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First published online December 5, 2007
doi: 10.1242/10.1242/jcs.020230

Journal of Cell Science 120, 4407-4415 (2007)
Published by The Company of Biologists 2007
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Munc18-1 prevents the formation of ectopic SNARE complexes in living cells

Claire N. Medine, Colin Rickman, Luke H. Chamberlain and Rory R. Duncan*

Centre for Integrative Physiology, University of Edinburgh, George Square, Edinburgh EH8 9XD, UK


Figure 1
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  		Fig. 1. SNAP-25 but not syntaxin traffics readily to the plasma membrane in live cells, but ectopic complexes can form. (A) Live N2a cells expressing Syx1-288, Syx1-288 coexpressed with munc18-1, Syx1-288[open] or Syx1-288[open] coexpressed with munc18-1. The FLIM data confirmed that Syx1-288 and Syx1-288[open] interact with munc18-1. (B) Fields of PC12 cells expressing the same constructs were scored for syntaxin trafficking to the cell surface. The percentage of cells with surface syntaxin was plotted. (C) N2a cells expressing SNAP-251-206 or truncated SNAP-251-121 were imaged by CLSM. Shown are representative equatorial sections; n>20 images. Bar, 10 µm. (D) Wild-type or mutant SNAP-251-206 (green) and Syx1-288 (red) were expressed in live N2a cells and imaged by CLSM, as before. The merged image shows areas of coincidence in yellow hues. The two-dimensional histogram represents the intensity of each channel in each voxel, with a colour scale representing frequency. The residual map corresponds to weighted residuals from the linear regression of the histogram, thus indicating fluorescent channel covariance. The hue is from –1 to 1, with cyan corresponding to a zero residual. Shown are representative equatorial sections; n>20 images. Bar, 5 µm.

 

Figure 2
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  		Fig. 2. Syntaxin and SNAP-25 colocalise and can interact in the Golgi complex in live cells. Syx1-288 and SNAP-251-206 coexpressed in live N2a cells and imaged by CLSM. (A) Residual map corresponds to weighted residuals indicating fluorescent channel covariance. The hue is from –1 to 1, with cyan corresponding to a zero residual. Bar, 10 µm. (B) The Golgi complex marker GRASP65-mCherry was coexpressed in live N2a cells and imaged by CLSM (left panel). Residual map of Syx1-288 and SNAP-251-206 covariance in the Golgi complex, as defined by a GRASP65-mCherry mask (middle panel). The weighted residuals of Syx1-288 and SNAP-251-206 contained within the Golgi complex, as defined by GRASP65-mCherry (black circles), alongside residual values from elsewhere in the cell (grey circles), were plotted as a frequency distribution histogram (right panel). (C) mCerulean-Syx1-288 (donor) fluorescence in the absence of EYFP-SNAP-251-206 (acceptor) exhibited an intracellular distribution. The colour scale in the FLIM map represents the fluorescence lifetime [1900 pseconds (red) – 2400 pseconds (blue)]. The fluorescence lifetime values were plotted as a frequency distribution histogram, showing the 99% confidence interval of the Syx1-288 fluorescence lifetime distribution (red line). Syx1-288 alone has a single fluorescence lifetime of 2388±47 pseconds. The excited-state fluorescence decay of Syx1-288 in the absence of an energy acceptor followed a mono-exponential decay (light grey circles) (right panel). (D) mCerulean-Syx1-288 (donor) fluorescence in the presence of SNAP-251-206 (acceptor) exhibited an intracellular distribution. The colour scale in the FLIM map represents the fluorescence lifetime. The fluorescence lifetime values were plotted as a frequency distribution histogram, showing the 99% confidence interval of the Syx1-288 fluorescence lifetime distribution (non-FRET; red line). The fluorescence lifetime of mCerulean-Syx1-288 was shortened significantly to 2176 ±134 pseconds (Mann-Whitney, n=5, P<0.01) when coexpressed with EYFP-SNAP-251-206. In the presence of an energy acceptor, the fluorescence decay of Syx1-288 was fit by a bio-exponential decay function (dark grey circles) (C, right panel). Syx1-288 (donor) fluorescence in the presence of SNAP-251-206 (acceptor) showing in red the pixels containing donor fluorescence lifetimes below than the 99% confidence interval (right panel). Data are expressed as the mean±s.e.m.

 

Figure 3
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  		Fig. 3. Munc18-1 prevents ectopic t-SNARE interactions. (A) Wild-type or mutants of Syx1-288 (red), SNAP-25 (green) and munc18-1 were coexpressed in live HEK293 cells and imaged by CLSM. The merged image shows areas of coincidence in yellow hues. The two-dimensional histogram represents the intensity of each channel in each voxel, with a colour scale representing frequency. The residual map corresponds to weighted residuals from the line fit to the histogram, thus indicating fluorescent channel covariance. The hue is from –1 to 1, with cyan corresponding to a zero residual. (B) Similar results were obtained using N2a cells. Bars, 5 µm. (C) The covariance data from HEK293 cells coexpressing wild-type or mutants of Syx and SNAP251-206, in the absence (filled bars) or presence of munc18-1 (open bars) were quantified and expressed as Pearson's coefficient values. These data showed that the covariance between the t-SNAREs is unaffected by the presence of munc18-1, but that the location of the colocalisation is altered (see panel A; n>=4). Data are expressed as the mean±s.e.m.

 

Figure 4
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  		Fig. 4. SNARE clusters are heterogeneous. Intensity images and FLIM maps showing mCerulean-Syx1-288, in the presence of EYFP-SNAP-251-206 and munc18-1 in live N2a cells, imaged by FLIM and two-photon microscopy. (A) mCerulean-Syx1-288 (donor) fluorescence exhibited a plasma membrane distribution. The colour scale [1900 (red) – 2400 pseconds (blue)] in the FLIM map represents the donor fluorescence lifetime in resting cells (top) and after depolarisation with 55 mM KCl (lower panels). These data were plotted as a frequency distribution histogram, showing the 99% confidence interval of the donor lifetime distribution (red dashed line; bars are mean±s.e.m., n=5 experiments). Bar, 5 µm. (B) Similar experiments performed in the presence of ionomycin, but a Ca2+-free environment also revealed clusters at the base of the cell. The colour scale in the FLIM map represents the donor fluorescence lifetime. Bar, 5 µm. [Colour scale: 1500 (red) – 2000 pseconds (blue).] The boxed region of interest is shown in the lower panels and illustrates clusters at the plasma membrane. Pixels containing fluorescence lifetimes below the 99% confidence interval of the non-FRET distribution in panel A are shown in red. Bar, 1 µm. SNARE clusters where no FRET could be detected are highlighted with a dashed circle. (C) In the presence of ionomycin and Ca2+, mCerulean-Syx1-288 (donor) fluorescence in the presence of EYFP-SNAP-251-206 (acceptor) and munc18-1 showed clusters at the base of the cell. The colour scale [1500 (red) – 2000 pseconds (blue)] in the FLIM map represents the donor fluorescence lifetime in the presence of ionomycin and Ca2+. The donor fluorescence lifetime was significantly shortened, indicative of FRET between the t-SNAREs. Bar, 5 µm. The boxed region of interest is shown in the lower panels and illustrates clusters at the plasma membrane. Pixels containing fluorescence lifetimes below the 99% confidence interval of the non-FRET distribution in panel A are shown in red. SNARE clusters where no FRET could be detected are highlighted with a dashed circle. Bar, 1 µm. (D) Similar results were obtained using KCl depolarization. The boxed region of interest is shown in the right-hand panels as a zoomed image, showing that the proportion of FRET-positive t-SNARE clusters increased after KCl-induced depolarization. (E) The donor fluorescence lifetime data for each sample were plotted as a frequency distribution histogram. The fluorescence lifetimes in the KCl-treated samples (open circles, grey fit line) were significantly reduced compared with those from resting cells (filled circles, black fit line).

 

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© The Company of Biologists Ltd 2007