STORM-TEM correlative imaging of replicative domains in optimally structurally preserved chromatin

Session

Poster Session 1

Authors

Ekaterina Ryumina (1, 3), Sergei Golyshev (1), Andrei Moiseenko (2), Igor Kireev (1, 2)

Affiliations

1. Belozersky Institute of Physico-chemical Biology, Lomonosov Moscow State University, Moscow 119234, Russia
2. Faculty of Biology, Lomonosov Moscow State University, Moscow 119234, Russia
3. Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow 119234, Russia

Keywords

chromatin, STORM, TEM, ET, CLEM, correlative microscopy, visualization, replicative labeling

Abstract text

The principles of spatial organization of eukaryotic genome remain unclear. Current hypotheses, based primarily on microscopic observations, range from the idea of hierarchical folding to the "polymer melt" model. Great amount of data about chromatin structure was obtained using electron microscopy. Although it allows for high resolution, it has several disadvantages over light microscopy, long multistep sample preparation that casts doubts on structure nativity being one of them. 
Chromatin is a very heterogeneous structure and it is sensitive to the experimental conditions. This requires development of sample preparation approaches combining good structure preservation with efficient selective labeling of structural-functional chromatin states. In this study we used a modified method of replicative labeling (1) adapted for strong aldehyde fixation. It includes Edu-labeling of newly synthesized DNA, label detection with Click-chemistry and biotin-streptavidin and subsequent Nanogold-Ag amplification for TEM. This approach was applied for correlative superresolution and TEM microscopy by using a mixture of biotinylated and fluorescent (AlexaFluor-647) azides and sequentially imaging the samples by STORM microscopy and labeling the same replicative domains with streptavidin-Nanogold. 
This protocol has several advantages over previously published ones. Glutaraldehyde fixation ensures optimal preservation of chromatin near-native structure. Click-chemistry provides simple and extremely selective labeling of replicated DNA, without the need of DNA denaturation prerequisite for BrdU detection with antibodies used in previous reports. The use of streptavidin-Nanogold conjugates provides better penetration efficiency even into glutaraldehyde-fixed samples due to the relatively small size of the probe. Overall our protocol allows for high contrast high-efficiency pre-embedding labeling compatible with various 3D-electron microscopy techniques. 

The study was supported by RFBR grant 19-015-00273, RSF grant 17-15-01290.

References

1. Deng, X., Zhironkina, O. A., Cherepanynets, V. D., Strelkova, O. S., Kireev, I. I., & Belmont, A. S. (2016). Cytology of DNA replication reveals dynamic plasticity of large-scale chromatin fibers. Current Biology, 26(18), 2527-2534.