3D Analysis of chromosome architecture: advantages and limitations with SEM

Abstract

Three-dimensional mitotic plant chromosome architecture can be investigated with the highest resolution with scanning electron microscopy compared to other microscopic techniques at present. Specific chromatin staining techniques making use of simultaneous detection of back-scattered electrons and secondary electrons have provided conclusive information on the distribution of DNA and protein in barley chromosomes through mitosis. Applied to investigate the structural effects of different preparative procedures, these techniques were the groundwork for the “dynamic matrix model” for chromosome condensation, which postulates an energy-dependent process of looping and bunching of chromatin coupled with attachment to a dynamic matrix of associated protein fibers. Data from SEM analysis shows basic higher order chromatin structures: chromomeres and matrix fibers. Visualization of nanogold-labeled phosphorylated histone H3 (ser10) with high resolution on chromomeres shows that functional modifications of chromatin can be located on structural elements in a 3D context.

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

“…The size of chromosomes would logically affect any physical forces during condensation, isolation, and spreading, in particular as observed in suspension preparations for which the influence of centrifugal forces are apparent ( fig. 2 C, D) [Wanner et al, 2005]. There is also evidence in wallaby (marsupial) hybrids that the length of the primary constriction is related to the number of centromere-specific DNA sequence repeats [Metcalfe et al, 2007].…”

“…We assume, according to the Dynamic Matrix Model for chromosome condensation [Wanner and Formanek, 2000], that parallel matrix fibers are integral to chromosome architecture, but only exposed under certain conditions. These conditions include inhibiting formation of the spindle apparatus by arresting of chromosomes [Wanner and Schroeder-Rei ter, 2008], isolation techniques (centrifugation, spreading of chromosomes) [Wanner et al, 2005], and, according to the data A B Fig. 7.…”

Ki-67 is a PP1-interacting protein that organises the mitotic chromosome periphery

“…When mitotic chromosomes are examined by whole mount microscopy the surface chromatin is obscured by a layer of proteins and RNA derived from the dense fibrillar component (DFC) and granular component (GC) of the nucleolus (Moyne and Garrido, 1976; Harrison et al, 1982; Adolph and Kreisman, 1983; Sumner, 1991; Gautier et al, 1992b; Wanner et al, 2005). This perichromosomal layer includes pre-rRNA, U3 snoRNAs, and over 20 ribosomal proteins, including nucleolin and Nopp140, NPM/B23, Bop1, Nop52, PM-Scl 100, and Ki-67 (Gautier et al, 1992a, 1994; Hernandez-Verdun and Gautier, 1994; Van Hooser et al, 2005; Fomproix et al, 1998; Angelier et al, 2005).…”

Focused ion beam (FIB) combined with high resolution scanning electron microscopy: A promising tool for 3D analysis of chromosome architecture

“…Classical studies elucidated chromosome topography, characterized by 30 nm fibril bunched into chromomeres, by secondary electron (SE) signal detection (Harrison et al, 1982;Allen et al, 1986;Sumner, 1991;Wanner et al, 1991Wanner et al, , 2005. Due to the compactness of metaphase chromosomes, only very limited insight into the chromosome architecture was possible with SE analysis.…”