title: 3D-CLEM Reveals that a Major Portion of Mitotic Chromosomes Is Not Chromatin.
abstract: Recent studies have revealed the importance of Ki-67 and the chromosome periphery in chromosome structure and segregation, but little is known about this elusive chromosome compartment. Here we used correlative light and serial block-face scanning electron microscopy, which we term 3D-CLEM, to model the entire mitotic chromosome complement at ultra-structural resolution. Prophase chromosomes exhibit a highly irregular surface appearance with a volume smaller than metaphase chromosomes. This may be because of the absence of the periphery, which associates with chromosomes only after nucleolar disassembly later in prophase. Indeed, the nucleolar volume almost entirely accounts for the extra volume found in metaphase chromosomes. Analysis of wild-type and Ki-67-depleted chromosomes reveals that the periphery comprises 30%-47% of the entire chromosome volume and more than 33% of the protein mass of isolated mitotic chromosomes determined by quantitative proteomics. Thus, chromatin makes up a surprisingly small percentage of the total mass of metaphase chromosomes.
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, EH9 3BF Edinburgh, UK. Electronic address: daniel.booth@ed.ac.uk.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Booth
authors.affiliation: Biomedical Electron Microscopy Unit, Division of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Crown Street, L69 3BX Liverpool, UK.
authors.forename: Alison J
authors.initials: AJ
authors.lastname: Beckett
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, EH9 3BF Edinburgh, UK.
authors.forename: Oscar
authors.initials: O
authors.lastname: Molina
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, EH9 3BF Edinburgh, UK.
authors.forename: Itaru
authors.initials: I
authors.lastname: Samejima
authors.affiliation: Department of Frontier Research, Laboratory of Cell Engineering, Kazusa DNA Research Institute, Kisarazu, 292-0818 Chiba, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Masumoto
authors.affiliation: Developmental Therapeutics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA.
authors.forename: Natalay
authors.initials: N
authors.lastname: Kouprina
authors.affiliation: Developmental Therapeutics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Larionov
authors.affiliation: Biomedical Electron Microscopy Unit, Division of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Crown Street, L69 3BX Liverpool, UK.
authors.forename: Ian A
authors.initials: IA
authors.lastname: Prior
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, EH9 3BF Edinburgh, UK. Electronic address: bill.earnshaw@ed.ac.uk.
authors.forename: William C
authors.initials: WC
authors.lastname: Earnshaw
mesh_terms: D002461:Cell Line, Transformed; D002466:Cell Nucleolus / Q000737:chemistry / Q000648:ultrastructure; D002843:Chromatin / Q000737:chemistry / Q000648:ultrastructure*; D002875:Chromosomes / Q000737:chemistry / Q000648:ultrastructure*; D015870:Gene Expression; D006367:HeLa Cells; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019394:Ki-67 Antigen / Q000235:genetics / Q000378:metabolism; D008677:Metaphase; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D011418:Prophase; D055213:Retinal Pigment Epithelium / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D002843:Chromatin; D006657:Histones; D019394:Ki-67 Antigen
keywords: 3D-CLEM; CLEM; Ki-67; chromosome periphery; electron microscopy