Dense chromatin plates in metaphase chromosomes

Abstract

In a previous work we observed multilayered plate-like structures surrounding partially denatured HeLa chromosomes at metaphase ionic conditions. This unexpected finding has led us to carry out an extensive investigation of these structures. Our results show that plates can also be found in metaphase chromosomes from chicken lymphocytes. We have used atomic force microscopy (AFM) to image and investigate the mechanical properties of plates in aqueous solution. Plates are thin (approximately 6.5 nm each layer) but compact and resistant to penetration by the AFM tip: their Young's modulus is approximately 0.2 GPa and the stress required for surface penetration is approximately 0.03 GPa in the presence of Mg(2+) (5-20 mM). Low-ionic strength conditions produce emanation of chromatin fibers from the edges of uncrosslinked plates. These observations and AFM results obtained applying high forces indicate that the chromatin filament is tightly tethered inside the plates. Images of metal-shadowed plates and cryo-electron microscopy images of frozen-hydrated plates suggest that nucleosomes are tilted with respect to the plate surface to allow an interdigitation between the successive layers and a thickness reduction compatible with the observed plate height. The similarities between denatured plates from chicken chromosomes and aggregates of purified chromatin from chicken erythrocytes suggest that chromatin has intrinsic structural properties leading to plate formation. Scanning electron micrographs and images obtained with the 200-kV transmission microscope show that plates are the dominant component of compact chromatids. We propose that metaphase chromosomes are formed by many stacked plates perpendicular to the chromatid axis.

The energy components of stacked chromatin layers explain the morphology, dimensions and mechanical properties of metaphase chromosomes

“…In these studies, it has to be taken into account that a significant part of the chromosome volume is occupied by water [67]. This and the relatively weak association between chromatin layers [15,16] suggest that chromosome structure may be dynamic. The rapid diffusion observed for topoisomerase II and other proteins in mitotic chromosomes in vivo [68][69][70] is consistent with the possibility that condensed chromatids are actually very dynamic structures.…”

“…Thus, it is very likely that the energy 1 nn that stabilizes the stacking of the successive layers in chromosomes is essentially due to face-to-face interactions between nucleosomes. The energy per nucleosome in internucleosome interactions has been obtained experimentally [21,22] and in modelling studies [25 -27] in several laboratories (see Introduction); taking into account all the values obtained in these studies, it is reasonable to consider that 1 nn % 3.4 -14 k B T. On the other hand, the easy sliding between layers in chromatin plates observed in electron microscopy experiments [15,16], suggested that the forces holding the chromatin filament within a layer are higher that the interactions between adjacent layers. This is consistent with the relatively large the Young modulus [15] and the high mechanical resistance [17] found for chromatin plates in AFM experiments in aqueous media, and indicates that the energy 1 wl stabilizing the structure of layers is higher than 1 nn .…”

“…Micromechanical studies [11], and immunofluorescence [12] and cryo-electron microscopy [13] results suggested models in which chromatin is irregularly folded within condensed chromatids (scheme b). The thinplate model (scheme c) was based on electron microscopy and AFM studies of the structure and mechanical properties of the multilayer plates that emanate from partially denatured chromosomes under metaphase ionic conditions [14,15]. Further studies using polarizing microscopy, cryo-electron microscopy and electron tomography [16] and AFM-based nanotribology [17] showed that in each layer the chromatin filament forms a flexible two-dimensional network (5-6 nm thick) in which nucleosomes are irregularly oriented, allowing the interdigitation of the successive layers that are stacked in the metaphase chromatids.…”

“…Chromosome dimensions and structural models for metaphase chromatin folding. (a) Schematic illustration of different models for chromatin structure in metaphase chromosomes: (a) chromatin fibres (grey lines) form loops that are bound to a central protein scaffold (brown) [8][9][10], (b) chromatin fibres are irregularly folded [11][12][13], and (c) chromatin has a laminar structure with many staked layers oriented perpendicular to the chromatid axis [2,[14][15][16][17][18]; parallel lines represent the side view of the stacked layers. (b) Examples showing the differences in metaphase chromosome sizes that exist among animal species: crested newt (T. cristatus) (a), human (b), and D. melanogaster (c); the three chromosome sets are represented at the same magnification; figure reproduced from [19] with permission from the Royal Society.…”

Electron microscopy and atomic force microscopy studies of chromatin and metaphase chromosome structure

“…6E-G (Caravaca et al, 2005;Gállego et al, 2009), metaphase chromosomes are very compact, even using buffers containing relatively low concentrations of Mg 2+ (5 mM) or polyamines (0.35 mM). Similar results were obtained in scanning electron microscopy experiments with chromosomes prepared in buffers containing 5 mM Mg 2+ (Adolph and Kreisman, 1983) or different concentrations of polyamines [0.7 mM (Sone et al, 2002); 0.35 mM (Gállego et al, 2009)]. Condensed metaphase chromosomes are compact but at the same time they are extremely soft structures that become easily distorted during the preparation process.…”