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IMAGING THE CELL AT HIGH RESOLUTION

par douarche - publié le , mis à jour le

 

 

IMAGING THE CELL AT HIGH RESOLUTION


 

 

Amélie Leforestier
Françoise Livolant

 

  • Chromosome structure in the nucleus of the living cell Our goal is to explore the structure of chromatin in the native eukaryotic cell. In collaboration with M. Eltsov (Frankfurt University), we use Cryo Electron Microscopy and Tomography Of VItreous Sections (CEMOVIS and CETOVIS) to obtain nm resolution imaging of the cell in its native state. We are thus able to visualise, recognise and analyse nucleosomes in their native nuclear environment, at a level of detail that allows us to measure the distance P between the DNA gyres wrapped around. We explored interphase nuclei of very different cell types - from human cell lines to embryonic insect cells – and detected the occurrence of multiple conformations. P-distances are on average larger than in the PDB canonical structure, and this parameter varies of more than 0.5 nm around the average. Further characterisation of the particles’ conformation, interactions and cartography is now needed in order to explore the relationship between this conformational variability, nucleosome local organisation and chromatin functional states. We also explore chromatin local supramolecular organization.

Visualization of nucleosomes inside the interphase cell nucleus (here KE37 human cell line) by CEMOVIS. (A) Overall view. The nuclear enveloppe (NE) and nuclear pore complex (NPC) are recognized. (B) Enlargements reveal nucleosomes seen in top (left) or side (right) views. The experimental patterns are compared with the nucleosome crystallographic canonical structure (from PDB ID 1EQZ)

 

  • Structure of biological membranes We analyzed the structure of a variety of cell membranes by CEMOVIS : the multilamellar bodies of the human cell line HT29, the membranes of the Golgi secretion system in the hypodermis of Caenorhabditis elegans, the membranes of the sub-plasmalemmal calcium storage sacs of Paramecium tetraurelia, and the thylakoids of the algae Euglena gracilis. The two bilayers of the membranes can be visualized, and membranes closely apposed at a distance less than 1.1 nm have been observed. Such close contact interactions, that were not observed yet because of a lack of spatial resolution, may be more frequent than expected in Eukaryotic cells, which rises new questions on the mechanisms of membrane fusion in living cell. Asymetry in membrane leaflets are also evidenced. Networks of transmembrane proteins are observed in Golgi and thylakoid membranes. The resolution of the technique allows us not only to solve topological problems but also to detect variations in membrane composition.

 

An Euglena gracillis cell observed by CEMOVIS with its nucleus (N), chromosomes (Chr), mitochondria (M), paramylon (P) and choloroplast (Chl).

 

Stack of thylakoid membranes in the chloroplast of Euglena gracilis. (1) Line profile of stacked mebranes with the Oxygen Evolving Complex (OEC). (2) Transmembrane proteins (photosystems I & II, red arrows) are visible within the membrane bilayers.

 

 

  • Multiscale imaging in biological tissue Cryo soft X ray microscopy (cryoTXM) is an imaging technique for cell and soft matter imaging, complementary to croEM. It provides structural information from vitrified specimens ten to hundred times thicker (typically 0.5 to 5 μm vs 40-75nm), but at lower resolution (20-50 nm). It was so far restricted to isolated small cells. We developed an experimental setup to apply it to large cells and tissue. We obtained thick (up to 3 μm) vitreous sections of Drosophila brain, by generating ultra slow cutting speeds (100 nm/sec). Regions of interest were targeted by coupling with fluorescent microscope. We imaged in parallel thick cryo-sections by cryoTXM at a resolution better than 25 nm, and ultrathin sections (40 nm) of the same region by cryoEM at the nm scale, providing information from the local molecular scale to the global tissue organisation  

Multiscale imaging of Drosophila brain by fluorescence microscopy, cryoTXM, cryoEM. (a) Observation of vitrified Drosophila brain at -140°C. Visualisation of synapses of the Kenyon cells (KC), labelled by GFP-synaptotagmin. (b) CryoTXM of a thick section and visualisation of a synapse (pre-synaptic vesicle *) charactrised by an increase of the inter-neurite distance (ds). (M : mitochondrion). (c) CEMOVIS of an ultrathin section and visualisation of a synapse with its post-synapitic density (PSD).

 
 

REFERENCES


 

ELTSOV M., GREWE D., LEMERCIER N., FRANGAKIS A., LIVOLANT F., LEFORESTIER A. (2018) Nucleosome conformational variability in solution and in interphase nuclei evidenced by cryo-electron microscopy of vitreous sections. Nucleic Acid Research, 46, 9189-9200. doi : 10.1093/nar/gky670.

LEFORESTIER A., P. LEVITZ, T. PREAT, P. GUTTMANN, L.J. MICHOT, TCHENIO P. (2014) Imaging Drosophila brain by combining cryo-soft X-ray microscopy of thick vitreous sections and cryo-electron microscopy of ultrathin vitreous sections. J Struct Biol. 188 (2), 177-82.

LEFORESTIER A, LEMERCIER N., LIVOLANT F. (2012) Contribution of cryo-electron microscopy of vitreous sections to the understanding of biological membrane structure. Proc. Natl. Acad. Sci. (USA) 109 (23) 8959-8964.