• Computer simulations based on single molecule dynamics to quantitatively interpret fluorescence microscopy experiments 

To accompany the recent progress in fluorescence live cell and super-resolution imaging methods, there is a pressing need for computer simulations allowing for the improved design and quantitative interpretation of experiments. To this aim, we have developed a unified real-time simulator of single molecule dynamics called FluoSim (Lagardère et al., Sci Rep 2020), which can reproduce a variety of imaging experiments performed on membrane proteins (Fig. 1). FluoSim was validated against SPT, PAF, FRAP, FCS, and STORM data obtained on the canonical neurexin-neuroligin complex in cell-cell contacts, using a small number of kinetic and photophysical parameters. This simulator considerably expands on our previous computer simulations of AMPA receptor trafficking at synapses (Czöndör et al., PNAS 2012), and was recently used to simulate LTP experiments (Letellier et al., eLife 2020). This software is a very interesting tool to improve experimental design and analysis, as well as to train users on the most challenging imaging methods. We used it recently to quantitatively analyze the dynamic behavior of extra-synaptic and synaptic neuroligin-1 over both short and long time ranges, and to provides an estimate of neuroligin-1 copy numbers in post-synaptic densities (Lagardère et al. Front Synaptic Neurosci 2022).

 

Figure 1. General principle of FluoSim

 

• Micropatterning assays to measure ligand-receptor interaction kinetics and screen for synapse differentiation

A few years ago, with the company CYTOO (cytoo.com) we developed an original biomimetic system consisting in culturing primary dissociated neurons on glass substrates micropatterned with purified Fc-tagged adhesion molecules (N-cadherin, Neurexin-1β, SynCAM1), to elicit axonal growth as well as pre- and post-synaptic differentiation (Fig. 2), depending on the protein ligand (Chazeau et al. MBoC 2015; Czöndör et al. Nat Commun 2013; Garcia et al. PNAS 2015). More recently, in collaboration with V. Studer at IINS, we are using micropatterned substrates coated with Fc-tagged ligands in combination with live-cell imaging (SPT & FRAP) of fluorescently tagged receptors in heterologous cells, to measure the kinetics of synaptic ligand-receptor interactions. We applied this strategy to study the heterophilic complex formed between neurexin-1β and neuroligin-1 and the homophilic SynCAM1 complex (Piette et al., in prep).

Figure 2. (Left) Hippocampal neuron expressing neuroligin-1 and PSD-95-GFP (green) cultured on micropatterned substrates coated with neurexin1β-Fc (blue dots). The inset shows the formation of PSD-95-GFP and AMPA receptor clusters at neurexin1β-Fc coated dots. (Right) COS-7 cells expressing neuroligin-1-GFP (green) plated on micropatterned lines coated with neurexin1β-Fc (red). To estimate ligand-receptor binding kinetics, FRAP experiments are performed on neuroligin-1-GFP accumulated on neurexin1β-Fc.

 

Selected references

• Lagardère M., Drouet A., Sainlos M., Thoumine O. (2022). High resolution fluorescence imaging combined with computer simulations to quantitate surface dynamics and nanoscale organization of neuroligin-1 at synapses. Front. Synaptic Neurosci. 14: 835427.
• Lagardère M., Chamma I., Bouilhol E., Nikolski M., Thoumine O. (2020). FluoSim: simulator of single molecule dynamics for fluorescence live-cell and super-resolution imaging of membrane proteins. Sci. Rep. 10, 19954.
• Garcia M., Leduc C., Lagardère M., Argento A., Sibarita J.-B., Thoumine O. (2015). Two-tiered coupling between flowing actin and immobilized N-cadherin/catenin complexes in neuronal growth cones. Proc. Natl. Acad. Sci. USA 112, 6997–7002.
• Chazeau A., Garcia M., Czöndör K., Perrais D., Tessier B., Giannone G., Thoumine O. (2015). Mechanical coupling between transsynaptic N-cadherin adhesions and actin flow stabilizes dendritic spines. Mol. Biol. Cell 26, 859–873.
• Czöndör K., Garcia M., Argento A., Constals A., Breillat C., Tessier B., Thoumine O. (2013). Micropatterned substrates coated with neuronal adhesion molecules for high-content study of synapse formation. Nat. Commun. 4, 2252.
• Czöndör K., Mondin M., Garcia M., Heine M., Frischknecht R., Choquet D., Sibarita J.-B., Thoumine O. (2012). Unified quantitative model of AMPA receptor trafficking at synapses. Proc. Natl. Acad. Sci. U.S.A. 109, 3522–3527.