Cryotomographie électronique

Résumé

La cryotomographie électronique, ou tomographie cryoélectronique, est un type de cryo-microscopie électronique utilisant les techniques de tomographies pour obtenir la restitution tridimensionnelle à partir d'images bidimensionnelles d'un échantillon vu sous différentes inclinaisons aux températures cryogéniques atteintes par refroidissement à l'azote liquide, voire à l'hélium liquide. Une cryotomographie électronique peut permettre d'obtenir des détails sur des structures cellulaires complexes à des résolutions de l'ordre de la dizaine de nanomètre. Le signal de cryotomographie électronique est assez faible, avec un rapport signal sur bruit typiquement de 0,01. Compte tenu de la longueur d'onde des électrons, la résolution est théoriquement limitée à environ , mais l'analyse de particules individuelles de subtomogrammes cryoélectroniques permet d'atteindre des résolutions de . Notes et références Catégorie:Microscope électronique Catégorie:Biologie cellulaire Catégori

Source officielle

https://fr.wikipedia.org/wiki/Cryotomographie_%C3%A9lectronique

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In this course we will study the cell (minimum unit of life) and its components. We will study several key cellular features: Membranes, genomes, channels and receptors. We will apply the laws of physics to develop models to make quantitative and predictive statements.

This course instructs students in the use of advanced computational models and simulations in cell biology. The importance of dimensionality, symmetry and conservation in models of self-assembly, membranes, and polymer/filament scaling laws reveals how cells exploit these principles in life.

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The centrosome, the major microtubule organizing center in animal cells, is composed of two orthogonally arranged centrioles and surrounding pericentriolar material. Centrioles are evolutionary conserved organelles characterized by a nine-fold symmetry of microtubules and are built around a cartwheel in their proximal region. The overall architecture at ~ 40 Å resolution of the exceptionally long proximal region of Trichonympha spp. basal body has been uncovered previously using cryo-electron tomography (cryo-ET) followed by subtomogram averaging. The resulting 3D map revealed not only a central cartwheel hub that can accommodate rings of the evolutionarily conserved SAS-6 proteins, but also novel features, whose identity is unknown. In this thesis, we used T. agilis, which is the only Trichonympha sp. with an assembled genome and transcriptomic data, to gain some more insight into the structure, composition and first steps of cartwheel assembly. With the aim of generating a high-resolution 3D model of the T. agilis proximal region, we used cryo-ET and subtomogram averaging to refine the structure of the cartwheel hub and cartwheel inner densities (CID). We uncovered that the CID is polarized, and discovered inter-cartwheel pillars (ICP) between superimposed cartwheel rings. Moreover, we discovered and analysed a new type of the cartwheel of Teranympha mirabilis with a novel type of cartwheel central densities (CCD). We also analyzed the composition of the proximal region of the T. agilis centriole using a proteomic approach. We optimized a density gradient centrifugation protocol for isolation of the T. agilis rostrum, which harbors ~ 1400 centrioles, and performed protein correlation profiling. In this manner, we have identified 47 candidates of the rostrum proteome, including three new T. agilis SAS-6 homologs, which were further characterized. Furthermore, we addressed the oligomerization properties of human and two T. agilis SAS-6 proteins (HsSAS-6, TaSAS-6_1 and TaSAS-6_2) in vitro. We found that HsSAS-6 requires an interaction partner in order to be stabilized and form rings and stacks. In contrast, TaSAS-6_1 did not oligomerize in our setup, and TaSAS-6_2 is able to oligomerize and form different types of oligomers, rings included. In conclusion, the structure and composition of the proximal region of the T. agilis centriole uncovered here are anticipated to be of a general significance for studies of the biogenesis and structure of centrioles.

EPFL2018

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Novel features of centriole polarity and cartwheel stacking revealed by cryo-tomography

Alexandra Bezler, Davide Demurtas, Paul Guichard, Georgios Hatzopoulos, Sergey Nazarov, Veronika Nemcíková Villímová

Centrioles are polarized microtubule-based organelles that seed the formation of cilia, and which assemble from a cartwheel containing stacked ring oligomers ofSAS-6 proteins. A cryo-tomography map of centrioles from the termite flagellateTrichonymphaspp. was obtained previously, but higher resolution analysis is likely to reveal novel features. Using sub-tomogram averaging (STA) inT. spp. andTrichonympha agilis, we delineate the architecture of centriolar microtubules, pinhead, and A-C linker. Moreover, we report similar to 25 angstrom resolution maps of the central cartwheel, revealing notably polarized cartwheel inner densities (CID). Furthermore,STAof centrioles from the distant flagellateTeranympha mirabilisuncovers similar cartwheel architecture and a distinct filamentousCID. Fitting the CrSAS-6 crystal structure into the flagellate maps and analyzing cartwheels generatedin vitroindicate thatSAS-6 rings can directly stack onto one another in two alternating configurations: with a slight rotational offset and in register. Overall, improvedSTAmaps in three flagellates enabled us to unravel novel architectural features, including of centriole polarity and cartwheel stacking, thus setting the stage for an accelerated elucidation of underlying assembly mechanisms.

2020

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Cell-free reconstitution reveals centriole cartwheel assembly mechanisms

Niccolo Banterle, Isabelle Fluckiger, Pierre Gönczy, Paul Guichard, Virginie Hamel, Mircea Ioan Iacovache, Veronika Nemcíková Villímová, Henning Paul-Julius Stahlberg

How cellular organelles assemble is a fundamental question in biology. The centriole organelle organizes around a nine-foldsymmetrical cartwheel structure typically 100 nm high comprising a stack of rings that each accommodates nine homodimers of SAS-6 proteins. Whether nine- fold symmetrical ring-like assemblies of SAS-6 proteins harbour more peripheral cartwheel elements is unclear. Furthermore, the mechanisms governing ring stacking are not known. Here we develop a cell-free reconstitution system for core cartwheel assembly. Using cryo-electron tomography, we uncover that the Chlamydomonas reinhardtii proteins CrSAS-6 and Bld10p together drive assembly of the core cartwheel. Moreover, we discover that CrSAS-6 possesses autonomous properties that ensure self-organized ring stacking. Mathematical fitting of reconstituted cartwheel height distribution suggests a mechanism whereby preferential addition of pairs of SAS- 6 rings governs cartwheel growth. In conclusion, we have developed a cell-free reconstitution system that reveals fundamental assembly principles at the root of centriole biogenesis.

Springer Nature2017

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EPFL2018