Chromosome Centromeres: Structural and Analytical Investigations with High Resolution Scanning Electron Microscopy in Combination with Focused Ion Beam Milling

Abstract

Whole mount mitotic metaphase chromosomes of different plants and animals were investigated with high resolution field emission scanning electron microscopy (FESEM) to study the ultrastructural organization of centromeres, including metacentric, acrocentric, telocentric, and holocentric chromosome variants. It could be shown that, in general, primary constrictions have distinctive ultrastructural features characterized by parallel matrix fibrils and fewer smaller chromomeres. Exposure of these structures depends on cell cycle synchronization prior to chromosome isolation, chromosome size, and chromosome isolation technique. Chromosomes without primary constrictions, small chromosomes, and holocentric chromosomes do not exhibit distinct ultrastructural elements that could be directly correlated to centromere function. Putative spindle structures, although rarely observed, spread over the primary constriction to the bordering pericentric regions. Analytical FESEM techniques, including specific DNA staining with Pt blue, staining of protein as a substance class with silver-colloid, and artificial loosening of fixed chromosomes with proteinase K, were applied, showing that centromere variants and ultrastructural elements in the centromere differ in DNA and protein distribution. Immunogold localization allowed high-resolution comparison between chromosomes with different centromere orientations of the distribution of centromere-related histone variants, phosphorylated histone H3 (ser10), and CENH3. A novel application of FESEM combined with focused ion beam milling (FIB) provided new insights into the spatial distribution of these histone variants in barley chromosomes.

Sperm Nuclear Architecture Is Locally Modified in Presence of a Robertsonian Translocation t(13;17)

“…Thus identical centromeric sequences may be specifically recognized by protein complexes and/or specific centromeric chromatin may be differentially marked allowing subtypes clustering. Thus, specific epigenetic marks have been described in plant acrocentric chromosomes [45] but also in mammals with specific H3K9me3 marks on pericentromeric and centromeric chromatin and with the presence of non coding transcripts and histone variants (for a review see [46]). In vivo data also support this hypothesis.…”

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

“…Using these data sets, 3D reconstructions with high resolution can be calculated, opening doors to analysis and quantification of structures that were inaccessible until now. This FIB/FESEM approach has been successfully applied in our lab for investigating chromosome architecture as a means to more precise chromatin analysis in mitotic chromosomes (Schroeder‐Reiter et al , 2009; Schroeder‐Reiter & Wanner, 2009; Heckmann et al , 2011).…”

“…Functionally, the centromere is primarily responsible for ordered chromosome segregation by sister chromatid cohesion, kinetochore and spindle assembly and checkpoint regulation in mitosis and meiosis. Centromeres are structurally distinct from the chromosome arms, exhibiting exposed parallelly oriented chromatin fibers and fewer, smaller chromomeres than the chromosomes arms (Wanner & Formanek, 2000; Metcalfe et al , 2007; Schroeder‐Reiter & Wanner, 2009).…”

Relating Centromeric Topography in Fixed Human Chromosomes to α-Satellite DNA and CENP-B Distribution

“…Centromeric ridges may be formed at highly compacted chromatin fibres and appear as 50 nm thick, globular structures , which is consistent with our own observations of denatured chromosomes. Such prominent rounded projections at the centromere [Jack et al, 1985] and the pericentromeric region of C-banded chromosomes have been observed by SEM [Jack et al, 1985;Schroeder-Reiter and Wanner, 2009] and AFM [Tan et al, 2001]. Interestingly, compaction in the pericentromeric region was affected to a lesser degree than along the chromosome arms [Jack et al, 1985].…”