Denaturation of metaphase chromatin plates observed by transmission electron microscopy and atomic force microscopy in aqueous solution

Abstract

It is currently considered that metaphase chromosomes are formed by folded chromatin fibers [1]. Nevertheless, chromatin fibers are only observed when metaphase chromosomes are treated with solutions of very low ionic strength; in metaphase ionic conditions, chromosomes are very dense and chromatin is apparently folded forming plate-like structures [2]. Metaphase chromosomes from HeLa cells were purified on sucrose gradients containing 5 mM Mg2+, dialyzed extensively to remove sucrose and finally mechanically disrupted by rapid passage through a syringe needle. A TEM micrograph corresponding to typical chromatin plates from metaphase chromosomes prepared following this procedure is presented in Figure 1A. When the plates spread on carbon-coated grids were treated with high concentrations of NaCl, before crosslinking with glutaraldehyde, we observed denaturation of these planar structures leading to different levels of unfolding; in some micrographs we observed small granules having the diameter corresponding to single nucleosomes or aggregates that may contain few nucleosomes (see Figure 1B). AFM imaging was performed using an Asylum MFP-3D system working in AC mode. Uncrosslinked samples were adsorbed on freshly cleaved mica and observed in the presence of a solution containing 5 mM Mg2+. The best images were obtained under a low resonant frequency (∼12 kHz) and increasing the setpoint after engaging, in order to retract the piezo at maximum without losing the interaction between tip and plate. An example of an AFM image of chromatin plates is presented in Figure 2A. After the addition of approximately 1 volume of 4 M NaCl, the same structures can be seen in Figure 2B completely denatured.