Synapsin2a tetramerization selectively controls the presynaptic nanoscale organisation of reserve synaptic vesicles

Neurotransmitter release relies on the regulated fusion of synaptic vesicles (SVs) that are tightly packed within the presynaptic bouton of neurons. The mechanism by which SVs are clustered at the presynapse, while preserving their ability to dynamically recycle to support neuronal communication, remains unknown. Synapsin 2a (Syn2a) tetramerization has been suggested as a potential clustering mechanism. Here, we used Dual-pulse sub-diffractional Tracking of Internalised Molecules (DsdTIM) to simultaneously track single SVs fromthe recycling and the reserve pools, in live hippocampal neurons. The reserve pool displays a lower presynaptic mobility compared to the recycling pool and exhibits a mobile axonal pool. Triple knockout of Synapsin 1-3 genes (SynTKO) increased the mobility of reserve pool SVs. Re-expression of wild type Syn2a (Syn2aWT), but not the tetramerization-deficient mutant K337Q (Syn2aK337Q), fully rescued these effects. Single-particle tracking revealed that Syn2aK337QmEos3.2 exhibited altered activity-dependent presynaptic translocation and nanoclustering. Therefore, Syn2a tetramerization controls its own presynaptic nanoclustering and thereby contributes to the dynamic immobilisation of the SV reserve pool.