Synaptic PI(3,4,5)P3 Is Required for Syntaxin1A Clustering and Neurotransmitter Release

Summary PI(3,4,5)P 3 is a low-abundance lipid thought to play a role in the regulation of synaptic activity; however, the mechanism remains obscure. We have constructed novel split Venus-based probes and used superresolution imaging to localize PI(3,4,5)P 3 at Drosophila larval neuromuscular synapses. We find the lipid in membrane domains at neurotransmitter release sites, where it concentrates with Syntaxin1A, a protein essential for vesicle fusion. Reducing PI(3,4,5)P 3 availability disperses Syntaxin1A clusters and increasing PI(3,4,5)P 3 levels rescues this defect. In artificial giant unilamellar vesicles, PI(3,4,5)P 3 also induces Syntaxin1A domain formation and this clustering, in vitro and in vivo, is dependent on positively charged residues in the Syntaxin1A-juxtamembrane domain. Functionally, reduced PI(3,4,5)P 3 causes temperature-sensitive paralysis and reduced neurotransmitter release, a phenotype also seen in animals expressing a Syntaxin1A with a mutated juxtamembrane domain. Thus, our data indicate that PI(3,4,5)P 3 , based on electrostatic interactions, clusters Syntaxin1A at release sites to regulate neurotransmitter release.