Project

Discipline

actin; synapsin; nanodisc; SNARE; synapse; time-resolved; membrane fusion; liposomes; cryo-electron tomography; synaptosome; dynamin; neurotransmitter release

Lead
La communication entre les neurones est à la base du fonctionnement du système nerveux. Cette communication a lieu à la synapse, c’est-à-dire la zone de contact entre deux neurones. Là, de petites molécules - les neurotransmetteurs - sont libérées par un des deux neurones et sont détectées par le deuxième neurone. Avant d’être libérées ces substances sont stockées dans des vésicules (des sphères délimitées par une membrane) au sein de la cellule. En réponse à un stimulus provocant l’élévation intracellulaire de la concentration en calcium, ces vésicules fusionnent avec la membrane cellulaire, qui délimite le neurone, libérant ainsi leur contenu hors de la cellule. Même si ce processus - l’exocytose - a été très étudié, nombre de ses aspects restent mal compris. Le but de notre projet de recherche est de mieux comprendre comment l’exocytose fonctionne au niveau moléculaire et comme elle est régulée.
Lay summary
Pour ce faire, nous allons employer la technique de cryomicroscopie électronique, qui a valu le Prix Nobel de Chimie à Jacques Dubochet. Ainsi nous visualiserons des synapses dans leur état naturel à très haute résolution. Nous allons focaliser notre analyse sur la façon dont l’actine, une molécule formant le squelette des cellules, influence l’exocytose. On sait en effet que celle-ci est présente à la synapse et qu’elle a des conséquences sur la façon dont les neurones libèrent leurs neurotransmetteurs mais on ne comprend pas encore bien comment cela se passe au plan moléculaire. Nous allons visualiser des synapses en état d’exocytose en présence de certaines molécules modifiant les propriétés du squelette d’actine. Nous allons aussi modifier l’activité d’autres protéines impliquées dans l’exocytose et observer quelles conséquences cela aura sur les structures synaptiques, en particulier lors d’activité d’exocytose.  Notre projet s’inscrit dans la recherche fondamentale sur les systèmes nerveux. Une meilleure compréhension pourrait contribuer à l’établissement de nouveaux traitements contre des maladies tels le syndrome de Parkinson, la dépression, la schizophrénie ou encore l’autisme.

Direct link to Lay Summary

Last update: 19.02.2019

Number	Title	Start	Funding scheme

Exocytosis is an ubiquitous and critical cellular mechanism leading to the secretion of the content of a membrane-bound vesicle into the extracellular space through the fusion of the vesicle membrane with the plasma membrane. Neurotransmitter release from presynaptic terminals depends on exocytosis. It is an extremely fast process. A few synaptic vesicles are docked to the presynaptic active zone plasma membrane and primed for fusion. When calcium concentration rises in the presynaptic cytoplasm after the influx of an action potential, primed vesicles fuse within millisecond. The mechanism of exocytosis has been extensively studied, yet many aspects remain unclear. For example the timing and extent of conformational changes of the proteins involved in exocytosis with respect to priming and exocytosis triggering are debated. What forces act on the membranes and how membrane shape evolve during exocytosis is not well known. This is in part due to the high speed of the reaction and to the fact that in most cases the proteins and membranes are not directly visualized but their structures are inferred from indirect measurements. To resolve these issues we have developed a time-resolved cryo-electron tomography approach where we can arrest vesicles while they fuse with the plasma membrane and reconstruct their three dimensional ultrastructure with nanometer resolution. We have observed that membranes get kinked right before exocytosis and that the fate of fusing vesicles can be twofold: either the vesicles fuse entirely or they get pinched off (kiss-and-run exocytosis). In this proposal we want to investigate in more detail how dynamin, a GTPase protein involved in endo- and exocytosis, affects this fate. Moreover actin dynamics have been shown to influence this process as well but the underlying mechanism is unclear. We will manipulate the activity of dynamin and the state of actin and we will monitor the structural changes occurring before, during, and after exocytosis onset in order to unravel the mechanism by which these proteins influence membrane fusion and fast retrieval. We will also use an in vitro reconstitution system to complement these experiments and address particular molecular issues.Vesicles are densely packed in presynaptic terminals. Many of them are connected to other vesicles by small filaments and some are bound to the plasma membrane. We have observed how this network of filaments, the so-called presynaptic cytomatrix, is modified shortly after the onset of exocytosis. These changes informed us on the way some of the vesicles are recruited to the readily-releasable pool. However many issues remain. Perhaps the most fundamental our lack of understanding of the molecular composition of the presynaptic cytomatrix. Several proteins have been proposed to be part but there is no consensus in the community. We will apply a ground-breaking in silico labelling procedure recently introduced by Grigorieff and Denk labs to determine the presynaptic cytomatrix. The role of the cytomatrix on the organization of the vesicle pools and vesicle mobility is debated. We will compare the cytomatrix structure in different types of synapses which have quite different synaptic vesicle mobilities. We will also characterize in detail the structural properties of the cytomatrix after several longer delays following the onset of excocytosis than what we have done so far. From these we anticipate to shed new light on the regulation of vesicle pool organization and on the mechanisms allowing synapses to sustained prolonged synaptic activity. The results gathered in this project will advance our understanding of synaptic functions and the methods developed will open new avenues of research in cellular and molecular structural biology.