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.
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References
Bajer AS, Mole-Bajer J (1972) Spindle dynamics and chromosome movements. Academic, New York, 271 pp
Blower MD, Sullivan BA, Karpen GH (2002) Conserved organization of centromeric chromatin in flies and humans. Developmental Cell 2:319–330
Braselton JP (1971) Ultrastructure of non-localized kinetochores of Luzula and Cyperus. Chromosoma 36:89–99
Cabral G, Marques A, Schubert V, Pedrosa-Harand A, Schlögelhofer P (2014) Chiasmatic and achiasmatic inverted meiosis of plants with holocentric chromosomes. Nature Commun 5:5070
Dawe RK, Richardson EA, Zhang X (2005) The simple ultrastructure of the maize kinetochore fits a two-domain model. Cytogenet Genome Res 109:128–133
Demidov D, Schubert V, Kumke K, Weiss O, Karimi-Ashtiyani R, Buttlar J, Heckmann S, Wanner G, Dong Q, Han F, Houben A (2014) Anti-phosphorylated histone H2AThr120: a universal microscopic marker for centromeric chromatin of mono- and holocentric plant species. Cytogenet Genome Res 143:150–156
Harrison CJ, Allen TD, Britch M, Harris R (1982) High-resolution scanning electron-microscopy of human metaphase chromosomes. J Cell Sci 56:409–422
Heckmann S, Jankowska M, Schubert V, Kumke K, Ma W, Houben A (2014) Alternative meiotic chromatid segregation in the holocentric plant Luzula elegans. Nature Commun 5:4979
Heckmann S, Macas J, Kumke K, Fuchs J, Schubert V, Ma L, Novak P, Neumann P, Taudien S, Platzer M, Houben A (2013) The holocentric species Luzula elegans shows interplay between centromere and large-scale genome organization. Plant J 73:555–565
Heckmann S, Schroeder-Reiter E, Kumke K, Ma L, Nagaki K, Murata M, Wanner G, Houben A (2011) Holocentric chromosomes of Luzula elegans are characterized by a longitudinal centromere groove, chromosome bending, and a terminal nucleolus organizer region. Cytogenet Genome Res 134:220–228
Heneen WK (1982) The centromeric region in the scanning electron-microscope. Hereditas 97:311–314
Houben A, Schroeder-Reiter E, Nagaki K, Nasuda S, Wanner G, Murata M, Endo TR (2007) CENH3 interacts with the centromeric retrotransposon cereba and GC-rich satellites and locates to centromeric substructures in barley. Chromosoma 116:275–283
Inaga S, Naguro T, Kameie T, Iino A (2000) Three-dimensional ultrastructure of in situ chromosomes and kinetochores of Tradescantia reflexa anther cells by scanning electron microscopy. 2. Whole mounted chromosomes and kinetochores of pollen moter cells and tapetal cells. Chromosome Science 4:11–20
Ishii T, Karimi-Ashtiyani R, Banaei-Moghaddam AM, Schubert V, Fuchs J, Houben A (2015) The differential loading of two barley CENH3 variants into distinct centromeric substructures is cell type- and development-specific. Chromosome Res 23:277–284
Jensen CG (1982) Dynamics of spindle microtubule organization: Kinetochore fiber microtubules of plant endosperm. J Cell Biol 92:540–558
Kawashima SA, Yamagishi Y, Honda T, Ishiguro K, Watanabe Y (2010) Phosphorylation of H2A by Bub1 prevents chromosomal instability through localizing shugoshin. Science 327(5962):172–177
Lambert AM (1971) Contribution a l'etude du centromere diffus chez les Luzules: Observations sur Luzula albida DC. C R Acad Sci, Ser D 272:403–406
Lermontova I, Schubert V, Fuchs J, Klatte S, Macas J, Schubert I (2006) Loading of Arabidopsis centromeric histone CENH3 occurs mainly during G2 and requires the presence of the histone fold domain. Plant Cell 18:2443–2451
Martin R, Busch W, Herrmann RG, Wanner G (1994) Efficient preparation of plant chromosomes for high-resolution scanning electron microscopy. Chromosome Res 2:411–415
McEwen BF, Chan GKT, Zubrowski B, Savoian MS, Sauer MT, Yen TJ (2001) CENP-E is essential for reliable bioriented spindle attachment, but chromosome alignment can be achieved via redundant mechanisms in mammalian cells. Mol Biol Cell 12:2776–2789
Metcalfe CJ, Bulazel KV, Ferreri GC, Schroeder-Reiter E, Wanner G, Rens W, Obergfell C, Eldridge MD, O'Neill RJ (2007) Genomic instability within centromeres of interspecific marsupial hybrids. Genetics 177:2507–2517
Nagaki K, Kashihara K, Murata M (2005) Visualization of diffuse centromeres with centromere-specific histone H3 in the holocentric plant Luzula nivea. Plant Cell 17:1886–1893
Nagaki K, Cheng Z, Ouyang S, Talbert PB, Kim M, Jones KM, Henikoff S, Buell CR, Jiang J (2004) Sequencing of a rice centromere uncovers active genes. Nat Genet 36:138–145
Neumann P, Navratilova A, Schroeder-Reiter E, Koblizkova A, Steinbauerova V, Chocholova E, Novak P, Wanner G, Macas J (2012) Stretching the rules: monocentric chromosomes with multiple centromere domains. Plos Genet 8, ARTN e1002777
Nordenskiöld H (1951) Cyto-taxonomical studies in the genus Luzula 1. Somatic chromosomes and chromosome numbers. Hereditas 37:325–355
Nordenskiöld H (1962) Studies of meiosis in Luzula purpurea. Hereditas 48:503–519
Pan WH, Houben A, Schlegel R (1993) Highly effective cell synchronization in plant roots by hydroxyurea and amiprophos-methyl or colchicine. Genome 36:387–390
Peterson JB, Ris H (1976) Electron-microscopic study of the spindle and chromosome movement in the yeast Saccharomyces cerevisiae. J Cell Sci 22:219–242
Ribeiro SA, Vagnarelli P, Dong Y, Hori T, McEwen BF, Fukagawa T, Flors C, Earnshaw WC (2010) A super-resolution map of the vertebrate kinetochore. Proc Nat Acad Sci USA 107:10484–10489
Ris H, Witt PL (1981) Structure of the mammalian kinetochore. Chromosoma 82:153–170
San Martin JA, Andrade CG, Vanzela AL (2009) Early meiosis in Rhynchospora pubera L. (Cyperaceae) is marked by uncommon ultrastructural features. Cell Biol International 33:1118–1122
Sanei M, Pickering R, Kumke K, Nasuda S, Houben A (2011) Loss of centromeric histone H3 (CENH3) from centromeres precedes uniparental chromosome elimination in interspecific barley hybrids. Proc Nat Acad Sci USA 108:E498–505
Schermelleh L, Heintzmann R, Leonhardt H (2010) A guide to super-resolution fluorescence microscopy. J Cell Biol 190:165–175
Schrader F (1947) The role of the kinetochore in the chromosomal evolution of the heteroptera and homoptera. Evolution 1:134–142
Schroeder-Reiter E, Houben A, Wanner G (2003) Immunogold labeling of chromosomes for scanning electron microscopy: a closer look at phosphorylated histone H3 in mitotic metaphase chromosomes of Hordeum vulgare. Chromosome Res 11:585–596
Schroeder-Reiter E, Sanei M, Houben A, Wanner G (2012) Current SEM techniques for de- and re-construction of centromeres to determine 3D CENH3 distribution in barley mitotic chromosomes. J Microscopy 246:96–106
Schroeder-Reiter E, Perez-Willard F, Zeile U, Wanner G (2009) Focused ion beam (FIB) combined with high resolution scanning electron microscopy: a promising tool for 3D analysis of chromosome architecture. J Struct Biol 165:97–106
Schroeder-Reiter E, Wanner G (2009) Chromosome centromeres: Structural and analytical investigations with high resolution scanning electron microscopy in combination with focused ion beam milling. Cytogenet Genome Res 124:239–250
Schubert V, Lermontova I, Schubert I (2014) Loading of the centromeric histone H3 variant during meiosis - How does it differ from mitosis? Chromosoma 123:491–497
Screpanti E, De Antoni A, Alushin GM, Petrovic A, Melis T, Nogales E, Musacchio A (2011) Direct binding of Cenp-C to the Mis12 complex joins the inner and outer kinetochore. Curr Biol 21:391–398
Steiner FA, Henikoff S (2014) Holocentromeres are dispersed point centromeres localized at transcription factor hotspots. Elife 3. doi:ARTN e02025
Stellfox ME, Bailey AO, Foltz DR (2013) Putting CENP-A in its place. Cell Mol Life Sci 70:387–406
Sullivan BA, Karpen GH (2004) Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchromatin and heterochromatin. Nat Struct Mol Biol 11:1076–1083
Sumner AT (1991) Scanning electron microscopy of mammalian chromosomes from prophase to telophase. Chromosoma 100:410–418
Wanner G, Formanek H (2000) A new chromosome model. J Struct Biol 132(2):147–161
Zhang H, Dawe RK (2012) Total centromere size and genome size are strongly correlated in ten grass species. Chromosome Res 20:403–412
Acknowledgments
We thank Karin Lipfert for artwork, Katrin Kumke and Jennifer Grünert for excellent technical assistance. This work was supported by the IPK Gatersleben and the China Scholarship Council.
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The authors declare that they have no competing interests.
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This article does not contain any studies with human participants or animals performed by any of the authors.
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Suppl. Table 1
BSE penetration and exiting depths (in nm) from carbon at different accelerating voltages (kV). From Monte Carlo simulations with 200 000 electrons in carbon with a density of 1.1 g/cm3. Average exiting depth in nanometers according to normalized z-max calculation. (PDF 21 kb)
Suppl. Figure 1
Fluorescent light micrographs merged with phase contrast images (greyscale) of (A) spelt metaphase chromosomes labeled with anti-H2AThr120ph (green) showing a signal band in the centromeric region, (B) barley chromosomes labeled with anti-CENH3 (green) showing two distinct signal spots laterally in the centromeric region, and (C) spelt chromosomes labeled with anti-α-tubulin (red) showing microtubule bundles attached at the centromeric regions. (GIF 127 kb)
Suppl. Figure 2
SEM micrographs of holocentric Luzula elegans chromosomes at prometaphase (A), early metaphase (B) and metaphase (C) showing whole chromosomes (left) and corresponding topographic details of the holocentromeric region (right, details of framed areas). Up to prometaphase, chromatin exhibits loose chromomeres but no groove (A). At early metaphase, chromosomes exhibit aligned indentations (B) that fuse at late metaphase to form a continuous groove (C) exhibiting a fibrillar network. (GIF 750 kb)
Suppl. Figure 3
SEM stereo micrograph of a complete holocentric L. elegans metaphase cell similarly immunolabeled for H2AThr120ph (yellow) as in Fig. 7a. At the sides without centromeric grooves a vague longitudinal indentation occurs (arrows), indicating the border between sister chromatids. (GIF 179 kb)
Suppl. Movie 1
SIM image stack of barley anaphase chromosomes labelled by CENH3 and H2AThr120ph, and attached at the monocentromeres by microtubules (α-tubulin). (AVI 2334 kb)
Suppl. Movie 2
Luzula metaphase chromosomes analysed by SIM. Microtubules (α-tubulin) attach along the entire holocentromere labelled by H2AThr120ph. (AVI 7779 kb)
Suppl. Movie 3
SIM image stack of Luzula metaphase chromosomes labelled by H2AThr120ph, and attached along the entire holocentromeres by microtubules (α-tubulin). (AVI 2035 kb)
Suppl. Movie 4
Rendering of the SIM image stack shown in Suppl. Movie 3. (MP4 2808 kb)
Suppl. Movie 5
3D distribution of H2AThr120ph in holocentric Luzula elegans chromosomes (same as in Fig. 7A) showing the H2AThr120ph localization predominantly in the longitudinal grooves, with only a minor fraction in the chromosome interior (H2AThr120ph = yellow labels; chromatin = blue). The exposed (upper) groove is more strongly labeled than the groove lying on the glass slide, presumably due to a diffusion gradient. The 3D image was reconstructed from 700 FIB/FESEM tomographic images. (MOV 10586 kb)
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Wanner, G., Schroeder-Reiter, E., Ma, W. et al. The ultrastructure of mono- and holocentric plant centromeres: an immunological investigation by structured illumination microscopy and scanning electron microscopy. Chromosoma 124, 503–517 (2015). https://doi.org/10.1007/s00412-015-0521-1
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DOI: https://doi.org/10.1007/s00412-015-0521-1