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Proc Natl Acad Sci U S A. 2013 Jul 23;110(30):E2812-20. doi: 10.1073/pnas.1310654110. Epub 2013 Jul 2.

Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ.

Author information

1
Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. PertsinA@mskcc.org

Abstract

Membrane fusion is mediated by complexes formed by SNAP-receptor (SNARE) and Secretory 1 (Sec1)/mammalian uncoordinated-18 (Munc18)-like (SM) proteins, but it is unclear when and how these complexes assemble. Here we describe an improved two-color fluorescence nanoscopy technique that can achieve effective resolutions of up to 7.5-nm full width at half maximum (3.2-nm localization precision), limited only by stochastic photon emission from single molecules. We use this technique to dissect the spatial relationships between the neuronal SM protein Munc18-1 and SNARE proteins syntaxin-1 and SNAP-25 (25 kDa synaptosome-associated protein). Strikingly, we observed nanoscale clusters consisting of syntaxin-1 and SNAP-25 that contained associated Munc18-1. Rescue experiments with syntaxin-1 mutants revealed that Munc18-1 recruitment to the plasma membrane depends on the Munc18-1 binding to the N-terminal peptide of syntaxin-1. Our results suggest that in a primary neuron, SNARE/SM protein complexes containing syntaxin-1, SNAP-25, and Munc18-1 are preassembled in microdomains on the presynaptic plasma membrane. Our superresolution imaging method provides a framework for investigating interactions between the synaptic vesicle fusion machinery and other subcellular systems in situ.

KEYWORDS:

active stabilization; colocalization; exocytosis; neurotransmission; single-molecule

PMID:
23821748
PMCID:
PMC3725074
DOI:
10.1073/pnas.1310654110
[Indexed for MEDLINE]
Free PMC Article

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