CE11 - Caractérisation des structures et relations structure-fonctions des macromolécules biologiques

Atlas of synapse morphology and nanoscale organization using computational super-resolution microscopy – NANO-SYNATLAS

Submission summary

Synapses are complex protein-crowded micron-scale compartments allowing neuronal communication, whose dynamic changes in structure and molecular composition underlie essential brain functions. Among its key players, the adhesion protein neuroligin-1 (NLG1) is of special importance as it is critically placed at the synapse to interact with pre- and post-synaptic partners. Modulating NLG1 is known to perturb specific protein-protein interactions, resulting for instance in disorganizing the pre- and post-synaptic alignment. Mapping the nanoscale distribution of the proteins building the synapse in relation with dynamic morphological changes, especially when physiological modifications are induced, is important to truly understand how synapses work.

We recently developed an innovative correlative nanoscopy platform allowing both Single-Molecule Localization Microscopy (SMLM) and extracellular space super-resolution microscopy (SUSHI-STED) to be performed on the same living biological sample. SMLM allows monitoring proteins organization and dynamics in living cells with down to a few nanometers spatial resolution while SUSHI-STED is a volumetric imaging technique particularly suited for imaging nanoscale cellular morphology. This correlative approach allows for mapping the nanoscale protein organization and dynamics within the densely arranged cellular structures, such as the connected pre- & post-synaptic compartments. Yet, several challenges still remain: i) multi-color single molecule localization and tracking is currently limited, ii) SUSHI-STED images cannot be easily segmented and iii) correlating and datamining the SMLM and SUSHI-STED quantifications necessitate heavy manual interaction.

NANO-SYNATLAS aims at addressing these important issues by combining i) our recent correlative nanoscopy platform with ii) multicolor synaptic protein labelling for super-resolution microscopy, iii) genetic, optical, and pharmacological manipulation of synaptic components, and iv) advanced image segmentation and quantitative analysis.

Good access to the dense synaptic cleft will be ensured by labeling proteins with a monomeric version of streptavidin that displays a small size (3 nm) and is compatible with 2-colors super-resolution microscopy. Three strategies will be used to perturb NLG1: i) genetic manipulation and ii) optogenetic control of NGL1; and iii) chemical protocols inducing long term potentiation or long term depression. Our correlative super-resolution microscopy platform will be operated to collect data of the (co)organization and dynamics of key synaptic components (adhesion, scaffold, cytoskeleton) with synapse morphology, for WT and perturbed NLG1. Deep-learning, geometry processing, and parametrization will be applied to automatically segment and classify synapses from the SUSHI-STED images. In parallel, cluster analysis and single molecule dynamics will be computed from the SMLM data. For each condition, an analytical model of the synapse will be created by projecting all the segmented SUSHI-STED synapses and SMLM quantifications. Each model will depict an atlas categorizing the diverse synapse states both in terms of morphology and internal structure. Their comparison will provide a better understanding on how specific protein-protein interactions regulate synapse dynamics and organization, with important insights on the underlying molecular mechanisms of neurodevelopmental pathologies such as autism spectrum disorders.

The consortium is composed of the interdisciplinary teams “Quantitative Imaging of the Cell” (F. Levet/JB. Sibarita) and “Cell Adhesion Molecules In Synapse Assembly” (O. Thoumine), which have workd-wide recognized expertise in the fields of super-resolution microscopy, quantitative analysis, synapse biology, and fluorescent labelling approaches. This proposal captures a unique strategy to integrate our combined expertise to develop a next generation of quantitative tools for synapse biology.

Project coordinator


The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.



Help of the ANR 374,286 euros
Beginning and duration of the scientific project: December 2020 - 36 Months

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