Figure 1. SNARE complex formation by FRET-FLIM.

(A) Model of the neuronal SNAREs (crystal structure; protein database 3HD7 [Stein et al., 2009]) with the C-termini of syntaxin 1 (red) conjugated to mCitrine (3DQ1 [Ho et al., 2008]; yellow) and VAMP2 (blue) conjugated to mCherry (2H5Q [Shu et al., 2006]; magenta). mCitrine (donor fluorophore) and mCherry (acceptor) are within 3 nm proximity resulting in FRET. Green: SNAP25. (B) Scheme of membrane fusion resulting in FRET. (C) Representative confocal microscopy (left) and FLIM (right) images of dendritic cells expressing Stx3-mCitrine (green in merge; upper panels), Stx3-mCitrine with VAMP3-mCherry (magenta; middle panels), or Stx3 conjugated to both mCitrine and mCherry (Stx3-mCitrine-mCherry; lower panels). Apparent fluorescence lifetimes of Stx3-mCitrine with VAMP3-mCherry were lowest at the cell membrane (yellow arrowheads). Scale bars, 10 µm. Full lifetime/intensity lookup table and lifetime images are in Figure 1—figure supplement 1D–E.

DOI: http://dx.doi.org/10.7554/eLife.23525.003

Figure 1—figure supplement 1. Lifetime images, confocal images and phasor analysis belonging to main Figure 1.

(A) The excitation (open curves) and emission (filled curves) spectra of mCitrine (yellow) and mCherry (pink) (data from Chroma Technology). (B) Representative confocal microscopy images of antibody staining of endogenous VAMP3 (top; magenta in merge) and VAMP8 (bottom) in dendritic cells. Blue: DAPI. BF: bright-field. (C) Distribution of total photon counts for all fluorescence lifetime recordings. (D) Full lookup table belonging to the FLIM images. Plotted is the fluorescence intensity (y-axis) versus the fluorescence lifetime (x-axis). (E) Fluorescence lifetime images belonging to main Figure 1C. FLIM images were generated by convolution of these lifetime images with the fluorescence intensities (i.e., the mCitrine images shown in the main figure). (F) Phasor plots for the cells shown in main Figure 1C. Cyan circles: center of the phasor location for Stx3-mCitrine in absence of acceptor fluorophore (upper graph); purple circles: center for Stx3-mCitrine with VAMP3-mCherry (middle graph); pink circle: center for Stx3-mCitrine-mCherry (lower graph). (G) Representative confocal microscopy images of live (top) and fixed (bottom) dendritic cells expressing the Stx3-mCitrine-mCherry tandem construct (mCitrine: green in merge; mCherry: magenta). (H) Pearson correlation coefficients for colocalization of the fluorophores of the Stx3-mCitrine-mCherry tandem construct for 3 donors (average ± SEM; paired two-sided Student’s t-test). (I–J) Same as panel G, but now for live (I) and fixed (J) cells co-expressing VAMP3-mCitrine with VAMP3-mCherry (left) or VAMP8-mCitrine with VAMP8-mCherry (right). (K) Pearson correlation coefficients for panels I–J (average ± SEM; VAMP3: p=0.046, paired two-sided Student’s t-test). Scale bars, 10 µm.

DOI: http://dx.doi.org/10.7554/eLife.23525.004