Cryo-EM reveals chromosomes are formed by stacked layers
New cryo-electron microscopy studies indicate that during mitosis, chromosome DNA is packed in stacked layers of chromatin.
The latest results from Professor Joan-Ramon Daban at the Chromatin Laboratory, Autonomous University of Barcelona, confirm a surprising structure proposed by UAB researchers more than a decade ago.
In the cell nuclei, the DNA is bound to histone proteins and forms long chains of nucleosomes called chromatin fibers.
In 2005, Daban and colleagues discovered the chromatin of mitotic chromosomes forms multilaminar plates, surprising many researchers that had not expected that linear fibres of chromatin could give rise to planar structures.
Critics also questioned the experimental approach, pointing out that the methods used - conventional electron microscopy and atomic force microscopy - demanded the sample to be adsorbed on flat surfaces of carbon and mica.
What's more, they also highlighted that in the case of electron microscopy, the sample had to be fixed with chemical crosslinkers, treated with contrasting agents, and dehydrated.
However, a new study based on cryo-electron microscopy under cryogenic conditions and synchrotron X-ray scattering, confirms that in mitotic chromosomes, the DNA is densely packed, forming stacked sheets of chromatin that are stabilised by interactions between nucleosomes.
Left: the perpendicular orientation of the chromatin layers within the mitotic chromosomes. Right: organisation of the nucleosomes in two adjacent layers, and the distances obtained in the cryotomograms (in blue) and in the X-ray scattering experiments (in red). [J.R.Daban]
As the researchers say, the advantage of the cryo-electron microscopy techniques used in this new study is that the sample (uncrosslinked and untreated with contrasting agents) is suspended in an aqueous solution that is kept frozen at -180 ° C, even during imaging.
The researchers went on to use cryo-electron tomography to capture images at different tilt angles, and obtain 3D reconstructions of the structures.
Reconstructions revealed that the chromatin emanating from human chromosomes maintained under physiological ionic conditions is planar and forms multilaminar plates.
The thickness measurements (single layer 7.5 nm; two layers in contact 13 nm) suggest that the plates are formed by mononucleosome layers, which are interdigitated between them.
Complementary X-ray scattering experiments showed a dominant peak at 6 nm, which can be correlated with the distance between layers and between nucleosomes associated through their lateral faces.
Analyses showed multilaminar plates with dimensions corresponding to that a human chromosome (600 nm).
According to the researchers, this suggests that the chromosomes are formed by stacked layers of chromatin that are oriented perpendicular to the axis of the chromosome.
This structure is very compact and probably has the function of protecting the integrity of genomic DNA during cell division.