title: Cross-linking mass spectrometry identifies new interfaces of Augmin required to localise the γ-tubulin ring complex to the mitotic spindle.
abstract: The hetero-octameric protein complex, Augmin, recruits γ-Tubulin ring complex (γ-TuRC) to pre-existing microtubules (MTs) to generate branched MTs during mitosis, facilitating robust spindle assembly. However, despite a recent partial reconstitution of the human Augmin complexin vitro, the molecular basis of this recruitment remains unclear. Here, we used immuno-affinity purification ofin vivoAugmin fromDrosophilaand cross-linking/mass spectrometry to identify distance restraints between residues within the eight Augmin subunits in the absence of any other structural information. The results allowed us to predict potential interfaces between Augmin and γ-TuRC. We tested these predictions biochemically and in theDrosophilaembryo, demonstrating that specific regions of the Augmin subunits, Dgt3, Dgt5 and Dgt6 all directly bind the γ-TuRC protein, Dgp71WD, and are required for the accumulation of γ-TuRC, but not Augmin, to the mitotic spindle. This study therefore substantially increases our understanding of the molecular mechanisms underpinning MT-dependent MT nucleation.
authors.affiliation: Department of Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK.
authors.forename: Jack W C
authors.identifier: http://orcid.org/0000-0002-8421-2443
authors.initials: JWC
authors.lastname: Chen
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Max Born Crescent, Edinburgh EH9 3BF, UK.
authors.forename: Zhuo A
authors.identifier: http://orcid.org/0000-0003-0811-8348
authors.initials: ZA
authors.lastname: Chen
authors.affiliation: Department of Statistics, University of Oxford, South Parks Road, Oxford OX1 3TG, UK.
authors.forename: Kacper B
authors.identifier: http://orcid.org/0000-0001-5997-7770
authors.initials: KB
authors.lastname: Rogala
authors.affiliation: Department of Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Metz
authors.affiliation: Department of Statistics, University of Oxford, South Parks Road, Oxford OX1 3TG, UK.
authors.forename: Charlotte M
authors.identifier: http://orcid.org/0000-0003-1388-2252
authors.initials: CM
authors.lastname: Deane
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Max Born Crescent, Edinburgh EH9 3BF, UK j.g.wakefield@exeter.ac.uk juri.rappsilber@ed.ac.uk.|Chair of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, 13355 Berlin, Germany.
authors.forename: Juri
authors.identifier: http://orcid.org/0000-0001-5999-1310
authors.initials: J
authors.lastname: Rappsilber
authors.affiliation: Department of Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK j.g.wakefield@exeter.ac.uk juri.rappsilber@ed.ac.uk.
authors.forename: James G
authors.identifier: http://orcid.org/0000-0003-3616-2346
authors.initials: JG
authors.lastname: Wakefield
keywords: Augmin; Drosophila; Microtubule; Mitosis; Spindle; γ-TuRC