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


Authors: Pertsinidis, A.; Mukherjee, K.; Sharma, M.; Pang, Z. P.; Park, S. R.; Zhang, Y.; Brunger, A. T.; Südhof, T. C.; Chu, S.
Article Title: Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ
Abstract: Membrane fusion is mediated by complexes formed by SNAPreceptor (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 synaptosomeassociated 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: nonhuman; protein conformation; protein localization; mouse; complex formation; cell structure; uvomorulin; cell membrane; imaging; feedback system; pheochromocytoma; fluorescence imaging; exocytosis; neurotransmission; neurotransmitter release; snare protein; syntaxin; cell ultrastructure; single-molecule; syntaxin 1; colocalization active stabilization; munc18 protein; protein sm; synaptobrevin; synaptosomal associated protein 25; syntaxin 1a; ultrahigh resolution imaging
Journal Title: Proceedings of the National Academy of Sciences of the United States of America
Volume: 110
Issue: 30
ISSN: 0027-8424
Publisher: National Academy of Sciences  
Date Published: 2013-07-23
Start Page: E2812
End Page: E2820
Language: English
DOI: 10.1073/pnas.1310654110
PROVIDER: scopus
PMCID: PMC3725074
PUBMED: 23821748
DOI/URL:
Notes: --- - "Export Date: 4 September 2013" - "CODEN: PNASA" - "Source: Scopus"
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