Emanuele Roscioli

Toscana Life Sciences

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Andrew D. McAinsh

University of Warwick

Daniela Cimini

Virginia Tech

Nigel J. Burroughs

University of Warwick

Patrizia Lavia

Institute of Molecular Biology and Pathology

Tsvetelina E. Germanova

University of Warwick

Peter Embacher

University College London

Brent Bowden

Virginia Tech

Claudia Sala

Toscana Life Sciences

Rino Rappuoli

University of Siena

Dario Cardamone

Toscana Life Sciences

Cooperative Institutions

Toscana Life Sciences

University of Warwick

Sapienza University of Rome

University of Siena

National Research Council

Institute of Molecular Biology and Pathology

Max Planck Institute of Molecular Cell Biology and Genetics

GlaxoSmithKline (Italy)

Virginia Tech

Rudjer Boskovic Institute

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RANBP1 localizes a subset of mitotic regulatory factors on spindle microtubules and regulates chromosome segregation in human cells

Journal of Cell Science (2007)

Antonio Tedeschi Marilena Ciciarello Rosamaria Mangiacasale Emanuele Roscioli Wilhelmina Maria Rensen

The GTPase RAN has an established role in spindle assembly and in mitotic progression, although not all mechanisms are fully understood in somatic cells. Here, we have downregulated RAN-binding protein 1 (RANBP1), a RAN partner that has highest abundance in G2 and mitosis, in human cells. RANBP1-depleted cells underwent prolonged prometaphase delay often followed by apoptosis. Cells that remained viable assembled morphologically normal spindles; these spindles, however, were hyperstable and failed to recruit cyclin B1 or to restrict the localization of HURP (DLG7), a microtubule-stabilizing factor, to plus-ends. RANBP1 depletion did not increase the frequency of unattached chromosomes; however, RANBP1-depleted cells frequently showed lagging chromosomes in anaphase, suggesting that merotelic attachments form and are not efficiently resolved. These data indicate that RANBP1 activity is required for the proper localization of specific factors that regulate microtubule function; loss of this activity contributes to the generation of aneuploidy in a microtubule-dependent manner.

Prometaphase

Spindle checkpoint

Multipolar spindles

Ran

10.1242/jcs.009308

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Citations (57)

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

bioRxiv (Cold Spring Harbor Laboratory) (2021)

Peter Embacher Tsvetelina E. Germanova Emanuele Roscioli Andrew D. McAinsh Nigel J. Burroughs

A bstract 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 (≲ 50nm). 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 markers and has several advantages over these previous methods. Specifically it improves confidence intervals for small lengths, estimates measurement errors of each fluorescent marker 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 cell division. We examine the conformational change induced by microtubule attachment using triple fluorophore marked data and demonstrate for the first time that in metaphase kinetochore conformation is heterogeneous.

Polygon (computer graphics)

10.1101/2021.08.22.457266

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Deep Learning for Classifying Anti-Shigella Opsono- Phagocytosis-Promoting Monoclonal Antibodies

Lecture notes in computer science (2025)

Elena Pianfetti Dario Cardamone Emanuele Roscioli Giorgio Ciano Giuseppe Maccari

10.1007/978-3-031-77786-8_3

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

STAR Protocols (2021)

Tsvetelina E. Germanova Emanuele Roscioli Jonathan U. Harrison Andrew D. McAinsh Nigel J. Burroughs

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).

10.1016/j.xpro.2021.100774

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Laser microsurgery reveals conserved viscoelastic behavior of the kinetochore

The Journal of Cell Biology (2016)

Gheorghe Cojoc Emanuele Roscioli Lijuan Zhang Alfonso García-Ulloa Jagesh V. Shah

Accurate chromosome segregation depends on proper kinetochore-microtubule attachment. Upon microtubule interaction, kinetochores are subjected to forces generated by the microtubules. In this work, we used laser ablation to sever microtubules attached to a merotelic kinetochore, which is laterally stretched by opposing pulling forces exerted by microtubules, and inferred the mechanical response of the kinetochore from its length change. In both mammalian PtK1 cells and in the fission yeast Schizosaccharomyces pombe, kinetochores shortened after microtubule severing. Interestingly, the inner kinetochore-centromere relaxed faster than the outer kinetochore. Whereas in fission yeast all kinetochores relaxed to a similar length, in PtK1 cells the more stretched kinetochores remained more stretched. Simple models suggest that these differences arise because the mechanical structure of the mammalian kinetochore is more complex. Our study establishes merotelic kinetochores as an experimental model for studying the mechanical response of the kinetochore in live cells and reveals a viscoelastic behavior of the kinetochore that is conserved in yeast and mammalian cells.

Schizosaccharomyces

10.1083/jcb.201506011

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Citations (28)

Overlap microtubules link sister k-fibres and balance the forces on bi-oriented kinetochores

Nature Communications (2016)

Janko Kajtez Anastasia I. Solomatina Maja Novak Bruno Polak Kruno Vukušić

Abstract During metaphase, forces on kinetochores are exerted by k-fibres, bundles of microtubules that end at the kinetochore. Interestingly, non-kinetochore microtubules have been observed between sister kinetochores, but their function is unknown. Here we show by laser-cutting of a k-fibre in HeLa and PtK1 cells that a bundle of non-kinetochore microtubules, which we term ‘bridging fibre’, bridges sister k-fibres and balances the interkinetochore tension. We found PRC1 and EB3 in the bridging fibre, suggesting that it consists of antiparallel dynamic microtubules. By using a theoretical model that includes a bridging fibre, we show that the forces at the pole and at the kinetochore depend on the bridging fibre thickness. Moreover, our theory and experiments show larger relaxation of the interkinetochore distance for cuts closer to kinetochores. We conclude that the bridging fibre, by linking sister k-fibres, withstands the tension between sister kinetochores and enables the spindle to obtain a curved shape.

10.1038/ncomms10298

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Citations (151)

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

Cell Reports (2020)

Emanuele Roscioli Tsvetelina E. Germanova Christopher A. Smith Peter Embacher Muriel Erent

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.

BUB1

Mad2

Spindle checkpoint

10.1016/j.celrep.2020.107535

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Anti-capsule human monoclonal antibodies protect against hypervirulent and pandrug-resistant Klebsiella pneumoniae

Research Square (Research Square) (2024)

Anna Kabanova Emanuele Roscioli V. Fonseca Soraya S. Bosch Ida Paciello

Abstract The silent pandemic caused by antimicrobial resistance (AMR) requires innovative therapeutic approaches. Human monoclonal antibodies (mAbs), which are among the most transformative, safe and effective drugs in oncology and autoimmunity, are rarely used for infectious diseases and not yet used for AMR. Here we applied an antigen-agnostic strategy to isolate extremely potent human mAbs against Klebsiella pneumoniae (Kp) sequence type 147 (ST147), a hypervirulent and pandrug-resistant clonotype which is spreading globally. Isolated mAbs target the bacterial capsule and the O-antigen. Surprisingly, although both capsule- and O-antigen-specific mAbs displayed bactericidal activity in the picomolar range in vitro, only the capsule-specific mAbs were protective against fulminant ST147 bloodstream infection. Protection correlated with in vitro bacterial uptake by macrophages and enchained bacterial growth. Our study describes the only therapeutic able to protect against pandrug-resistant Kp and provides a strategy to isolate mAbs and identify correlates of protection against AMR bacteria.

Capsule

10.21203/rs.3.rs-4631260/v1

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Citations (1)

Dynamic bonds and polar ejection force distribution explain kinetochore oscillations in PtK1 cells

The Journal of Cell Biology (2013)

Gul Civelekoglu‐Scholey Bin He Muyao Shen Xiaohu Wan Emanuele Roscioli

10.1083/jcb.20130102207162013c

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

The Journal of Cell Biology (2020)

Philip Auckland Emanuele Roscioli Helena L.E. Coker Andrew D. McAinsh

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.

10.1083/jcb.201905018

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Citations (35)