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Methodological developments at the METi stem in large part from the research activity of the Laboratory of Eucaryotic Molecular Biology (LBME), which has been using transmission electron microscopy (TEM) for over 30 years to understand the mechanisms of gene expression, in particular the synthesis of ribonucleoprotein particles and the functional organization of the nucleus. Electron microscopy is being involved at different scales from the ultrastructural description of nuclear domains and the intracellular localization of proteins and RNAs involved in these mechanisms to the observation of individual molecules or macromolecular complexes and the determination of their three-dimensional structure.
The LBME has been gradually implementing advanced electron microscopy methods to study the functional organization of the cell nucleus. The laboratory was among the first in France to acquire a unit for ultra-fast freezing for improving the preparation of cell samples through cryofixation and cryoembedding. Hence, the use of ultra-fast freezing revealed the internal structures of the nucleolus in the yeast Schizosaccharomyces pombe and Saccharomyces cerevisiae (1-3). With the JEM 2100-CRP 200 keV TEM installed in 2007 came the possibility to perform electron tomography, which allows visualization of samples in three dimensions and improves the axial resolution when observing cell sections. Using this technique, it is possible to detect macromolecular complex in their cellular environment, as illustrated by the recent observation of pre-ribosomes, the maturation intermediates of ribosomal subunits, in pre-nucleolar bodies in human cells (4). Installation of this microscope has also paved the way for the implementation of cryo-electron microscopy. This technique is now at the heart of projects aiming at determining the structure of pre-ribosomes. Our ultimate goal is to visualize the structure of these complexes in situ by integrating these structural biology data into the observation of cell sections by tomography in order to understand the intracellular organization of ribosome biogenesis at the molecular level. The use of correlative light-electron microscopy combines the resolution and contextual nature of electron microscopy with the sensitivity and flexibility of fluorescence microscopy, which lends itself to the observation of living cells. It is then possible, by fluorescence microscopy, to perform electron microscopic observations of rare events or of structures difficult to recognize by their morphology, or to identify in living cells the appropriate time window for sample preparation.
Electron microscopy at the LBME was developed in conjunction with genome sequencing, which has ushered in a new era in the study of gene expression. The yeast S. cerevisiae was the first organism whose genome was completely sequenced in 1997. It is amenable to molecular genetics and has become an important model organism for studying mechanisms such as ribosome assembly or chromosome dynamics. Electron microscopy at the LBME has been involved in the study of several of the 150+ factors required for the synthesis of ribosomal subunits and identified by proteomics. The subnuclear localization of these proteins in yeast has been determined by immunodetection, sometimes in association with the detection of associated RNAs by EM in situ hybridization. Electron microscopy has also been instrumental for characterizing the changes induced by the mutation of these factors on the organization of the nucleus or the transport of pre-ribosomal particles (5-7). Along the same line, the characterization of Diamond-Blackfan anemia, a rare inherited pathology associated with the mutation of genes encoding ribosomal proteins, has highlighted disruption of the nucleolus associated with ribosome synthesis defects in patient cells (8). Ribosomal gene transcription can be directly visualized by performing chromatin spreading according to Miller, or "Christmas trees", thus allowing direct assessment of parameters like the number of active polymerases per gene (9) or the size of the genes (10). The current development of new genome editing techniques opens up new possibilities for tagging genes, RNAs and proteins. Combining these new tools with advanced methods in electron microscopy (CEMOVIS, template matching…) should allow exploring the ultrastructure of gene expression in unprecedented ways.
