Superresolution Imaging Reveals the Internal Organization of Syntaxin Clusters in Hippocampal Processes

Abstract

N‐ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins are the universal molecular machinery used to mediate membrane fusion events. Syntaxins are SNARE proteins that reside in the target membrane and determine the allowable sites for membrane fusion. Clusters of syntaxin molecules release reactive syntaxin molecules for interactions with their partners in SNARE formation. In hippocampal neurons, syntaxin‐1a (Stx1a) mediates neurotransmitter release from synaptic vesicles whereas syntaxin‐3 (Stx3) facilitates activity‐dependent α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid(AMPA) receptor exocytosis. To explore the single‐molecule distribution of these syntaxin variants in hippocampal neurons, we implemented photoactivated localization microscopy (PALM), a superresolution fluorescence microscopy technique. PALM revealed single Stx1a molecules as well as clusters of Stx1a distributed throughout the hippocampal processes. The clusters of Stx1a varied markedly in size and the density of Stx1a molecules within the clusters was nonuniform with the greatest density of Stx1a molecules observed in the core of the clusters. The largest Stx1a clusters contained multiple density gradients of Stx1a within the cluster. PALM images revealed that Stx3 also forms clusters in hippocampal processes, and these Stx3 clusters likewise contained a density gradient of syntaxin molecules with Stx3 density most pronounced in the cluster core. This work is supported by National Institutes of Health grant 2 R15 NS40425‐03.