title: Microtubule organization within mitotic spindles revealed by serial block face scanning electron microscopy and image analysis.
abstract: Serial block face scanning electron microscopy (SBF-SEM) is a powerful method to analyze cells in 3D. Here, working at the resolution limit of the method, we describe a correlative light-SBF-SEM workflow to resolve microtubules of the mitotic spindle in human cells. We present four examples of uses for this workflow that are not practical by light microscopy and/or transmission electron microscopy. First, distinguishing closely associated microtubules within K-fibers; second, resolving bridging fibers in the mitotic spindle; third, visualizing membranes in mitotic cells, relative to the spindle apparatus; and fourth, volumetric analysis of kinetochores. Our workflow also includes new computational tools for exploring the spatial arrangement of microtubules within the mitotic spindle. We use these tools to show that microtubule order in mitotic spindles is sensitive to the level of TACC3 on the spindle.
authors.affiliation: Centre for Mechanochemical Cell Biology, Warwick Medical School, Coventry CV4 7AL, UK.|Institute for Translational Medicine, University of Liverpool L69 3BX, UK.
authors.forename: Faye M
authors.identifier: 0000-0002-7029-2649
authors.initials: FM
authors.lastname: Nixon
authors.affiliation: Department of Statistics, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Honnor
authors.affiliation: Centre for Mechanochemical Cell Biology, Warwick Medical School, Coventry CV4 7AL, UK.
authors.forename: Nicholas I
authors.initials: NI
authors.lastname: Clarke
authors.affiliation: Centre for Mechanochemical Cell Biology, Warwick Medical School, Coventry CV4 7AL, UK.
authors.forename: Georgina P
authors.initials: GP
authors.lastname: Starling
authors.affiliation: Institute for Translational Medicine, University of Liverpool L69 3BX, UK.
authors.forename: Alison J
authors.initials: AJ
authors.lastname: Beckett
authors.affiliation: Department of Statistics, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Adam M
authors.initials: AM
authors.lastname: Johansen
authors.affiliation: Department of Statistics, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Julia A
authors.initials: JA
authors.lastname: Brettschneider
authors.affiliation: Institute for Translational Medicine, University of Liverpool L69 3BX, UK.
authors.forename: Ian A
authors.identifier: 0000-0002-4055-5161
authors.initials: IA
authors.lastname: Prior
authors.affiliation: Centre for Mechanochemical Cell Biology, Warwick Medical School, Coventry CV4 7AL, UK s.j.royle@warwick.ac.uk.
authors.forename: Stephen J
authors.identifier: 0000-0001-8927-6967
authors.initials: SJ
authors.lastname: Royle
mesh_terms: D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional; D018386:Kinetochores / Q000378:metabolism / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D008870:Microtubules / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D008941:Spindle Apparatus / Q000378:metabolism* / Q000648:ultrastructure
keywords: 3D cell biology; Electron microscopy; Microtubules; Mitosis; Mitotic spindle; Statistics