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Cited by 46Cellular transport and secretionExtracellular vesicles in diseaseAdvanced Fluorescence Microscopy Techniques

Two-photon fluorescence lifetime imaging of primed SNARE complexes in presynaptic terminals and β cells

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Takahashi, Noriko, Sawada, Wakako, Noguchi, Jun, Watanabe, Satoshi, Ucar, Hasan, Hayashi-Takagi, Akiko, Yagishita, Sho, Ohno, Mitsuyo +2 more·2015·Nature Communications
DOI 10.1038/ncomms9531PMC PMC4600761
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Abstract

It remains unclear how readiness for Ca 2+-dependent exocytosis depends on varying degrees of SNARE complex assembly. Here we directly investigate the SNARE assembly using two-photon fluorescence lifetime imaging (FLIM) of Förster resonance energy transfer (FRET) between three pairs of neuronal SNAREs in presynaptic boutons and pancreatic β cells in the islets of Langerhans. These FRET probes functionally rescue their endogenous counterparts, supporting ultrafast exocytosis. We show that trans-SNARE complexes accumulated in the active zone, and estimate the number of complexes associated with each docked vesicle. In contrast, SNAREs were unassembled in resting state, and assembled only shortly prior to insulin exocytosis, which proceeds slowly. We thus demonstrate that distinct states of fusion readiness are associated with SNARE complex formation. Our FRET/FLIM approaches enable optical imaging of fusion readiness in both live and chemically fixed tissues.

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Authors
Takahashi, N., Sawada, W., Noguchi, J., Watanabe, S., Ucar, H., Hayashi-Takagi, A., Yagishita, S., Ohno, M., Tokumaru, H., & Kasai, H.
Affiliations
  • Japan Science and Technology Agency (JP)
  • Center for Disease Biology and Integrative Medicine, The University of Tokyo (JP)
  • Tokushima Bunri University (JP)
Corresponding author
Kasai, Haruo
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2015-10-06
Volume
6
Issue
1
Pages
8531-8531
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/ncomms9531
PMC
PMC4600761
OpenAlex
W2255151838
Cited by
46
References
6
Citations by year
2025: 4 · 2024: 3 · 2023: 5 · 2022: 2 · 2021: 9 · 2020: 3 · 2019: 3 · 2018: 6 · 2017: 8 · 2016: 3
FWCI
2.72
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
Funders
Japan Society for the Promotion of Science (26430005, 24116001, 26293260, 26221001)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
Keywords
SNARE complex assembly, exocytosis, presynaptic terminals, pancreatic beta cells, insulin exocytosis, active zone, docked vesicles, two-photon fluorescence lifetime imaging, FRET probes, calcium-dependent exocytosis, pancreatic islets
MeSH terms
Animals, Exocytosis, Mice, Inbred C57BL, Presynaptic Terminals, Fluorescence Resonance Energy Transfer, Insulin-Secreting Cells, SNARE Proteins, Mice, Optical Imaging

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