For this purpose, we combined live cell imaging and high-resolution imaging techniques to investigate the fate of vesicular proteins upon exocytosis and the molecular organisation of the presynapse. First, using pH-sensitive optical probes targeted to synaptic vesicles, we visualized single vesicle fusion events and found that vesicular proteins are retrieved extremely rapidly (t = 575 ms) consistent with an ultra-fast mode of endocytosis. Second, we developed the hemi-synapse model where the presynaptic nerve terminals are formed on a micro-patterned substrate comprising thousands of micron-scale dots coated with the adhesion protein SynCAM1 to mimic the postsynapse and induce presynaptic differentiation. The favourable geometry of this model provides a tight control on the site of presynapse formation and enables high resolution mapping of the presynaptic exo- and endocytosis sites. The hemi-synapses are functional and show similar properties compared to normal synapses, such as the size of synaptic vesicle pools and the exo-endocytosis dynamics. Third, to use super-resolution microscopy techniques on these models we needed to develop new improved pH-sensitive fluorescent reporters in order to detect a brighter signal upon single vesicle fusion event. This work have been performed in collaboration with the groups of Luke Lavis (Janelia Research Campus, Howard Hughes Medical Institute, USA) and Justin Taraska (National Heart, Lung, and Blood Institute, National Institutes of Health, USA). Two pH-sensitive dyes, carbofluorescein and Virginia Orange have been produced and characterized. They notably enable the labeling of endogenous vesicular proteins and the detection of a brighter signal upon vesicular fusion compared to previously described reporters. This work is now accepted for publication in Nature Communications. This unexpected technical development delayed the project for almost a year. At present we are using these new sensors to locate at high spatial resolution the exocytosis and endocytosis sites. Correlating the organization of exocytosis and endocytosis sites with the location of key proteins such as calcium channels and scaffold proteins will reveal the functional microarchitecture of the presynaptic nerve terminal.
