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

The folding of the chromatin filament and, in particular, the organization of genomic DNA within metaphase chromosomes has attracted the interest of many laboratories during the last five decades. This review discusses our current understanding of chromatin higher-order structure based on results obtained with transmission electron microscopy (TEM), cryo-electron microscopy (cryo-EM), and different atomic force microscopy (AFM) techniques. Chromatin isolated from different cell types in buffers without cations form extended filaments with nucleosomes visible as separated units. In presence of low concentrations of Mg(2+), chromatin filaments are folded into fibers having a diameter of ∼ 30 nm. Highly compact fibers were obtained with isolated chromatin fragments in solutions containing 1-2mM Mg(2+). The high density of these fibers suggested that the successive turns of the chromatin filament are interdigitated. Similar results were obtained with reconstituted nucleosome arrays under the same ionic conditions. This led to the proposal of compact interdigitated solenoid models having a helical pitch of 4-5 nm. These findings, together with the observation of columns of stacked nucleosomes in different liquid crystal phases formed by aggregation of nucleosome core particles at high concentration, and different experimental evidences obtained using other approaches, indicate that face-to-face interactions between nucleosomes are very important for the formation of dense chromatin structures. Chromatin fibers were observed in metaphase chromosome preparations in deionized water and in buffers containing EDTA, but chromosomes in presence of the Mg(2+) concentrations found in metaphase (5-22 mM) are very compact, without visible fibers. Moreover, a recent cryo-electron microscopy analysis of vitreous sections of mitotic cells indicated that chromatin has a disordered organization, which does not support the existence of 30-nm fibers in condensed chromosomes. TEM images of partially denatured chromosomes obtained using different procedures that maintain the ionic conditions of metaphase showed that bulk chromatin in chromosomes is organized forming multilayered plate-like structures. The structure and mechanical properties of these plates were studied using cryo-EM, electron tomography, AFM imaging in aqueous media, and AFM-based nanotribology and force spectroscopy. The results obtained indicated that the chromatin filament forms a flexible two-dimensional network, in which DNA is the main component responsible for the mechanical strength observed in friction force measurements. The discovery of this unexpected structure based on a planar geometry has opened completely new possibilities for the understanding of chromatin folding in metaphase chromosomes. It was proposed that chromatids are formed by many stacked thin chromatin plates oriented perpendicular to the chromatid axis. Different experimental evidences indicated that nucleosomes in the plates are irregularly oriented, and that the successive layers are interdigitated (the apparent layer thickness is 5-6 nm), allowing face-to-face interactions between nucleosomes of adjacent layers. The high density of this structure is in agreement with the high concentration of DNA observed in metaphase chromosomes of different species, and the irregular orientation of nucleosomes within the plates make these results compatible with those obtained with mitotic cell cryo-sections. The multilaminar chromatin structure proposed for chromosomes allows an easy explanation of chromosome banding and of the band splitting observed in stretched chromosomes.