Dictyostelium Myosin-IE Is a Fast Molecular Motor Involved in Phagocytosis

Class I myosins are single-headed motor proteins, implicated in various motile processes including organelle translocation, ion-channel gating, and cytoskeleton reorganization. Here we describe the cellular localization of myosin-IE and its role in the phagocytic uptake of solid particles and cells. A complete analysis of the kinetic and motor properties of Dictyostelium discoideum myosin-IE was achieved by the use of motor domain constructs with artificial lever arms. Class I myosins belonging to subclass IC like myosin-IE are thought to be tuned for tension maintenance or stress sensing. In contrast to this prediction, our results show myosin-IE to be a fast motor. Myosin-IE motor activity is regulated by myosin heavy chain phosphorylation, which increases the coupling efficiency between the actin and nucleotide binding sites tenfold and the motile activity more than fivefold. Changes in the level of free Mg(2+) ions, which are within the physiological range, are shown to modulate the motor activity of myosin-IE by inhibiting the release of adenosine diphosphate

Probing microtubule polymerisation state at single kinetochores during metaphase chromosome motion

Kinetochores regulate the dynamics of attached microtubule bundles (kinetochore-fibres, K-fibres) to generate the forces necessary for chromosome movements in mitosis. Current models suggest that poleward-moving kinetochores are attached to depolymerising K-fibres and anti-poleward-moving kinetochores to polymerising K-fibres. How the dynamics of individual microtubules within the K-fibre relate to poleward and anti-poleward movements is poorly understood. To investigate this, we developed a live-cell imaging assay combined with computational image analysis that allows eGFP-tagged EB3 (also known as MAPRE3) to be quantified at thousands of individual metaphase kinetochores as they undergo poleward and anti-poleward motion. Surprisingly, we found that K-fibres are incoherent, containing both polymerising and depolymerising microtubules – with a small polymerisation bias for anti-poleward-moving kinetochores. K-fibres also display bursts of EB3 intensity, predominantly on anti-poleward-moving kinetochores, equivalent to more coherent polymerisation, and this was associated with more regular oscillations. The frequency of bursts and the polymerisation bias decreased upon loss of kinesin-13, whereas loss of kinesin-8 elevated polymerisation bias. Thus, kinetochores actively set the balance of microtubule polymerisation dynamics in the K-fibre while remaining largely robust to fluctuations in microtubule polymerisation

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. [Abstract copyright: Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

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

Potential association of acidogenic bacteria and highly effective sulfate reducers for bioconversion of gypsum industrial by-product

The kinesins-8 were originally thought to be microtubule depolymerases, but are now emerging as more versatile catalysts of microtubule dynamics. We show here that S. pombe Klp5-436 and Klp6-440 are non-processive plus-end-directed motors whose in vitro velocities on S. pombe microtubules at 7 and 23 nm s−1 are too slow to keep pace with the growing tips of dynamic interphase microtubules in living S. pombe. In vitro, Klp5 and 6 dimers exhibit a hitherto-undescribed combination of strong enhancement of microtubule nucleation with no effect on growth rate or catastrophe frequency. By contrast in vivo, both Klp5 and Klp6 promote microtubule catastrophe at cell ends whilst Klp6 also increases the number of interphase microtubule arrays (IMAs). Our data support a model in which Klp5/6 bind tightly to free tubulin heterodimers, strongly promoting the nucleation of new microtubules, and then continue to land as a tubulin-motor complex on the tips of growing microtubules, with the motors then dissociating after a few seconds residence on the lattice. In vivo, we predict that only at cell ends, when growing microtubule tips become lodged and their growth slows down, will Klp5/6 motor activity succeed in tracking growing microtubule tips. This mechanism would allow Klp5/6 to detect the arrival of microtubule tips at cells ends and to amplify the intrinsic tendency for microtubules to catastrophise in compression at cell ends. Our evidence identifies Klp5 and 6 as spatial regulators of microtubule dynamics that enhance both microtubule nucleation at the cell centre and microtubule catastrophe at the cell ends

Calmodulin-containing substructures of the centrosomal matrix released by microtubule perturbation

Calmodulin redistribution in MDCK and HeLa cells subjected to microtubule perturbations by antimitotic drugs was followed using a calmodulin-EGFP fusion protein that preserves the Ca2+ affinity, target binding and activation properties of native calmodulin. CaM-EGFP targeting to spindle structures in normal cell division and upon spindle microtubule disruption allows evaluation of the dynamic redistribution of calmodulin in cell division. Under progressive treatment of stably transfected mammalian cells with nocodazole or vinblastine, the centrosomal matrix at the mitotic poles subdivides into numerous small star-like structures, with the calmodulin concentrated centrally, and partially distinct from the reduced microtubule mass to which kinetochores and chromosomes are attached. Prolonged vinblastine treatment causes the release of localised calmodulin into a uniform cytoplasmic distribution, and tubulin paracrystal formation. By contrast, paclitaxel treatment of metaphase cells apparently causes limited disassembly of the pericentriolar material into a number of multipolar ring-like structures containing calmodulin, each one having multiple attached microtubules terminating in the partially disordered kinetochore/chromosome complex. Thus drugs with opposite effects in either destabilising or stabilising mitotic microtubules cause subdivision of the centrosomal matrix into two distinctive calmodulin-containing structures, namely small punctate stars or larger polar rings respectively. The star-like structures may represent an integral subcomponent for the attachment of kinetochore microtubules to the metaphase centrosome complex. The results imply that microtubules have a role in stabilising the structure of the pericentriolar matrix, involving interaction, either direct or indirect, with one or more proteins that are targets for binding of calmodulin. Possible candidates include the pericentriolar matrix-associated coiled-coil proteins containing calmodulin-binding motifs, such as myosin V, kendrin (PCNT2) and AKAP450

Klp6₄₄₀His coated beads move processively on microtubules.

<p>Video-Enhanced Differential Interference Contrast microscopy showing a Klp6₄₄₀His coated bead moving along a taxol-stabilised pig brain microtubule adsorbed to a coverslip. Bead velocity was 47±7 nm s⁻¹ (5) (mean ± SEM (n)) during translocation. At the microtubule tip (dashed line) the bead dwelled for several seconds before detaching. Time (minutes : seconds), scale bar:1 µm.</p

Model for the role of Klp5/6 in microtubule dynamics in vivo.

<p>Cartoon of an interphase <i>S. pombe</i> cell with the nucleus (grey disc) and microtubule nucleation sites (green discs) in the cell centre. The right-hand side shows wild type cells with microtubules (purple) nucleating at the cell centre, growing into the end zone of the cell and undergoing catastrophe in contact with the cell end. Klp5/6 (turquoise/red) heterodimers help promote nucleation, crosslink microtubules in the IMAs and accelerate catastrophe at the cell end, possibly by physical linkage of the microtubule tip to the cell cortex. Klp5/6 continuously incorporate at the microtubule tip but rapidly detach as the tubulin dimers are incorporated into the microtubule lattice. Thus, Klp5/6 continuously sample the location of the microtubule within the cell. Microtubule growth slows after the microtubule contacts the cell end, allowing accumulation of Klp5/6 by incorporation and short-range translocation. The left-hand side shows a <i>klp5/6</i> deletion mutant, in which the absence of Klp5 or Klp6 reduces nucleation activity, weakens cross-linking in the IMAs and increases the dwell time of the microtubule tips at the cell end before they undergo catastrophe, leading to some microtubule overgrowth.</p

Microtubule and tubulin stimulated Klp5/6 ATPase activity.

<p>Microtubules were assembled from either pig brain tubulin and stabilised with Taxol or <i>S. pombe</i> tubulin stabilised with GMPCPP. Microtubule or tubulin heterodimer stimulated ATPase activities were determined in linked assays at 25°C. Non-linear fits of plots of ATPase activity against tubulin concentration were used to determine Vmax and Km values ± standard error of mean.</p