Deciphering the role of adhesion dynamics in synapse development using micropatterning and multimodal microscopy – Synaptoligation

Adhesion is a fundamental property in cell biology, regulating many processes including cell shape and differentiation. At the molecular level, adhesion is mediated by specific membrane-associated ligand-receptor pairs. Several families of adhesion molecules, including the neurexin-neuroligin complex, have been shown to play critical roles in synapse formation in neurons. Genetic mutations in these molecules are associated with neurodevelopmental diseases in humans, highlighting their importance in brain function. Despite these advances, many issues remain unclear regarding the role of adhesion in synapse organization and function. In particular, NRXs and NLGs contain multiple isoforms and splice variants, known to associate through a specific code that specifies synapse types, but how the intrinsic binding properties between the different partners govern synapse structure, dynamics, and strength is still unresolved.

Our central research hypothesis is that by regulating the interaction kinetics between adhesion complexes, neurons can modulate the morphology and strength of their synaptic contacts, with implications in neuronal physiology. To explore this concept, we will manipulate synaptic complexes through the expression of a repertoire of neurexin and neuroligin isoforms/splice variants and their competitors (MDGAs, LRRTMs), and search for correlations between adhesion dynamics and synaptic parameters including structure, morphology, and functional differentiation.

To answer the scientific question formulated above, we raise four specific objectives:

Objective 1: To characterize the adhesion kinetics of membrane-bound synaptic complexes
We will micropattern adhesion protein ectodomains and characterize their interaction kinetics with fluorescently tagged counter receptors in heterologous cells.

Objective 2: To determine the effects of ligand-receptor adhesion on synapse differentiation in vitro
We will design the dual micropatterning of different protein ligands to study the role of adhesion synergy and competition on synapse differentiation in cultured neurons.

Objective 3: To explore the dynamics and nanoscale distribution of synaptic adhesion complexes
We will monitor adhesion protein dynamics by single molecule tracking and their localization in extra-synaptic and synaptic compartments by correlative light-electron microscopy (CLEM).

Objective 4: To characterize the role of adhesion molecule dynamics on synapse morphology and function ex vivo
We will examine the effects of neurexin and neuroligin isoforms/splice variants on synapse morphology and physiology by 3D live imaging and electrophysiology in brain slices.


To reach these objectives, we are building a highly interdisciplinary consortium of four partners:
Partner 1: O. THOUMINE (DR1 CNRS, Bordeaux) will bring his knowledge of neuronal adhesion, genetic tools, cultures from transgenic mice, labelling strategies with small monomeric probes, single molecule imaging, and computer simulations.
Partner 2: V. STUDER (DR2 CNRS, Bordeaux) will bring his expertise in micro-engineering and microscopy, notably 2D photopatterning and 3D structured illumination.
Partner 3: ALVEOLE S.A. (Paris) will provide its latest prototypes and patterning protocols, and dedicated software development toolkits.
Partner 4. J.-M. VERBAVATZ (Pr, Institut Jacques Monod, Paris) will bring his strong expertise in membrane proteins, TEM and CLEM. He is also co-director of the ImagoSeine imaging core facility (member of France BioImaging), where the EM will be performed.

Overall, we expect to unravel striking correlations between adhesion dynamics and synapse development. This project will yield important new insights into relevant and timely scientific questions underlying synaptogenesis, as well as new microscopy solutions that will be commercialized by the industrial partner.