2), may contribute to this phenomenon. Our data indicate that SGO2 preserves sister chromatid cohesion in meiosis by protecting a "cohesin bridge" between sister chromatids. In human oocytes, SGO2 localizes to both sub-centromere cups and the pericentromeric bridge, which spans the sister chromatid junction. SGO2 normally colocalizes with cohesin; however, in meiosis II oocytes from older women, SGO2 is frequently lost from the pericentromeric bridge and sister chromatid cohesion is weakened. MPS1 and BUB1 kinase activities maintain SGO2 at sub-centromeres and the pericentromeric bridge. Removal of SGO2 throughout meiosis I by MPS1 inhibition reduces cohesion protection, increasing the incidence of single chromatids at meiosis II. Therefore, SGO2 deficiency in human oocytes can exacerbate the effects of maternal age by rendering residual cohesin at pericentromeres vulnerable to loss in anaphase I. Our data show that impaired SGO2 localization weakens cohesion integrity and may contribute to the increased incidence of aneuploidy observed in human oocytes with advanced maternal age.

Principles and dynamics of spindle assembly checkpoint signalling.

The transmission of a complete set of chromosomes to daughter cells during cell division is vital for development and tissue homeostasis. The spindle assembly checkpoint (SAC) ensures correct segregation by informing the cell cycle machinery of potential errors in the interactions of chromosomes with spindle microtubules prior to anaphase. To do so, the SAC monitors microtubule engagement by specialized structures known as kinetochores and integrates local mechanical and chemical cues such that it can signal in a sensitive, responsive and robust manner. In this Review, we discuss how SAC proteins interact to allow production of the mitotic checkpoint complex (MCC) that halts anaphase progression by inhibiting the anaphase-promoting complex/cyclosome (APC/C). We highlight recent advances aimed at understanding the dynamic signalling properties of the SAC and how it interprets various naturally occurring intermediate attachment states. Further, we discuss SAC signalling in the context of the mammalian multisite kinetochore and address the impact of the fibrous corona. We also identify current challenges in understanding how the SAC ensures high-fidelity chromosome segregation.

Bayesian inference of multi-point macromolecular architecture mixtures at nanometre resolution.

Gaussian spot fitting methods have significantly extended the spatial range where fluorescent microscopy can be used, with recent techniques approaching nanometre (nm) resolutions. However, small inter-fluorophore distances are systematically over-estimated for typical molecular scales. This bias can be corrected computationally, but current algorithms are limited to correcting distances between pairs of fluorophores. Here we present a flexible Bayesian computational approach that infers the distances and angles between multiple fluorophores and has several advantages over these previous methods. Specifically it improves confidence intervals for small lengths, estimates measurement errors of each fluorophore individually and infers the correlations between polygon lengths. The latter is essential for determining the full multi-fluorophore 3D architecture. We further developed the algorithm to infer the mixture composition of a heterogeneous population of multiple polygon states. We use our algorithm to analyse the 3D architecture of the human kinetochore, a macro-molecular complex that is essential for high fidelity chromosome segregation during cell division. Using triple fluorophore image data we unravel the mixture of kinetochore states during human mitosis, inferring the conformation of microtubule attached and unattached kinetochores and their proportions across mitosis. We demonstrate that the attachment conformation correlates with intersister tension and sister alignment to the metaphase plate.

The first mitotic division of human embryos is highly error prone.

Human beings are made of ~50 trillion cells which arise from serial mitotic divisions of a single cell - the fertilised egg. Remarkably, the early human embryo is often chromosomally abnormal, and many are mosaic, with the karyotype differing from one cell to another. Mosaicism presumably arises from chromosome segregation errors during the early mitotic divisions, although these events have never been visualised in living human embryos. Here, we establish live cell imaging of chromosome segregation using normally fertilised embryos from an egg-share-to-research programme, as well as embryos deselected during fertility treatment. We reveal that the first mitotic division has an extended prometaphase/metaphase and exhibits phenotypes that can cause nondisjunction. These included multipolar chromosome segregations and lagging chromosomes that lead to formation of micronuclei. Analysis of nuclear number and size provides evidence of equivalent phenotypes in 2-cell human embryos that gave rise to live births. Together this shows that errors in the first mitotic division can be tolerated in human embryos and uncovers cell biological events that contribute to preimplantation mosaicism.

The Four Causes: The Functional Architecture of Centromeres and Kinetochores.

Kinetochores are molecular machines that power chromosome segregation during the mitotic and meiotic cell divisions of all eukaryotes. Aristotle explains how we think we have knowledge of a thing only when we have grasped its cause. In our case, to gain understanding of the kinetochore, the four causes correspond to questions that we must ask: (a) What are the constituent parts, (b) how does it assemble, (c) what is the structure and arrangement, and (d) what is the function? Here we outline the current blueprint for the assembly of a kinetochore, how functions are mapped onto this architecture, and how this is shaped by the underlying pericentromeric chromatin. The view of the kinetochore that we present is possible because an almost complete parts list of the kinetochore is now available alongside recent advances using in vitro reconstitution, structural biology, and genomics. In many organisms, each kinetochore binds to multiple microtubules, and we propose a model for how this ensemble-level architecture is organized, drawing on key insights from the simple one microtubule-one kinetochore setup in budding yeast and innovations that enable meiotic chromosome segregation.

Evidence for a HURP/EB free mixed-nucleotide zone in kinetochore-microtubules.

Current models infer that the microtubule-based mitotic spindle is built from GDP-tubulin with small GTP caps at microtubule plus-ends, including those that attach to kinetochores, forming the kinetochore-fibres. Here we reveal that kinetochore-fibres additionally contain a dynamic mixed-nucleotide zone that reaches several microns in length. This zone becomes visible in cells expressing fluorescently labelled end-binding proteins, a known marker for GTP-tubulin, and endogenously-labelled HURP - a protein which we show to preferentially bind the GDP microtubule lattice in vitro and in vivo. We find that in mitotic cells HURP accumulates on the kinetochore-proximal region of depolymerising kinetochore-fibres, whilst avoiding recruitment to nascent polymerising K-fibres, giving rise to a growing "HURP-gap". The absence of end-binding proteins in the HURP-gaps leads us to postulate that they reflect a mixed-nucleotide zone. We generate a minimal quantitative model based on the preferential binding of HURP to GDP-tubulin to show that such a mixed-nucleotide zone is sufficient to recapitulate the observed in vivo dynamics of HURP-gaps.

Kinetochore tracking in 3D from lattice light-sheet imaging data with KiT.

MOTIVATION: Lattice light-sheet microscopy (LLSM) is revolutionizing cell biology since it enables fast, high-resolution extended imaging in three dimensions combined with a drastic reduction in photo-toxicity and bleaching. However, analysis of such datasets still remains a major challenge. RESULTS: Automated tracking of kinetochores, the protein complex facilitating and controlling microtubule attachment of the chromosomes within the mitotic spindle, provides quantitative assessment of chromosome dynamics in mitosis. Here, we extend existing open-source kinetochore tracking software (KiT) to track (and pair) kinetochores throughout prometaphase to anaphase in LLSM data. One of the key improvements is a regularization term in the objective function to enforce biological information about the number of kinetochores in a human mitotic cell, as well as improved diagnostic tools. This software provides quantitative insights into how kinetochores robustly ensure congression and segregation of chromosomes during mitosis. AVAILABILITY AND IMPLEMENTATION: KiT is free, open-source software implemented in MATLAB and can be downloaded as a package from https://github.com/cmcb-warwick/KiT. The source repository is available at https://bitbucket.org/jarmond/kit (tag v2.4.0) and under continuing development. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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.

Subcellular Euclidean distance measurements with multicolor fluorescence localization imaging in cultured cells.

This protocol measures the 3D Euclidean distance (Δ3D) between two/three fluorescently labeled kinetochore components in fixed samples using Kinetochore Delta software (KiDv1.0.1, MATLAB based). Overestimation of mean Δ3D is corrected through a Bayesian algorithm, with ΔEC distances reflecting the ensemble average positions of fluorophores within a kinetochore population. This package also enables kinetochore categorization, which can be used to sub-sample kinetochores and measure ΔEC. Together, this allows the dynamic architecture of human kinetochores to be investigated (tested in hTERT-RPE1 cells). For complete details on the use and execution of this protocol, please refer to Roscioli et al. (2020).

Ensemble-Level Organization of Human Kinetochores and Evidence for Distinct Tension and Attachment Sensors.

Kinetochores are multi-protein machines that form dynamic attachments to microtubules and control chromosome segregation. High fidelity is ensured because kinetochores can monitor attachment status and tension, using this information to activate checkpoints and error-correction mechanisms. To explore how kinetochores achieve this, we used two- and three-color subpixel fluorescence localization to define how proteins from six major complexes (CCAN, MIS12, NDC80, KNL1, RZZ, and SKA) and the checkpoint proteins Bub1, Mad1, and Mad2 are organized in the human kinetochore. This reveals how the outer kinetochore has a high nematic order and is largely invariant to the loss of attachment or tension, except for two mechanical sensors. First, Knl1 unravels to relay tension, and second, NDC80 undergoes jackknifing and loss of nematic order under microtubule detachment, with only the latter wired up to the checkpoint signaling system. This provides insight into how kinetochores integrate mechanical signals to promote error-free chromosome segregation.

CENP-F stabilizes kinetochore-microtubule attachments and limits dynein stripping of corona cargoes.

Accurate chromosome segregation demands efficient capture of microtubules by kinetochores and their conversion to stable bioriented attachments that can congress and then segregate chromosomes. An early event is the shedding of the outermost fibrous corona layer of the kinetochore following microtubule attachment. Centromere protein F (CENP-F) is part of the corona, contains two microtubule-binding domains, and physically associates with dynein motor regulators. Here, we have combined CRISPR gene editing and engineered separation-of-function mutants to define how CENP-F contributes to kinetochore function. We show that the two microtubule-binding domains make distinct contributions to attachment stability and force transduction but are dispensable for chromosome congression. We further identify a specialized domain that functions to limit the dynein-mediated stripping of corona cargoes through a direct interaction with Nde1. This antagonistic activity is crucial for maintaining the required corona composition and ensuring efficient kinetochore biorientation.

Bub1 is not essential for the checkpoint response to unattached kinetochores in diploid human cells.

Error-free chromosome segregation during mitosis depends on a functional spindle assembly checkpoint (SAC). The SAC is a multi-component signalling system that is recruited to unattached or incorrectly attached kinetochores to catalyse the formation of a soluble inhibitor, known as the Mitotic Checkpoint Complex (MCC), which binds and inhibits the anaphase promoting complex (APC/C) [1]. We have previously proposed that two separable pathways, composed of KNL1-Bub3-Bub1 (KBB) and Rod-Zwilch-Zw10 (RZZ), recruit Mad1-Mad2 complexes to human kinetochores to activate the SAC [2]. Although Bub1 is absolutely required for checkpoint signalling in yeast (which lack RZZ), there is conflicting evidence as to whether this is the case in human cells based on siRNA studies [2-5]. Here we show that, while Bub1 is required for recruitment of BubR1, it is not strictly required for the checkpoint response to unattached kinetochores in diploid human cells.

Reconstitution of a 26-Subunit Human Kinetochore Reveals Cooperative Microtubule Binding by CENP-OPQUR and NDC80.

The approximately thirty core subunits of kinetochores assemble on centromeric chromatin containing the histone H3 variant CENP-A and connect chromosomes with spindle microtubules. The chromatin proximal 16-subunit CCAN (constitutive centromere associated network) creates a mechanically stable bridge between CENP-A and the kinetochore's microtubule-binding machinery, the 10-subunit KMN assembly. Here, we reconstituted a stoichiometric 11-subunit human CCAN core that forms when the CENP-OPQUR complex binds to a joint interface on the CENP-HIKM and CENP-LN complexes. The resulting CCAN particle is globular and connects KMN and CENP-A in a 26-subunit recombinant particle. The disordered, basic N-terminal tail of CENP-Q binds microtubules and promotes accurate chromosome alignment, cooperating with KMN in microtubule binding. The N-terminal basic tail of the NDC80 complex, the microtubule-binding subunit of KMN, can functionally replace the CENP-Q tail. Our work dissects the connectivity and architecture of CCAN and reveals unexpected functional similarities between CENP-OPQUR and the NDC80 complex.

Mitotic live-cell imaging at different timescales.

Mitosis is a highly dynamic and choreographed process in which chromosomes are captured by the mitotic spindle and physically segregated into the two daughter cells to ensure faithful transmission of the genetic material. Live-cell fluorescence microscopy enables these dynamics to be analyzed over diverse temporal scales. Here we present the methodologies to study chromosome segregation at three timescales: we first show how automated tracking of kinetochores enables investigation of mitotic spindle and chromosome dynamics in the seconds-to-minutes timescale; next we highlight how new DNA live dyes allow the study of chromosome segregation over a period of several hours in any cell line; finally, we demonstrate how image sequences acquired over several days can reveal the fate of whole cell populations over several consecutive cell divisions.

Complete microtubule-kinetochore occupancy favours the segregation of merotelic attachments.

Kinetochores are multi-protein complexes that power chromosome movements by tracking microtubules plus-ends in the mitotic spindle. Human kinetochores bind up to 20 microtubules, even though single microtubules can generate sufficient force to move chromosomes. Here, we show that high microtubule occupancy at kinetochores ensures robust chromosome segregation by providing a strong mechanical force that favours segregation of merotelic attachments during anaphase. Using low doses of the microtubules-targeting agent BAL27862 we reduce microtubule occupancy and observe that spindle morphology is unaffected and bi-oriented kinetochores can still oscillate with normal intra-kinetochore distances. Inter-kinetochore stretching is, however, dramatically reduced. The reduction in microtubule occupancy and inter-kinetochore stretching does not delay satisfaction of the spindle assembly checkpoint or induce microtubule detachment via Aurora-B kinase, which was so far thought to release microtubules from kinetochores under low stretching. Rather, partial microtubule occupancy slows down anaphase A and increases incidences of lagging chromosomes due to merotelically attached kinetochores.

Kif15 functions as an active mechanical ratchet.

Kif15 is a kinesin-12 that contributes critically to bipolar spindle assembly in humans. Here we use force-ramp experiments in an optical trap to probe the mechanics of single Kif15 molecules under hindering or assisting loads and in a variety of nucleotide states. While unloaded Kif15 is established to be highly processive, we find that under hindering loads, Kif15 takes <∼10 steps. As hindering load is increased, Kif15 forestep:backstep ratio decreases exponentially, with stall occurring at 6 pN. In contrast, under assisting loads, Kif15 detaches readily and rapidly, even from its AMPPNP state. Kif15 mechanics thus depend markedly on the loading direction. Kif15 interacts with a binding partner, Tpx2, and we show that Tpx2 locks Kif15 to microtubules under both hindering and assisting loads. Overall, our data predict that Kif15 in the central spindle will act as a mechanical ratchet, supporting spindle extension but resisting spindle compression.

Rapid production of pure recombinant actin isoforms in Pichia pastoris.

Actins are major eukaryotic cytoskeletal proteins, and they are involved in many important cell functions, including cell division, cell polarity, wound healing and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively for biochemical studies of the non-muscle actin cytoskeleton. Here, we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris Actin is expressed as a fusion with the actin-binding protein thymosin ß4 and purified by means of an affinity tag introduced in the fusion. Following cleavage of thymosin ß4 and the affinity tag, highly purified functional full-length actin is liberated. We purify actins from Saccharomycescerevisiae and Schizosaccharomycespombe, and the ß- and γ-isoforms of human actin. We also report a modification of the method that facilitates expression and purification of arginylated actin, a form of actin thought to regulate dendritic actin networks in mammalian cells. The methods we describe can be performed in all laboratories equipped for molecular biology, and should greatly facilitate biochemical and cell biological studies of the actin cytoskeleton.

The Enterohemorrhagic Escherichia coli Effector EspW Triggers Actin Remodeling in a Rac1-Dependent Manner.

Enterohemorrhagic Escherichia coli (EHEC) is a diarrheagenic pathogen that colonizes the gut mucosa and induces attaching-and-effacing lesions. EHEC employs a type III secretion system (T3SS) to translocate 50 effector proteins that hijack and manipulate host cell signaling pathways, which allow bacterial colonization and subversion of immune responses and disease progression. The aim of this study was to characterize the T3SS effector EspW. We found espW in the sequenced O157:H7 and non-O157 EHEC strains as well as in Shigella boydii Furthermore, a truncated version of EspW, containing the first 206 residues, is present in EPEC strains belonging to serotype O55:H7. Screening a collection of clinical EPEC isolates revealed that espW is present in 52% of the tested strains. We report that EspW modulates actin dynamics in a Rac1-dependent manner. Ectopic expression of EspW results in formation of unique membrane protrusions. Infection of Swiss cells with an EHEC espW deletion mutant induces a cell shrinkage phenotype that could be rescued by Rac1 activation via expression of the bacterial guanine nucleotide exchange factor, EspT. Furthermore, using a yeast two-hybrid screen, we identified the motor protein Kif15 as a potential interacting partner of EspW. Kif15 and EspW colocalized in cotransfected cells, while ectopically expressed Kif15 localized to the actin pedestals following EHEC infection. The data suggest that Kif15 recruits EspW to the site of bacterial attachment, which in turn activates Rac1, resulting in modifications of the actin cytoskeleton that are essential to maintain cell shape during infection.

Congressing kinetochores progressively load Ska complexes to prevent force-dependent detachment.

Kinetochores mediate chromosome congression by either sliding along the lattice of spindle microtubules or forming end-on attachments to their depolymerizing plus-ends. By following the fates of individual kinetochores as they congress in live cells, we reveal that the Ska complex is required for a distinct substep of the depolymerization-coupled pulling mechanism. Ska depletion increases the frequency of naturally occurring, force-dependent P kinetochore detachment events, while being dispensable for the initial biorientation and movement of chromosomes. In unperturbed cells, these release events are followed by reattachment and successful congression, whereas in Ska-depleted cells, detached kinetochores remain in a futile reattachment/detachment cycle that prevents congression. We further find that Ska is progressively loaded onto bioriented kinetochore pairs as they congress. We thus propose a model in which kinetochores mature through Ska complex recruitment and that this is required for improved load-bearing capacity and silencing of the spindle assembly checkpoint.