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

Nat Commun. 2015 Oct 6;6:8531. doi: 10.1038/ncomms9531.

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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Exocytosis
  • Fluorescence Resonance Energy Transfer / methods*
  • Insulin-Secreting Cells / chemistry*
  • Insulin-Secreting Cells / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Optical Imaging / methods*
  • Presynaptic Terminals / chemistry*
  • Presynaptic Terminals / metabolism
  • SNARE Proteins / genetics
  • SNARE Proteins / metabolism*

Substances

  • SNARE Proteins