The interplay between SNARE complex components is of central importance for synaptic vesicle exocytosis at active zones. At present, few appropriate in vivo optical methods are available to capture the transient and fast interactions of synaptic proteins residing on the vesicle and the presynaptic membrane. There are several other approaches closing in on the study of protein dynamics and interactions at the synapse, e.g. electrophysiological or ultrastructural imaging techniques. However, the characterization of protein interactions using optical measurements in living specimens is challenging given the low protein copy number and the poor signal-to-noise ratio at the presynaptic membrane-vesicle interface. Electrophysiology, by contrast, is limited in spatial resolution and in assessing protein-protein interactions.

To address these difficulties we generated a molecular coincidence detection system based on the split GFP technique, where a fully functional GFP chromophore is reconstituted from two non-fluorescent GFP hemi-fragments (Feinberg et al., 2008). In order to visualize different stages of synaptic vesicle turnover at active zones we designed "docking" and "fusion sensor" protein pairs with split GFP fragments fused to the vesicular proteins n-Synaptobrevin, Synaptotagmin, the presynaptic membrane protein Syntaxin and the active zone marker Bruchpilot/CAST/ELKS. Using the larval neuromuscular junction of the fruitfly Drosophila melanogaster we present data on how this assay is used to determine subcellular domains of presynaptic protein interactions in vivo.

Feinberg EH, Vanhoven MK, Bendesky A, Wang G, Fetter RD, Shen K, and Bargmann CI. GFP Reconstitution Across Synaptic Partners (GRASP) defines cell contacts and synapses in living nervous systems.Neuron57: 353–363, 2008.