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Teaser, summary, work performed and final results

Periodic Reporting for period 1 - VesHemiSyn (Exo- endocytosis cycle of individual synaptic vesicles in synapses and hemi-synapses)

Teaser

Synapses in the central nervous system are highly specialized structures dedicated to fast chemical transfer of information from one neuron to another. Synaptic transmission is initiated when an action potential triggers synaptic vesicle fusion with the plasma membrane, a...

Summary

Synapses in the central nervous system are highly specialized structures dedicated to fast chemical transfer of information from one neuron to another. Synaptic transmission is initiated when an action potential triggers synaptic vesicle fusion with the plasma membrane, a process called exocytosis that thus leads to neurotransmitter release from a presynaptic nerve terminal. After exocytosis, vesicular components are retrieved by endocytosis for maintaining efficient synaptic transmission. The mechanism of vesicle fusion and retrieval has been subjected to intense interest but also fierce debate for the last 40 years. Several modes of synaptic vesicle cycling have been described that rely on distinct kinetics, associated proteins and spatial organisation of the exo- and endocytosis sites. Yet, the mechanisms underlying endocytosis initiation and coupling to exocytosis are still largely unknown because the detailed dissection of the single vesicle cycle remains a major challenge. Thus, the aim of the VesHemiSyn project was to characterize at high temporal and spatial resolution the functional microarchitecture of the presynaptic nerve terminal.

Work performed

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.

Final results

Overall, our study will provide new insights into the mechanisms of exo-endocytosis cycle of individual synaptic vesicles. Upon completion, it will offer a more refined scientific view on the organisation and dynamics of the presynaptic nerve terminal. Thus, it has the great potential to accumulate new important evidence to understand the physiology of the presynaptic nerve terminal and how it contributes to synaptic transmission and plasticity. The significance of our study is reinforced by the role for altered synapse structure and function in the aetiology of cognitive disorders that represent a substantial financial and social burden. Therefore, elucidating temporal and spatial features of the synaptic vesicle cycle will help shedding light onto the influence of the synapse architecture on neurotransmitter release and thus on synaptic transmission. In conclusion, our approach considers basic questions related to synaptic vesicle cycle. It is our hope that the successfully completed project attracts considerable interest among other specialists of this and related fields and that the new knowledge stimulates further research towards understanding the impact of the functional organisation of the presynapse on the function of integrated systems.

Website & more info

More info: http://www.iins.u-bordeaux.fr/Membrane-Trafficking.