Figure 4. FRET-FLIM of Stx3 and Stx4 with VAMP3 and VAMP8.

(A) Representative confocal microscopy (left) and convoluted FLIM (right) images of dendritic cells expressing Stx3-mCitrine or Stx4-mCitrine (green in merge) with VAMP3-mCherry or VAMP8-mCherry (magenta). The apparent fluorescence lifetimes of Stx3-mCitrine with VAMP3-mCherry were lowest at the cell membrane, whereas the lifetimes with VAMP8-mCherry were lowest at intracellular compartments (yellow arrowheads). Scale bars, 10 µm. Lifetime images are in Figure 4—figure supplement 1A. (B) Whole-cell apparent fluorescence lifetimes for the conditions from panel A, both in presence or absence of NEM. Shown are individual cells pooled from at least 3 donors (mean ± SEM shown; one-way ANOVA with Bonferroni correction; n: number of cells; individual donors in Figure 4—figure supplement 1B).

DOI: http://dx.doi.org/10.7554/eLife.23525.010

Figure 4—figure supplement 1. Fluorescence lifetime images belonging to main Figure 4.

(A) Fluorescence lifetime images belonging to main Figure 4A. FLIM images were generated by convolution of these lifetime images with the fluorescence intensities (i.e., the mCitrine images shown in the main figure). Scale bars, 10 µm. (B) Same as main Figure 4B, but now with the averages for individual donors. Shown are donor-averaged whole-cell apparent fluorescence lifetimes of dendritic cells expressing Stx3-mCitrine or Stx4-mCitrine with or without VAMP3-mCherry or VAMP8-mCherry and in absence or presence of NEM treatment (mean ± SEM shown; one-way ANOVA with Bonferroni correction; n: number of donors).

DOI: http://dx.doi.org/10.7554/eLife.23525.011