Single-Pull

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> Objectives of the project

The main objective of this project is to build a commercial instrument able to perform single-molecule pull-down of fluorescently tagged proteins from a cell extract in a simple ans automated fashion. To reach this goal, we will apply single-molecule, TIRF and FRAP imaging modalities and develop quantitative analysis software in order to extract statistical information of the composition and stoichiometry of individual protein complexes.

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> Main results

During the first year of the project we managed to :

• Pattern the bait protein at precise locations with a controlled density
• Define a protocol to specifically target the bait protein with dedicated anchors
• Develop a dedicated software able to analyze multicolor single molecule fluorescence, co-localization and photobleaching steps (quantification)

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> During the second year of the project

• We optimized our multilayer protein coating to elicit specific recognition between purified adhesion proteins on the patterned substrate and the recombinant counter receptors expressed in living cultured cells (N-cadherins, SynCam, Neurexin/Neuroligin complexes)
• We managed to perform reproducible FRAP (Fluorescence Recovery After Photobleaching) on GFP tagged receptors accumulated at the micro-patterns, providing a way to quantify ligand-receptor interaction rates
• Alveole our industrial partner commercialized a fluid handling robot based on our prototype

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> Working plan to continue

• We will establish a bank of interaction rates characterizing various synaptic adhesion complexes. These results are expected to be published within a year
• Increase the throughput of the system (software and liquid handling)

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