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The ultrastructure of mono- and holocentric plant centromeres: an immunological investigation by structured illumination microscopy and scanning electron microscopy

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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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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.

Conflict of Interest

The authors declare that they have no competing interests.

Compliance with Ethical Standard

This article does not contain any studies with human participants or animals performed by any of the authors.

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Authors and Affiliations

  1. Ultrastructural Research, Department Biology I, Biozentrum der Ludwig-Maximillians-Universität München, Großhadernerstr. 2-4, D-82152, Planegg-Martinsried, Germany

    Gerhard Wanner & Elizabeth Schroeder-Reiter

  2. Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Correnstraße 3, D-06466, Stadt Seeland, Germany

    Wei Ma, Andreas Houben & Veit Schubert

Authors
  1. Gerhard Wanner
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  2. Elizabeth Schroeder-Reiter
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  3. Wei Ma
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  4. Andreas Houben
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  5. Veit Schubert
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Corresponding author

Correspondence to Veit Schubert.

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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)

High Resolution Image (TIFF 2506 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)

High Resolution Image (TIFF 8196 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)

High Resolution Image (TIFF 6537 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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