title: The ultrastructure of mono- and holocentric plant centromeres: an immunological investigation by structured illumination microscopy and scanning electron microscopy.
abstract: The spatial distribution of the three centromere-associated proteins α-tubulin, CENH3, and phosphorylated histone H2A (at threonine 120, H2AThr120ph) was analysed by indirect immunodetection at monocentric cereal chromosomes and at the holocentric chromosomes of Luzula elegans by super-resolution light microscopy and scanning electron microscopy (SEM). Using structured illumination microscopy (SIM) as the super-resolution technique on squashed specimens and SEM on uncoated isolated specimens, the three-dimensional (3D) distribution of the proteins was visualized at the centromeres. Technical aspects of 3D SEM are explained in detail. We show that CENH3 forms curved "pads" mainly around the lateral centromeric region in the primary constriction of metacentric chromosomes. H2AThr120ph is present in both the primary constriction and in the pericentromere. α-tubulin-labeled microtubule bundles attach to CENH3-containing chromatin structures, either in single bundles with a V-shaped attachment to the centromere or in split bundles to bordering pericentromeric flanks. In holocentric L. elegans chromosomes, H2AThr120ph is located predominantly in the centromeric groove of each chromatid as proven by subsequent FIB/FESEM ablation and 3D reconstruction. α-tubulin localizes to the edges of the groove. In both holocentric and monocentric chromosomes, no additional intermediate structures between microtubules and the centromere were observed. We established models of the distribution of CENH3, H2AThr120ph and the attachment sites of microtubules for metacentric and holocentric plant chromosomes.
authors.affiliation: Ultrastructural Research, Department Biology I, Biozentrum der Ludwig-Maximillians-Universität München, Großhadernerstr. 2-4, D-82152, Planegg-Martinsried, Germany.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Wanner
authors.affiliation: Ultrastructural Research, Department Biology I, Biozentrum der Ludwig-Maximillians-Universität München, Großhadernerstr. 2-4, D-82152, Planegg-Martinsried, Germany.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Schroeder-Reiter
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Correnstraße 3, D-06466, Stadt Seeland, Germany.
authors.forename: Wei
authors.initials: W
authors.lastname: Ma
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Correnstraße 3, D-06466, Stadt Seeland, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Houben
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Correnstraße 3, D-06466, Stadt Seeland, Germany. schubertv@ipk-gatersleben.de.
authors.forename: Veit
authors.initials: V
authors.lastname: Schubert
mesh_terms: D001324:Autoantigens / Q000378:metabolism; D002503:Centromere / Q000378:metabolism / Q000648:ultrastructure*; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D032461:Chromosomes, Plant / Q000378:metabolism / Q000648:ultrastructure*; D006657:Histones / Q000378:metabolism; D019684:Magnoliopsida / Q000235:genetics* / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D008870:Microtubules / Q000648:ultrastructure*; D010940:Plant Proteins / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism
chemical_list: D001324:Autoantigens; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D010940:Plant Proteins; D014404:Tubulin
keywords: Barley; CENH3; H2AThr120ph; Luzula; Tubulin; Wheat