Synaptic vesicle fusion is mediated by SNARE proteins forming in between synaptic vesicle (v-SNARE) and plasma membrane (t-SNARE), one of which is Syntaxin-1A. Although exocytosis mainly occurs at active zones, Syntaxin-1A appears to cover the entire neuronal membrane. By using STED super-resolution light microscopy and image analysis of Drosophila neuro-muscular junctions, we show that Syntaxin-1A clusters are more abundant and have an increased size at active zones. A computational particle-based model of syntaxin cluster formation and dynamics is developed. The model is parametrized to reproduce Syntaxin cluster-size distributions found by STED analysis, and successfully reproduces existing FRAP results. The model shows that the neuronal membrane is adjusted in a way to strike a balance between having most syntaxins stored in large clusters, while still keeping a mobile fraction of syntaxins free or in small clusters that can efficiently search the membrane or be traded between clusters. This balance is subtle and can be shifted toward almost no clustering and almost complete clustering by modifying the syntaxin interaction energy on the order of only 1 kBT. This capability appears to be exploited at active zones. The larger active-zone syntaxin clusters are more stable and provide regions of high docking and fusion capability, whereas the smaller clusters outside may serve as flexible reserve pool or sites of spontaneous ectopic release. Author Summary For the communication between two nerve cells, a synaptic vesicle containing neurotransmitters has to fuse with the neuronal membrane at a specific fusion site, releasing its signaling molecules. The vesicle fusion is mediated by a specific family of proteins (SNAREs) that are located on the vesicle as well as on the neuronal membrane. As many other membrane proteins, SNARE proteins are not uniformly distributed over the membrane but rather exist in complexes or clusters. Syntaxin, one of the SNARE proteins, is known to form such clusters through attractive protein- protein interactions. With the help of light microscopy techniques we show that the actual size and abundance of these clusters depends on its proximity to the fusion site on the membrane. We developed a computational model of Syntaxin cluster formation that can explain the observed differences in clustering and allow us to speculate on their potential role in the process of docking and fusion of synaptic vesicles. The formation of clusters through weak protein-protein interactions allow for a highly dynamic behavior of proteins, being able to easily switch between a state with stable and almost immobile clusters and a more dynamic situation with clusters exchanging particles at high rates.
