The structure of nervous tissue and of Gram-positive bacteria studied by cryo-electron microscopy of vitreous sections

Resume La structure, ou l'architecture, des etres vivants definit le cadre dans lequel la physique de la vie s'accomplit. La connaissance de cette structure dans ses moindres details est un but essentiel de la biologie. Son etude est toutefois entravee par des limitations techniques. Malgre son potentiel theorique, la microscopie electronique n'atteint pas une resolution atomique lorsqu'elle est appliquee a la matiexe biologique. Cela est du en grande partie au fait qu'elle contient beaucoup d'eau qui ne resiste pas au vide du microscope. Elle doit donc etre deshydratee avant d'etre introduite dans un microscope conventionnel. Des artefacts d'agregation en decoulent inevitablement. La cryo-microscopie electronique des sections vitreuses (CEMOVIS) a ete developpee afin de resoudre cela. Les specimens sont vitrifies, c.-a-d. que leur eau est immobilisee sans cristalliser par le froid. Ils sont ensuite coupes en sections ultrafines et celles-ci sont observees a basse temperature. Les specimens sont donc observes sous forme hydratee et non fixee; ils sont proches de leur etat natif. Durant longtemps, CEMOVIS etait tres difficile a executer mais ce n'est plus le cas. Durant cette these, CEMOVIS a ete applique a differents specimens. La synapse du systeme nerveux central a ete etudiee. La presence dans la fente synaptique d'une forte densite de molecules organisees de maniere periodique a ete demontree. Des particules luminales ont ete trouvees dans Ies microtubules cerebraux. Les microtubules ont servi d'objets-test et ont permis de demontrer que des details moleculaires de l'ordre du nm sont preserves. La comprehension de la structure de l'enveloppe cellulaire des bacteries Grampositives aete amelioree. Nos observations ont abouti a l'elaboration d'un nouveau modele hypothetique de la synthese de la paroi. Nous avons aussi focalise notre attention sur le nucleoide bacterien et cela a suscite un modele de la fonction des differents etats structuraux du nucleoide. En conclusion, cette these a demontre que CEMOVIS est une excellente methode poux etudier la structure d'echantillons biologiques a haute resolution. L'etude de la structure de divers aspects des etres vivants a evoque des hypotheses quant a la comprehension de leur fonctionnement. Summary The structure, or the architecture, of living beings defines the framework in which the physics of life takes place. Understanding it in its finest details is an essential goal of biology. Its study is however hampered by technical limitations. Despite its theoretical potential, electron microscopy cannot resolve individual atoms in biological matter. This is in great part due to the fact. that it contains a lot of water that cannot stand the vacuum of the microscope. It must therefore be dehydrated before being introduced in a conventional microscope. Aggregation artefacts unavoidably happen. Cryo-electron microscopy of vitreous sections (CEMOVIS) has been developed to solve this problem. Specimens are vitrified, i.e. they are rapidly cooled and their water is immobilised without crystallising by the cold. They are then. sectioned in ultrathin slices, which are observed at low temperatures. Specimens are therefore observed in hydrated and unfixed form; they are close to their native state. For a long time, CEMOVIS was extremely tedious but this is not the case anymore. During this thesis, CEMOVIS was applied to different specimens. Synapse of central nervous system was studied. A high density of periodically-organised molecules was shown in the synaptic cleft. Luminal particles were found in brain microtubules. Microtubules, used as test specimen, permitted to demonstrate that molecular details of the order of nm .are preserved. The understanding of the structure of cell envelope of Gram-positive bacteria was improved. Our observations led to the elaboration of a new hypothetic model of cell wall synthesis. We also focused our attention on bacterial nucleoids and this also gave rise to a functional model of nucleoid structural states. In conclusion, this thesis demonstrated that CEMOVIS is an excellent method for studying the structure of biological specimens at high resolution. The study of the structure of various aspects of living beings evoked hypothesis for their functioning.