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.

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.

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.

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.

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: () What are the constituent parts, () how does it assemble, () what is the structure and arrangement, and () 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.

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.

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.

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

This protocol measures the 3D Euclidean distance (Δ) between two/three fluorescently labeled kinetochore components in fixed samples using Kinetochore Delta software (KiDv1.0.1, MATLAB based).

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 >10 kinetochores.

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.

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.

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].

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.

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.

Rapid production of pure recombinant actin isoforms in .

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 Actin is expressed as a fusion with the actin-binding protein thymosin β4 and purified by means of an affinity tag introduced in the fusion.

Loss of E-cadherin provides tolerance to centrosome amplification in epithelial cancer cells.

Centrosome amplification is a common feature of human tumors. To survive, cancer cells cluster extra centrosomes during mitosis, avoiding the detrimental effects of multipolar divisions. However, it is unclear whether clustering requires adaptation or is inherent to all cells.

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

Enterohemorrhagic (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.

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.

Mps1 Regulates Kinetochore-Microtubule Attachment Stability via the Ska Complex to Ensure Error-Free Chromosome Segregation.

The spindle assembly checkpoint kinase Mps1 not only inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1's error correction-relevant substrates are unknown. Using a chemically tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K fibers) epistatically to Aurora B, the other major error-correcting kinase.

Human kinetochores are swivel joints that mediate microtubule attachments.

Chromosome segregation is a mechanical process that requires assembly of the mitotic spindle - a dynamic microtubule-based force-generating machine. Connections to this spindle are mediated by sister kinetochore pairs, that form dynamic end-on attachments to microtubules emanating from opposite spindle poles. This bi-orientation generates forces that have been reported to stretch the kinetochore itself, which has been suggested to stabilise attachment and silence the spindle checkpoint.

KiT: a MATLAB package for kinetochore tracking.

Unlabelled: During mitosis, chromosomes are attached to the mitotic spindle via large protein complexes called kinetochores. The motion of kinetochores throughout mitosis is intricate and automated quantitative tracking of their motion has already revealed many surprising facets of their behaviour. Here, we present 'KiT' (Kinetochore Tracking)-an easy-to-use, open-source software package for tracking kinetochores from live-cell fluorescent movies.

Kinesin-12 motors cooperate to suppress microtubule catastrophes and drive the formation of parallel microtubule bundles.

Human Kinesin-12 (hKif15) plays a crucial role in assembly and maintenance of the mitotic spindle. These functions of hKif15 are partially redundant with Kinesin-5 (Eg5), which can cross-link and drive the extensile sliding of antiparallel microtubules. Although both motors are known to be tetramers, the functional properties of hKif15 are less well understood.