Current SEM techniques for de‐ and re‐construction of centromeres to determine 3D CENH3 distribution in barley mitotic chromosomes

Schroeder-Reiter, Elizabeth; Sanei, Maryam; Houben, Andreas; Wanner, Gerhard

doi:10.1111/j.1365-2818.2011.03592.x

Cited by 20 publications

(13 citation statements)

References 38 publications

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“…As it is crucial to define the coordinates of a target area for re-localization in SEM we developed a variety of slides with coordinates, successively improved for different demands (Schroeder-Reiter et al 2012). Our aim was to embed cells on slides or cover slips within an ultra-thin resin layer (i) for rapid and precise correlation to LM micrographs and (ii) to allow FIB-milling in any desired direction.…”

Section: Introductionmentioning

confidence: 99%

Precise and economic FIB/SEM for CLEM: with 2 nm voxels through mitosis

Luckner

Wanner

2018

Histochem Cell Biol

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A portfolio is presented documenting economic, high-resolution correlative focused ion beam scanning electron microscopy (FIB/SEM) in routine, comprising: (i) the use of custom-labeled slides and coverslips, (ii) embedding of cells in thin, or ultra-thin resin layers for correlative light and electron microscopy (CLEM) and (iii) the claim to reach the highest resolution possible with FIB/SEM in xyz. Regions of interest (ROIs) defined in light microscope (LM), can be relocated quickly and precisely in SEM. As proof of principle, HeLa cells were investigated in 3D context at all stages of the cell cycle, documenting ultrastructural changes during mitosis: nuclear envelope breakdown and reassembly, Golgi degradation and reconstitution and the formation of the midzone and midbody.Electronic supplementary materialThe online version of this article (10.1007/s00418-018-1681-x) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Precise and economic FIB/SEM for CLEM: with 2 nm voxels through mitosis

Luckner

Wanner

2018

Histochem Cell Biol

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“…Dual beam focused ion beam/field emission scanning electron microscopy (FIB/FESEM) investigations were performed on a Zeiss Auriga CrossBeam Workstation, a field emission scanning electron microscope equipped with a Gallium ion beam, in-lens, chamber SE and EsB detectors (Carl Zeiss, Germany) as described in Schroeder-Reiter et al [37]. For FIB/FESEM sectioning the specimens were carbon-coated to 10 nm for stability.…”

Section: Methodsmentioning

confidence: 99%

Stretching the Rules: Monocentric Chromosomes with Multiple Centromere Domains

et al. 2012

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The centromere is a functional chromosome domain that is essential for faithful chromosome segregation during cell division and that can be reliably identified by the presence of the centromere-specific histone H3 variant CenH3. In monocentric chromosomes, the centromere is characterized by a single CenH3-containing region within a morphologically distinct primary constriction. This region usually spans up to a few Mbp composed mainly of centromere-specific satellite DNA common to all chromosomes of a given species. In holocentric chromosomes, there is no primary constriction; the centromere is composed of many CenH3 loci distributed along the entire length of a chromosome. Using correlative fluorescence light microscopy and high-resolution electron microscopy, we show that pea (Pisum sativum) chromosomes exhibit remarkably long primary constrictions that contain 3–5 explicit CenH3-containing regions, a novelty in centromere organization. In addition, we estimate that the size of the chromosome segment delimited by two outermost domains varies between 69 Mbp and 107 Mbp, several factors larger than any known centromere length. These domains are almost entirely composed of repetitive DNA sequences belonging to 13 distinct families of satellite DNA and one family of centromeric retrotransposons, all of which are unevenly distributed among pea chromosomes. We present the centromeres of Pisum as novel “meta-polycentric” functional domains. Our results demonstrate that the organization and DNA composition of functional centromere domains can be far more complex than previously thought, do not require single repetitive elements, and do not require single centromere domains in order to segregate properly. Based on these findings, we propose Pisum as a useful model for investigation of centromere architecture and the still poorly understood role of repetitive DNA in centromere evolution, determination, and function.

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“…However, centromere chromatin does not consist exclusively of CENP-A nucleosomes 2 – 4 , as canonical histone H3 nucleosomes are also present at the centromere. In all models of centromere chromatin structure in mitotic chromosomes, the CENP-A nucleosomes are found exclusively on the external face of the centromere, whereas the inner centromere contains exclusively H3 nucleosomes 2 , 4 – 7 . The N-terminal tail of CENP-A differs considerably from that of H3, and between species.…”

Section: Introductionmentioning

confidence: 99%

Aurora A-dependent CENP-A phosphorylation at inner centromeres protects bioriented chromosomes against cohesion fatigue

et al. 2018

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Sustained spindle tension applied to sister centromeres during mitosis eventually leads to uncoordinated loss of sister chromatid cohesion, a phenomenon known as “cohesion fatigue.” We report that Aurora A-dependent phosphorylation of serine 7 of the centromere histone variant CENP-A (p-CENP-AS7) protects bioriented chromosomes against cohesion fatigue. Expression of a non-phosphorylatable version of CENP-A (CENP-AS7A) weakens sister chromatid cohesion only when sister centromeres are under tension, providing the first evidence of a regulated mechanism involved in protection against passive cohesion loss. Consistent with this observation, p-CENP-AS7 is detected at the inner centromere where it forms a discrete domain. The depletion or inhibition of Aurora A phenocopies the expression of CENP-AS7A and we show that Aurora A is recruited to centromeres in a Bub1-dependent manner. We propose that Aurora A-dependent phosphorylation of CENP-A at the inner centromere protects chromosomes against tension-induced cohesion fatigue until the last kinetochore is attached to spindle microtubules.

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Current SEM techniques for de‐ and re‐construction of centromeres to determine 3D CENH3 distribution in barley mitotic chromosomes

Cited by 20 publications

References 38 publications

Precise and economic FIB/SEM for CLEM: with 2 nm voxels through mitosis

Precise and economic FIB/SEM for CLEM: with 2 nm voxels through mitosis

Stretching the Rules: Monocentric Chromosomes with Multiple Centromere Domains

Aurora A-dependent CENP-A phosphorylation at inner centromeres protects bioriented chromosomes against cohesion fatigue

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