Explore the vast range of titles available.

Key Points The assembly of a bipolar microtubule-based spindle is crucial for the accurate and timely segregation of the chromosomes into the two daughter cells during mitosis. Three microtubule nucleation pathways function in mitotic cells to contribute microtubules to the assembling spindle, namely the centrosome-, chromatin- and microtubule-mediated pathways. Microtubules nucleated from the centrosomes find the chromosomes via 'search and capture' — a process that relies on the dynamic instability of microtubules — which allows them to search space to contact the chromosomes. When a microtubule contacts a kinetochore, it is captured and becomes stabilized to form kinetochore fibres (K-fibres). The speed and efficiency of chromosome capture is promoted by the chromatin-mediated pathway that biases microtubule nucleation and stabilization to the vicinity of the chromosomes. Microtubules are also generated from within the spindle itself via Augmin, which promotes microtubule nucleation from pre-existing microtubules. This nucleation increases the density of microtubules within the spindle and thus contributes to its robustness. Although the nucleation pathways are at least partially redundant, they are integrated to provide an intricate balance of microtubule nucleation that ensures the fidelity of chromosome segregation and the timely completion of mitosis. In the absence of any one of the pathways, spindle assembly still occurs — although with increased use of the remaining pathways — but mitosis takes longer and this can result in genome instability. Three microtubule nucleation pathways — initiated from centrosomes, chromatin and existing spindle microtubules — contribute to the assembly of a functional mitotic spindle in animal cells to ensure accurate chromosome segregation. Recent findings have shed light on their relative contributions to building the spindle and on adaptation of the spindle to variations in cell size and shape. The mitotic spindle has a crucial role in ensuring the accurate segregation of chromosomes into the two daughter cells during cell division, which is paramount for maintaining genome integrity. It is a self-organized and dynamic macromolecular structure that is constructed from microtubules, microtubule-associated proteins and motor proteins. Thirty years of research have led to the identification of centrosome-, chromatin- and microtubule-mediated microtubule nucleation pathways that each contribute to mitotic spindle assembly. Far from being redundant pathways, data are now emerging regarding how they function together to ensure the timely completion of mitosis. We are also beginning to comprehend the multiple mechanisms by which cells regulate spindle scaling. Together, this research has increased our understanding of how cells coordinate hundreds of proteins to assemble the dynamic, precise and robust structure that is the mitotic spindle.

In the dividing eukaryotic cell, the spindle assembly checkpoint (SAC) ensures that each daughter cell inherits an identical set of chromosomes. The SAC coordinates the correct attachment of sister chromatid kinetochores to the mitotic spindle with activation of the anaphase-promoting complex (APC/C), the E3 ubiquitin ligase responsible for initiating chromosome separation. In response to unattached kinetochores, the SAC generates the mitotic checkpoint complex (MCC), which inhibits the APC/C and delays chromosome segregation. By cryo-electron microscopy, here we determine the near-atomic resolution structure of a human APC/C–MCC complex (APC/C MCC ). Degron-like sequences of the MCC subunit BubR1 block degron recognition sites on Cdc20, the APC/C coactivator subunit responsible for substrate interactions. BubR1 also obstructs binding of the initiating E2 enzyme UbcH10 to repress APC/C ubiquitination activity. Conformational variability of the complex enables UbcH10 association, and structural analysis shows how the Cdc20 subunit intrinsic to the MCC (Cdc20 MCC ) is ubiquitinated, a process that results in APC/C reactivation when the SAC is silenced. A high-resolution structure of a complex between the anaphase-promoting complex (APC/C) and the mitotic checkpoint complex (MCC) reveals how MCC interacts with and represses APC/C by obstructing substrate recognition and suppressing E3 ligase activity. Basis of mitotic regulation The spindle assembly checkpoint (SAC) is a surveillance mechanism that detects incorrect chromatid kinetochore attachments and delays chromosome segregation by generating a 'wait anaphase' signal. It is activated via the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex (APC/C), a multimeric E3 ligase. Here, David Barford and colleagues use cryo-electron microscopy to determine near-atomic resolution structures of the APC/C–MCC complex. The structures reveal how MCC interacts with and represses APC/C by obstructing substrate recognition and suppressing E3 ligase activity.

ASPM (known as Asp in fly and ASPM-1 in worm) is a microcephaly-associated protein family that regulates spindle architecture, but the underlying mechanism is poorly understood. Here, we show that ASPM forms a complex with another protein linked to microcephaly, the microtubule-severing ATPase katanin. ASPM and katanin localize to spindle poles in a mutually dependent manner and regulate spindle flux. X-ray crystallography revealed that the heterodimer formed by the N- and C-terminal domains of the katanin subunits p60 and p80, respectively, binds conserved motifs in ASPM. Reconstitution experiments demonstrated that ASPM autonomously tracks growing microtubule minus ends and inhibits their growth, while katanin decorates and bends both ends of dynamic microtubules and potentiates the minus-end blocking activity of ASPM. ASPM also binds along microtubules, recruits katanin and promotes katanin-mediated severing of dynamic microtubules. We propose that the ASPM–katanin complex controls microtubule disassembly at spindle poles and that misregulation of this process can lead to microcephaly. Jiang et al.  show that the microcephaly-associated protein ASPM and katanin form a complex that binds microtubule minus ends and can sever microtubules and block microtubule minus-end elongation to control spindle pole dynamics.

Muscle spindle proprioceptive receptors play a primary role in encoding the effects of external mechanical perturbations to the body. During externally-imposed stretches of passive, i.e. electrically-quiescent, muscles, the instantaneous firing rates (IFRs) of muscle spindles are associated with characteristics of stretch such as length and velocity. However, even in passive muscle, there are history-dependent transients of muscle spindle firing that are not uniquely related to muscle length and velocity, nor reproduced by current muscle spindle models. These include acceleration-dependent initial bursts, increased dynamic response to stretch velocity if a muscle has been isometric, and rate relaxation, i.e., a decrease in tonic IFR when a muscle is held at a constant length after being stretched. We collected muscle spindle spike trains across a variety of muscle stretch kinematic conditions, including systematic changes in peak length, velocity, and acceleration. We demonstrate that muscle spindle primary afferents in passive muscle fire in direct relationship to muscle force-related variables, rather than length-related variables. Linear combinations of whole muscle-tendon force and the first time derivative of force (dF/dt) predict the entire time course of transient IFRs in muscle spindle Ia afferents during stretch (i.e., lengthening) of passive muscle, including the initial burst, the dynamic response to lengthening, and rate relaxation following lengthening. Similar to acceleration scaling found previously in postural responses to perturbations, initial burst amplitude scaled equally well to initial stretch acceleration or dF/dt, though later transients were only described by dF/dt. The transient increase in dF/dt at the onset of lengthening reflects muscle short-range stiffness due to cross-bridge dynamics. Our work demonstrates a critical role of muscle cross-bridge dynamics in history-dependent muscle spindle IFRs in passive muscle lengthening conditions relevant to the detection and sensorimotor response to mechanical perturbations to the body, and to previously-described history-dependence in perception of limb position.

The anti-tumor effects of chemotherapy and radiation are thought to be mediated by triggering G1/S or G2/M cell cycle checkpoints, while spindle poisons, such as paclitaxel, block metaphase exit by initiating the spindle assembly checkpoint. In contrast, we have found that 150 kilohertz (kHz) alternating electric fields, also known as Tumor Treating Fields (TTFields), perturbed cells at the transition from metaphase to anaphase. Cells exposed to the TTFields during mitosis showed normal progression to this point, but exhibited uncontrolled membrane blebbing that coincided with metaphase exit. The ability of such alternating electric fields to affect cellular physiology is likely to be dependent on their interactions with proteins possessing high dipole moments. The mitotic Septin complex consisting of Septin 2, 6 and 7, possesses a high calculated dipole moment of 2711 Debyes (D) and plays a central role in positioning the cytokinetic cleavage furrow, and governing its contraction during ingression. We showed that during anaphase, TTFields inhibited Septin localization to the anaphase spindle midline and cytokinetic furrow, as well as its association with microtubules during cell attachment and spreading on fibronectin. After aberrant metaphase exit as a consequence of TTFields exposure, cells exhibited aberrant nuclear architecture and signs of cellular stress including an overall decrease in cellular proliferation, followed by apoptosis that was strongly influenced by the p53 mutational status. Thus, TTFields are able to diminish cell proliferation by specifically perturbing key proteins involved in cell division, leading to mitotic catastrophe and subsequent cell death.

Purified microtubules have been shown to align along the static magnetic field (SMF) in vitro because of their diamagnetic anisotropy. However, whether mitotic spindle in mammalian cells can be aligned by magnetic field has not been experimentally proved. In particular, the biological effects of SMF of above 20 T (Tesla) on mammalian cells have never been reported. Here we found that in both CNE-2Z and RPE1 human cells spindle orients in 27 T SMF. The direction of spindle alignment depended on the extent to which chromosomes were aligned to form a planar metaphase plate. Our results show that the magnetic torque acts on both microtubules and chromosomes, and the preferred direction of spindle alignment relative to the field depends more on chromosome alignment than microtubules. In addition, spindle morphology was also perturbed by 27 T SMF. This is the first reported study that investigated the mammalian cellular responses to ultra-high magnetic field of above 20 T. Our study not only found that ultra-high magnetic field can change the orientation and morphology of mitotic spindles, but also provided a tool to probe the role of spindle orientation and perturbation in developmental and cancer biology. Nowadays, a number of methods can be used to ‘look’ inside the body to investigate potential health problems. One of these is a technique called magnetic resonance imaging (MRI) that uses magnetic fields that are several hundred times stronger than a fridge magnet (or over 10,000 times stronger than the Earth’s natural magnetic field) to generate images of the inside of the body. In general, stronger magnetic fields enable higher quality images to be obtained. However, the effects of exposing the body’s cells to these magnetic fields have not been fully determined. Like most other biological materials, protein polymers called microtubules can respond to high magnetic fields – for example, by aligning with the field. Microtubules play a number of roles inside cells. This includes forming the mitotic spindle that separates copies of chromosomes – the structures in which the majority of a cell’s genetic material is stored – equally between dividing cells. The orientation of the mitotic spindle determines the direction in which a cell will divide. This direction is important for generating different types of cells and tissues. Furthermore, many cancerous cells have incorrectly oriented spindles. Zhang, Hou et al. have now exposed cancerous and normal human cells to magnetic fields of varying strengths. The maximum magnetic field strength tested (27 Tesla – or around 10 times the highest field strengths produced by standard hospital MRI scanners) did not kill the cells after four hours of exposure, but the orientation of the spindles inside the cells did change. In addition, the 27 Tesla magnetic field caused spindles that were perpendicular to the direction of the field to widen. At an intermediate field strength (9 Tesla – a magnetic field strength that has been used in some experimental MRI scanners), the orientation of the spindle only changed after three days of continuous exposure to the magnetic field. Lower field strengths (such as those currently used in hospital MRI scanners) did not alter the orientation of the spindle even after seven days of exposure. Zhang, Hou et al. also observed that the magnetic field acts on both the microtubules and chromosomes. However, the alignment of the chromosomes in the cell was the greatest determinant of the direction in which the spindle would align itself in response to the magnetic field. The next step is to analyze the consequences of magnetic field-induced spindle orientation changes – can these lead to cancer or reduce cancer growth, or change how animal tissues develop? Understanding how to control the position of the spindle could also ultimately make it possible to use ultra-high magnetic fields to engineer tissues or stimulate their regeneration.

The spindle checkpoint is a mitotic surveillance system which ensures equal segregation of sister chromatids. It delays anaphase onset by inhibiting the action of the E3 ubiquitin ligase known as the anaphase promoting complex or cyclosome (APC/C). Mad3/BubR1 is a key component of the mitotic checkpoint complex (MCC) which binds and inhibits the APC/C early in mitosis. Mps1.sup.Mph1 kinase is critical for checkpoint signalling and MCC-APC/C inhibition, yet few substrates have been identified. Here we identify Mad3 as a substrate of fission yeast Mps1.sup.Mph1 kinase. We map and mutate phosphorylation sites in Mad3, producing mutants that are targeted to kinetochores and assembled into MCC, yet display reduced APC/C binding and are unable to maintain checkpoint arrests. We show biochemically that Mad3 phospho-mimics are potent APC/C inhibitors in vitro, demonstrating that Mad3p modification can directly influence Cdc20.sup.Slp1 -APC/C activity. This genetic dissection of APC/C inhibition demonstrates that Mps1.sup.Mph1 kinase-dependent modifications of Mad3 and Mad2 act in a concerted manner to maintain spindle checkpoint arrests.

Cell polarity and correct mitotic spindle positioning are essential for the maintenance of a proper prostate epithelial architecture, and disruption of the two biological features occurs at early stages in prostate tumorigenesis. However, whether and how these two epithelial attributes are connected in vivo is largely unknown. We herein report that conditional genetic deletion of E-cadherin, a key component of adherens junctions, in a mouse model results in loss of prostate luminal cell polarity and randomization of spindle orientations. Critically, E-cadherin ablation causes prostatic hyperplasia which progresses to invasive adenocarcinoma. Mechanistically, E-cadherin and the spindle positioning determinant LGN interacts with the PDZ domain of cell polarity protein SCRIB and form a ternary protein complex to bridge cell polarity and cell division orientation. These findings provide a novel mechanism by which E-cadherin acts an anchor to maintain prostate epithelial integrity and to prevent carcinogenesis in vivo.

Mitotic divisions depend on the timely assembly and proper orientation of the mitotic spindle. Malfunctioning of these processes can considerably delay mitosis, thereby compromising tissue growth and homeostasis, and leading to chromosomal instability. Loss of functional Mms19 drastically affects the growth and development of mitotic tissues in Drosophila larvae and we now demonstrate that Mms19 is an important factor that promotes spindle and astral microtubule (MT) growth, and MT stability and bundling. Mms19 function is needed for the coordination of mitotic events and for the rapid progression through mitosis that is characteristic of neural stem cells. Surprisingly, Mms19 performs its mitotic activities through two different pathways. By stimulating the mitotic kinase cascade, it triggers the localization of the MT regulatory complex TACC/Msps (Transforming Acidic Coiled Coil/Minispindles, the homolog of human ch-TOG) to the centrosome. This activity of Mms19 can be rescued by stimulating the mitotic kinase cascade. However, other aspects of the Mms19 phenotypes cannot be rescued in this way, pointing to an additional mechanism of Mms19 action. We provide evidence that Mms19 binds directly to MTs and that this stimulates MT stability and bundling.

Asymmetric cell division is essential to generate cellular diversity. In many animal cells, the cleavage plane lies perpendicular to the mitotic spindle, and it is the spindle positioning that dictates the size of the daughter cells. Although some properties of spindle positioning are conserved between distantly related model species and different cell types, little is known of the evolutionary robustness of the mechanisms underlying this event. We recorded the first embryonic division of 42 species of nematodes closely related to Caenorhabditis elegans, which is an excellent model system to study the biophysical properties of asymmetric spindle positioning. Our recordings, corresponding to 128 strains from 27 Caenorhabditis and 15 non-Caenorhabditis species (accessible at http://www.ens-lyon.fr/LBMC/NematodeCell/videos/), constitute a powerful collection of subcellular phenotypes to study the evolution of various cellular processes across species. In the present work, we analyzed our collection to the study of asymmetric spindle positioning. Although all the strains underwent an asymmetric first cell division, they exhibited large intra- and inter-species variations in the degree of cell asymmetry and in several parameters controlling spindle movement, including spindle oscillation, elongation, and displacement. Notably, these parameters changed frequently during evolution with no apparent directionality in the species phylogeny, with the exception of spindle transverse oscillations, which were an evolutionary innovation at the base of the Caenorhabditis genus. These changes were also unrelated to evolutionary variations in embryo size. Importantly, spindle elongation, displacement, and oscillation each evolved independently. This finding contrasts starkly with expectations based on C. elegans studies and reveals previously unrecognized evolutionary changes in spindle mechanics. Collectively, these data demonstrate that, while the essential process of asymmetric cell division has been conserved over the course of nematode evolution, the underlying spindle movement parameters can combine in various ways. Like other developmental processes, asymmetric cell division is subject to system drift.