4407Research Article

IntroductionEvery eukaryotic cell relies on the SNARE proteins to mediatethe vesicular targeting, docking and fusion processes thatunderlie membrane trafficking and exocytosis. Essential to theexocytotic process are the interactions of the plasma membraneproteins (t-SNAREs) syntaxin and SNAP-25 (synaptosome-associated protein 25 kDa) and the vesicular proteinsynaptobrevin (v-SNARE) (Burgoyne and Morgan, 2003;Sudhof, 1995). Membrane fusion requires the assembly of allthree proteins into a trimeric, four-helical complex to promotefusion of the two opposing bilayers in living cells (Sutton etal., 1998; Burgoyne and Morgan, 2003; Sudhof, 1995). Thisprocess is tightly regulated by a conserved set of accessoryproteins that operate throughout the trafficking pathway.Members of the Sec1p/munc18 (SM) protein family representone such set of modulators that might add to the specificity ofSNARE protein interactions. Indeed, mutations of SM proteinsare characterised by a severe disruption of general secretion orneurotransmitter release (Brenner, 1974; Harrison et al., 1994;Novick and Schekman, 1979; Verhage et al., 2000). Thepossible functions of SM proteins were obtained originallyfrom the observations that munc18-1 (STXB1) binds directlyto the monomeric form of syntaxin 1, rendering the t-SNAREunable to form the SDS-resistant ternary SNARE complex(Hata et al., 1993; Pevsner et al., 1994). However, owing to thediversity of SM proteins and SNARE interactions, thesefunctions have remained enigmatic.

Syntaxin can adopt two structurally distinct forms(Dulubova et al., 1999): a closed form in which the three-helical Habc domain is folded back on to the syntaxin SNARE

helix, and an open, elongated structure with an accessibleSNARE helix. Munc18-1 binds to the closed form of syntaxinwith high affinity, rendering the syntaxin unable to enter intoSNARE complexes (Hata et al., 1993; Pevsner et al., 1994;Rickman et al., 2007). In addition, munc18-1 can interact withan extreme N-terminal motif of syntaxin (Dulubova et al.,2007; Rickman et al., 2007; Shen et al., 2007). This interactioncan occur with syntaxin in the open conformation and mostlikely is also present in the closed-form mode of interaction.The association of munc18-1 with the syntaxin N-terminusallows this interaction to be maintained throughout theformation of both the binary (syntaxin–SNAP-25 dimer) andternary SNARE complex, both in vitro and in cells (Dulubovaet al., 2007; Rickman et al., 2007). The interaction of munc18-1 with the N-terminal peptide of syntaxin increases the rate ofmembrane fusion in an in vitro assay (Shen et al., 2007), butthe role of the closed-form interaction in the process ofexocytosis remains undefined (Burgoyne and Morgan, 2007).

The t-SNARE proteins localise in clusters or spots on theplasma membrane, of between 60-750 nm in diameter,depending on the imaging approach used in visualisation (Langet al., 2001; Lang et al., 2002; Ohara-Imaizumi et al., 2002;Rickman et al., 2004; Sieber et al., 2007). These clusters havebeen shown to define the site of vesicle docking and exocytosisin a variety of neuroendocrine cells (Lang et al., 2001; Lang etal., 2002; Ohara-Imaizumi et al., 2002). It is not clear, however,whether every cluster is fusion competent, and indeed it hasnever been demonstrated convincingly that the t-SNAREproteins contained within each cluster interact before the fusionevent occurs. Experiments performed using inside-out lysed

Membrane trafficking in eukaryotic cells must be strictlyregulated both temporally and spatially. The assembly atthe plasma membrane of the ternary SNARE complex,formed between syntaxin1a, SNAP-25 and VAMP, isessential for efficient exocytotic membrane fusion. Theseexocytotic SNAREs are known to be highly promiscuous intheir interactions with other non-cognate SNAREs. It istherefore an important cellular requirement to trafficexocytotic SNARE proteins through the endoplasmicreticulum and Golgi complex while avoiding ectopicinteractions between SNARE proteins. Here, we show thatsyntaxin1a traffics in an inactive form to the plasmamembrane, requiring a closed-form interaction, but not N-terminal binding, with munc18-1. If syntaxin is permitted

to interact with SNAP-25, both proteins fail to traffic to theplasma membrane, becoming trapped in intracellularcompartments. The munc18-1–syntaxin interactions mustform before syntaxin encounters SNAP-25 in the Golgicomplex, preventing the formation of intracellularexocytotic SNARE complexes there. Upon delivery to theplasma membrane, most SNARE clusters in resting cells donot produce detectable FRET between t-SNARE proteins.These observations highlight the crucial role that munc18-1 plays in trafficking syntaxin through the secretorypathway.

Key words: FLIM, FRET, SNARE, Exocytosis

Summary

Munc18-1 prevents the formation of ectopic SNAREcomplexes in living cellsClaire N. Medine, Colin Rickman, Luke H. Chamberlain and Rory R. Duncan*Centre for Integrative Physiology, University of Edinburgh, George Square, Edinburgh EH8 9XD, UK*Author for correspondence (e-mail: rduncan@staffmail.ed.ac.uk)

Accepted 25 September 2007Journal of Cell Science 120, 4407-4415 Published by The Company of Biologists 2007doi:10.1242/jcs.020230

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sheets of plasma membrane showed that exogenously addedVAMP or syntaxin (but interestingly not SNAP-25) interactedwith endogenous SNAREs in clusters in ‘aged’ preparationsbut not in freshly prepared membrane fragments (Lang et al.,2002); this work led to the current hypothesis that the SNAREsare constitutively active.

Several studies have suggested that binding of syntaxin tomunc18-1 is required for trafficking of syntaxin to the cellsurface (Rowe et al., 2001; Rowe et al., 1999). Thisrequirement has also been observed for the homologous pairof proteins in the yeast Saccharomyces cerevisiae, Vps45p andTlg2p (SM protein and cognate syntaxin, respectively),whereby ablation of Vps45p resulted in a marked reduction inTlg2p expression. However, others have reported that syntaxincan traffic to the plasma membrane in the absence of munc18-1 in a variety of different cell types (Schutz et al., 2005; Toonenet al., 2005). Studies using a munc18-1-null mousedemonstrated that syntaxin can be detected at the cell surface(Toonen et al., 2005; Verhage et al., 2000), but, in this system,syntaxin levels are reduced by 70% and other munc18 isoformsstill exist (Toonen et al., 2005). It has been suggested thatmunc18-1 is a ‘docking factor’, based on observations usingthe same null mouse (Voets et al., 2001). More recently,however, it was demonstrated that docking is actually syntaxindependent (de Wit et al., 2006). Under certain cultureconditions, it has been reported that PC12 cells possess anexcess of syntaxin over munc18-1 (Schutz et al., 2005), andthis could be used as an argument to suggest that munc18-1 isnot required for SNARE trafficking. Other studies (Liu et al.,2004; Rickman et al., 2007) have shown that syntaxin andmunc18-1 interact in intracellular membranes in intact cells.However, neither the functional significance, nor the mode(s)of interaction, of such intracellular interactions have beendefined.

Here, we show that syntaxin forms stable intracellularSNARE complexes with SNAP-25 if the proteins encounterone another before munc18-1 binding to syntaxin. Theformation of such ectopic exocytotic SNARE complexesoccurs predominantly in the Golgi complex, preventingtrafficking of both t-SNAREs. Using quantitative imagingapproaches including colocalisation and fluorescence lifetimeimaging (FLIM), we demonstrate that this intracellulartrapping of the t-SNAREs depends on their direct interactionand that munc18-1 binding to the closed form of syntaxin, butnot the N-terminal peptide motif, inhibits the formation of thiscomplex. Syntaxin thus traffics efficiently through the Golgicomplex while bound to munc18-1, and, once at the cellsurface, remains inactive even in the presence of SNAP-25colocalised in clusters. Plasma membrane clusters containingboth syntaxin1a and SNAP-25 are heterogeneous in theirinteraction status. However, the influx of Ca2+ significantlyincreased the number of SNARE clusters containinginteracting t-SNARE proteins. These findings provideimportant information on the role of munc18-1 in the SNAREtrafficking life cycle.

ResultsMunc18-1 facilitates syntaxin, but not SNAP-25,trafficking to the cell surface in neuroendocrine cellsIt has previously been shown that coexpression of munc18-1is required for efficient trafficking of syntaxin in

nonspecialised cells (Martinez-Arca et al., 2003; Rowe et al.,2001). It has not been demonstrated convincingly, however,whether a SNARE-trafficking function for munc18-1 exists inspecialised secretory cells such as neuroendocrine cell lines.To address this, we expressed fluorescent fusions of syntaxinor syntaxin mutants in Neuro2a (N2a) cells, both in isolationor in conjunction with fluorescent munc18-1 (Fig. 1A). Full-length syntaxin (Syx1-288) was never observed to traffic to thecell surface in either HEK293 cells or N2a cells in the absenceof coexpressed munc18-1. For all cell types, the expressionlevels were similar and only cells with the lowest detectableexpression levels were selected. Fluorescence correlationspectroscopy (FCS) was performed to estimate the fluorescentprotein concentration from multiple points on the plasmamembrane of N2a cells expressing fluorescent syntaxin andmunc18-1. These experiments used cells expressingheterologous proteins at the same levels as those used forFLIM and colocalisation analyses. FCS revealed that theabsolute number of molecules per excitation volume, on theplasma membrane of these cells, was approximately 10 (datanot shown). As the excitation volume of the diffraction-limitedfocal point is sub-femtolitre, the concentration of the proteinsunder study is in the nanomolar range in the plasma membrane.It should be noted that there are potential caveats to usingheterologous proteins expressed on a background ofendogenous proteins. We have taken steps, therefore, tominimise overexpression and avoid potential targeting ormislocalisation problems. Other work has utilised primaryembryonic cells from animal null models; these approaches arepowerful but also subject to caveats: chronic ablation of proteintargets might have undesired nonspecific effects on bothdevelopment and cellular function (de Wit et al., 2006; Gulyas-Kovacs et al., 2007; Toonen et al., 2005). In addition, theproteins studied are present in multiple isoforms in thesemodels and these might functionally substitute for the ablatedtarget. In the absence of a ‘clean’ cellular model, devoid of allmunc18 isoforms, and through careful experimental design,low-level acute expression of heterologous proteins in acellular environment provides a tractable system to investigatethe function of syntaxin 1a and munc18-1.

One argument against the role of munc18-1 as achaperone for syntaxin would be the ability of a mutantsyntaxin, spending more time in the ‘open’ conformation(SyxL165E, E166E; Syx1-288[open]) (Dulubova et al., 1999), totraffic to the cell surface. Although munc18-1 has been shownrecently to interact with this mutant through an alternative, N-terminal, binding site (Rickman et al., 2007; Shen et al., 2007),the recognised ability of the open mutant to form SNAREcomplexes while bound to munc18-1 (Rickman et al., 2007)would argue against a ‘protective’ role for munc18-1. Toaddress this, we performed similar experiments, where weexpressed Syx1-288[open] either alone or together with munc18-1. In the absence of munc18-1, Syx1-288[open] remainedtrapped in intracellular membranes (Fig. 1A). Whencoexpressed with munc18-1, however, Syx1-288[open]trafficked efficiently to the cell surface (Fig. 1A). BothSyx1-288 and Syx1-288[open] interact with munc18-1 inintracellular membranes and on the cell surface (Fig. 1A)(Rickman et al., 2007) (see text below and Figs 3-4 for details).While these findings could argue against munc18-1 being achaperone for syntaxin, Syx1-288[open] is not constitutively

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‘open’ – it is known to spend time in the closed state (Fasshauerand Margittai, 2004). In addition, this mutant has an affinityfor munc18-1 identical to that of wild-type Syx1-288 (5 nM)(Rickman et al., 2007); abolition of the N-terminal motif stillallows ‘open’ syntaxin to bind to munc18-1 with high affinity(44 nM) (Rickman et al., 2007). Finally, it was shown recentlythat Syx1-288[open] can interact with munc18-1 in living cells,but that the interaction in intracellular membranes wassignificantly decreased compared with wild-type Syx1-288(Rickman et al., 2007).

Syntaxin has been shown to traffic to the plasma membranein PC12 cells without coexpressed munc18-1. In addition, it isthought that syntaxin exists in a large excess over munc18-1in these cells (Schutz et al., 2005). Both these observationsargue against a role for munc18-1 in trafficking syntaxin. Toeliminate the possibility that the requirement for munc18-1 intrafficking syntaxin in N2a cells was specific to this cell line,we repeated the same experiments in PC12 cells (Fig. 1B). Inthis case, syntaxin was seen to traffic efficiently to the cellsurface, but in a minority of cells. Whereas 19±2%(mean±s.e.m., n=153 cells from at least four experiments) ofcells had syntaxin on the cell surface in the absence ofcoexpressed munc18-1, 93±5% (mean±s.e.m., n=122) ofco-transfected cells had both proteins on the cell surface.Also, 11±4% (mean±s.e.m., n=135) of cells expressing

Syx1-288[open] had syntaxin on the cell surface, whereas37±6% (mean±s.e.m., n=100; significantly lower than forSyx1-288; t test, P<0.001) had Syx1-288[open] and munc18-1.We conclude, therefore, that munc18-1 has an essential role inthe trafficking of syntaxin to the cell surface in intactneuroendocrine cells. SNAP-25 trafficking, however, did notrely on munc18-1, or on the presence of any specialised protein(Loranger and Linder, 2002), as it trafficked efficiently to thecell surface in both HEK293 and N2a cells.

Syntaxin and SNAP-25 colocalise in intracellularmembranes in the absence of munc18-1We hypothesised that the role of munc18-1 in traffickingsyntaxin might be dependent on spatio-temporal factors. Toaddress this issue, we performed similar experiments in bothN2a and HEK293 cells, where we coexpressed Syx1-288 andSNAP-25 or SNAP-25 mutants in the absence of additionalmunc18-1. These experiments were designed to test the abilityof syntaxin to form complexes with SNAP-25; are theSNAREs reactive or non-reactive in intact intracellularmembranes? Quantitative colocalisation analysesdemonstrated that neither SNARE trafficked to the cell surfacein this situation, even in the presence of the endogenousmunc18-1 in N2a cells (Fig. 1D). A two-dimensionalhistogram was generated by analysing the fluorescence

Fig. 1. SNAP-25 but not syntaxin trafficsreadily to the plasma membrane in livecells, 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 FLIMdata confirmed that Syx1-288 and Syx1-

288[open] interact with munc18-1. (B) Fieldsof PC12 cells expressing the sameconstructs were scored for syntaxintrafficking to the cell surface. Thepercentage of cells with surface syntaxinwas plotted. (C) N2a cells expressingSNAP-251-206 or truncated SNAP-251-121were imaged by CLSM. Shown arerepresentative equatorial sections; n>20images. Bar, 10 �m. (D) Wild-type ormutant SNAP-251-206 (green) and Syx1-288(red) were expressed in live N2a cells andimaged by CLSM, as before. The mergedimage shows areas of coincidence in yellowhues. The two-dimensional histogramrepresents the intensity of each channel ineach voxel, with a colour scale representingfrequency. The residual map corresponds toweighted residuals from the linearregression of the histogram, thus indicatingfluorescent channel covariance. The hue isfrom –1 to 1, with cyan corresponding to azero residual. Shown are representativeequatorial sections; n>20 images. Bar,5 �m.

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intensity, for each channel present in a single voxel, andplotting this on the histogram. This approach was applied toevery voxel in the three-dimensional image stack, with thecolour scale of the histogram corresponding to the frequencyof voxels for each pair of intensity values. This histogram isfit by linear least-squares regression, yielding a Pearson’scorrelation coefficient. If the concentrations of both proteinsare colinear, there will be a high degree of covariance betweenthem – as would be expected for two proteins that interact witha defined stoichiometry. From this fit, residuals for each voxelwere calculated and displayed as a ‘residual map’ to highlightareas of covariance (Fig. 1D). Syntaxin and SNAP-25exhibited a high degree of coincidence and of covariance inperinuclear membranes, reminiscent of the Golgi complex inthese cells. To improve our understanding of this effect,we coexpressed Syx1-288 alongside SNAP-251-121. It wasdemonstrated previously that the single N-terminal SNAREmotif of SNAP-25 is sufficient for interaction with syntaxin(Fasshauer et al., 1997). The construct used in our studyincludes this region necessary for SNARE interaction, inaddition to the flexible linker required for localisation to theplasma membrane (Gonzalo et al., 1999). In this case, SNAP-251-121 was seen to traffic to the cell surface, but most remainedcolocalised with Syx1-288 in intracellular membranes (Fig. 1D).These experiments provided evidence to support thehypothesis that the t-SNAREs are in fact reactive in living cells

and that another, spatially and temporally localised, factor isrequired to prevent them from forming SNARE complexes inintracellular membranes.

Syntaxin and SNAP-25 complexes form in the GolgicomplexNext, we determined the intracellular site of colocalisation andinteraction between Syx1-288 and SNAP-25. We performedsimilar experiments as before, where we coexpressedfluorescent fusions to the t-SNAREs, but this time alsocoexpressed a marker of the Golgi complex, GRASP-65. Ared-fluorescent (mCherry) protein fusion to this markerlocalises to the cis-Golgi (J. Lane, personal communication)and is spectrally distinct from both cerulean-Syx1-288 andEYFP-SNAP-25. Quantitative colocalisation analysis, asbefore, confirmed that the site of greatest t-SNARE covariancewas the Golgi complex (Fig. 2A). Frequency distributionhistograms were generated from these data, emphasising thatthe weighted residuals centered around zero (i.e. highestcovariance) were in the Golgi complex (Fig. 2A, right).

This quantitative analysis of colocalisation between mutantproteins in living cells provided evidence suggesting that thet-SNAREs are able to interact in intracellular membranes.However, colocalisation data are limited by the resolution ofthe microscope (maximally 200 nm) and are not a directindication of protein interactions. To increase our

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Fig. 2. Syntaxin and SNAP-25 colocaliseand can interact in the Golgi complex inlive cells. Syx1-288 and SNAP-251-206coexpressed in live N2a cells and imagedby CLSM. (A) Residual mapcorresponds to weighted residualsindicating fluorescent channelcovariance. The hue is from –1 to 1, withcyan corresponding to a zero residual.Bar, 10 �m. (B) The Golgi complexmarker GRASP65-mCherry wascoexpressed in live N2a cells and imagedby CLSM (left panel). Residual map ofSyx1-288 and SNAP-251-206 covariance inthe Golgi complex, as defined by aGRASP65-mCherry mask (middlepanel). The weighted residuals of Syx1-

288 and SNAP-251-206 contained withinthe Golgi complex, as defined byGRASP65-mCherry (black circles),alongside residual values from elsewherein the cell (grey circles), were plotted asa frequency distribution histogram (rightpanel). (C) mCerulean-Syx1-288 (donor)fluorescence in the absence of EYFP-SNAP-251-206 (acceptor) exhibited anintracellular 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 Syx1-288fluorescence lifetime distribution (red line). Syx1-288 alone has a single fluorescence lifetime of 2388±47 pseconds. The excited-statefluorescence 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 scalein 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 ofmCerulean-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 donorfluorescence lifetimes below than the 99% confidence interval (right panel). Data are expressed as the mean±s.e.m.

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understanding of the mechanism whereby SNAP-25 appearstrapped by coexpressed Syx1-288, we employed FLIM to detectand quantify directly Förster resonance energy transfer(FRET). FLIM quantifies the excited state fluorescencelifetime of a fluorophore, the duration of which is exquisitelysensitive to the microenvironment it inhabits (Medine et al.,2007). Thus FRET, to an interacting acceptor molecule,dramatically shortens the donor fluorescence lifetime(Lakowicz, 1999); this effect can be quantified directly in eachpixel of an image. Importantly, the amount of lifetimequenching is dependent on the distance between the proteinsand the fluorophores used; thus, the actual numerical valuesobtained for each protein pair will vary. Furthermore, the lowlight levels required for FLIM allowed us to use the same cellsas before, selected for the lowest detectable levels ofexpression. In contrast to other, intensity based, approaches fordetecting FRET, this approach has the advantage of beingdirectly quantitative and requires only one measurement. Nocomplex arithmetical adjustments to the pixel data arerequired, and two-photon excitation is inherently in focus,providing high-resolution data.

We used FLIM analysis to detect FRET between cerulean-Syx1-288 (the donor) and EYFP-SNAP-25 (the acceptor; Fig.2B). FLIM analysis of N2a cells expressing cerulean-Syx1-288alone revealed a mono-exponential fluorescence lifetime decayof 2388±47 pseconds (mean±s.d., n=5), in agreement withprevious studies of mCerulean (Rizzo et al., 2004) and ofcerulean-Syx1-288 (Rickman et al., 2007). In the presence ofcoexpressed EYFP-SNAP-25, however, this donor lifetimewas shortened significantly to 2176±134 pseconds (Mann-Whitney U non-parametric test; P<0.01; mean±s.d., n=5).These data are presented as intensity images, frequencydistribution histograms and as FLIM maps (Fig. 2B). FLIMmaps represent the measured fluorescence lifetime in eachpixel of an image as a false-colour to reveal the intracellularlocations where FRET occurs. In this case, statisticallysignificant donor fluorescence lifetime quenching wasrestricted to the perinuclear Golgi region, where the t-SNAREscolocalise, consistent with the conclusion that the t-SNAREsare highly reactive in the absence of the correct levels ofmunc18-1 in the appropriate intracellular location.

Munc18-1 interacts with syntaxin in intracellularmembranes, preventing formation of ectopic SNAREcomplexes and permitting efficient traffickingIf our hypothesis is accurate, that munc18-1 at differentintracellular locations can prevent Syx1-288 from entering intoectopic SNARE complexes, then coexpressing munc18-1 withthe t-SNAREs should allow both Syx1-288 and SNAP-25 totraffic efficiently to the cell surface. We have demonstratedpreviously that munc18-1 interacts with syntaxin1a inintracellular membranes (Rickman et al., 2007). To explore thisfurther, we coexpressed both t-SNAREs, as before withmunc18-1, in HEK293 cells. We used fibroblasts in thisexperiment as they do not express any endogenous munc18isoform (Rowe et al., 2001). Coexpression of Syx1-288, or amutant of Syx lacking the ability to bind to munc18-1 throughits N-terminal peptide motif (Syx1-288 �6) (Rickman et al.,2007) with SNAP-25 and munc18-1, resulted in the efficienttrafficking of both t-SNAREs to the cell surface (Fig. 3).However, when Syx1-288[open] (Rickman et al., 2007) or

Syx1-288[open] �6 was coexpressed with SNAP-25 andmunc18-1, the t-SNAREs remained colocalised in the Golgicomplex, as before. Trafficking to the cell surface was thussignificantly reduced; we hypothesise that Syx1-288[open] canpreferentially form complexes with SNAP-25 in cells even inthe presence of munc18-1, as shown previously in vitro(Rickman et al., 2007). These data indicate that munc18-1 canprevent the formation of intracellular t-SNARE complexesand, importantly, show that binding to the closed form ofSyx1-288 is required for this function.

SNARE clusters are heterogeneous in their interactionstatusIf munc18-1 binding to Syx1-288 is required to prevent formationof ectopic t-SNARE complexes, but this in turn inhibits Syx1-

288 from entering the ternary SNARE complex, how doesexocytosis proceed? The t-SNAREs are known to colocalise inclusters at the cell surface (Lang et al., 2001). Our model is thatSNAP-25 can traffic to the cell surface without the involvementof any other specialised factors, as long as closed Syx1-288 isfirst stabilised by munc18-1 binding. We again used FLIM todetermine whether the newly delivered t-SNAREs couldinteract on the plasma membrane in the presence of munc18-1(Fig. 4A). Colocalisation analyses as before confirmed that82±2% of Syx1-288 colocalised with SNAP-25 in these clusters,and that the clusters observed were indistinguishable fromendogenous clusters in terms of size, number and density(R.R.D., unpublished). In resting cells, we were unable to detectsignificant donor (cerulean-Syx1-288) lifetime quenching, evenin the presence of colocalised EYFP-SNAP-25. These data canbe interpreted in two ways; either the newly arrived t-SNAREsdo not interact at the surface before fusion, or the t-SNAREinteraction adopts a conformation where the fluorophores aresufficiently distant, or of an orientation, to preclude FRET. Weattempted to dissect these possibilities by using a SNAP-25construct with the fluorescent fusion to the C-terminus, and stillno FRET could be detected (R.R.D., unpublished). In eithercase, these data demonstrate heterogeneity in resting SNAREclusters not previously recognised, owing to alternative SNAREcomplex conformational states or a sequestration of closedsyntaxin by munc18-1.

Depolarisation of the cells with KCl induced FRET inpuncta on the cell surface (Fig. 4A, lower panels). To furtherour understanding of this effect, we performed FLIM imagingat the base of the cells, attempting to acquire FLIM-FRET datafrom individual SNARE clusters there. As a stimulationparadigm, ionomycin was used to elevate intracellular Ca2+

levels directly, circumventing any potential indirect effect ofCa2+ channels on SNARE interactions. In this case, in bothresting cells or in stimulated cells in a Ca2+-free, chelatingmedium, we saw heterogeneity in the SNARE clusterpopulation (Fig. 4B). After ionomycin-induced Ca2+ influx,this situation was reversed; almost every t-SNARE cluster(95±4%, mean±s.e.m., n=15) now contained interactingSNAREs (Fig. 4C). We investigated this effect further,comparing the donor fluorescence lifetimes of cerulean-Syxcolocalized with EYFP-SNAP-25 before and after treatmentwith KCl (Fig. 4D). Again, the proportion of FRET-positiveSNARE clusters increased. The frequency distribution of allthe donor fluorescence lifetimes in each sample was fitseparately by nonlinear regression (Fig. 4E). These nonlinear

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fits were significantly different (extra sum-of-squares F test,P<0.0001; Fig. 4E). These data suggest that the formation ofsuitable syntaxin–SNAP-25 dimeric binary complexes, whichcan act as acceptor sites for VAMP-containing vesicles, mightbe a regulatable step of SNARE function.

DiscussionWe have demonstrated that munc18-1 does indeed have anessential role in preventing formation of ectopic t-SNAREcomplexes during trafficking of syntaxin in neuroendocrinecells. The closed-form mode of binding to syntaxin is

necessary and sufficient for this function. While munc18-1clearly plays a crucial role in trafficking syntaxin to the plasmamembrane in neuroendocrine cells, as shown here (Figs 1, 3and 4), it is possible that a small percentage of syntaxin 1 isable to reach the plasma membrane independently of munc18-1. This possibility would reconcile our findings with previousobservations that syntaxin is severely depleted, but still presentat low levels, in organisms where a specific SM protein hasbeen ablated (Toonen et al., 2005). Importantly, directknockdown of expression of syntaxin 1 (de Wit et al., 2006)has been shown to result in a phenocopy of that observed in

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Fig. 3. Munc18-1 prevents ectopic t-SNARE interactions. (A) Wild-typeor mutants of Syx1-288 (red), SNAP-25 (green) and munc18-1 werecoexpressed in live HEK293 cellsand imaged by CLSM. The mergedimage shows areas of coincidence inyellow hues. The two-dimensionalhistogram represents the intensity ofeach channel in each voxel, with acolour scale representing frequency.The residual map corresponds toweighted residuals from the line fitto the histogram, thus indicatingfluorescent channel covariance. Thehue is from –1 to 1, with cyancorresponding to a zero residual.(B) Similar results were obtainedusing N2a cells. Bars, 5 �m.(C) The covariance data fromHEK293 cells coexpressing wild-type or mutants of Syx andSNAP251-206, in the absence (filledbars) or presence of munc18-1(open bars) were quantified andexpressed as Pearson’s coefficientvalues. These data showed that thecovariance between the t-SNAREsis unaffected by the presence ofmunc18-1, but that the location ofthe colocalisation is altered (seepanel A; n�4). Data are expressedas the mean±s.e.m.

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the munc18-1-knockout mouse (de Wit et al., 2006; Voets etal., 2001). This highlights the close interrelationship betweenSM proteins and their cognate syntaxin throughout their lifecycle. Once at the membrane, the newly delivered t-SNAREsform interaction-heterogeneous clusters, with interactionsmodulated by elevation of intracellular Ca2+ levels. This

enhancement of interaction between the t-SNAREs, illustratedin Fig. 4, could be a shift in the equilibrium from monomerict-SNAREs to the binary intermediate. However, we cannotexclude that these interacting clusters might also be reportinga conformational change induced by VAMP during theexocytotic events.

Fig. 4. SNARE clusters areheterogeneous. Intensityimages and FLIM mapsshowing mCerulean-Syx1-288,in the presence of EYFP-SNAP-251-206 and munc18-1in live N2a cells, imaged byFLIM and two-photonmicroscopy. (A) mCerulean-Syx1-288 (donor) fluorescenceexhibited a plasma membranedistribution. The colour scale[1900 (red) – 2400 pseconds(blue)] in the FLIM maprepresents the donorfluorescence lifetime in restingcells (top) and afterdepolarisation with 55 mMKCl (lower panels). These datawere plotted as a frequencydistribution histogram,showing the 99% confidenceinterval of the donor lifetimedistribution (red dashed line;bars are mean±s.e.m., n=5experiments). Bar, 5 �m.(B) Similar experimentsperformed in the presence ofionomycin, but a Ca2+-freeenvironment also revealedclusters at the base of the cell.The colour scale in the FLIMmap represents the donorfluorescence lifetime. Bar,5 �m. [Colour scale: 1500(red) – 2000 pseconds (blue).]The boxed region of interest isshown in the lower panels andillustrates clusters at theplasma membrane. Pixelscontaining fluorescencelifetimes below the 99%confidence interval of the non-FRET distribution in panel Aare shown in red. Bar, 1 �m.SNARE clusters where noFRET could be detected arehighlighted with a dashedcircle. (C) In the presence ofionomycin and Ca2+, mCerulean-Syx1-288 (donor) fluorescence in the presence of EYFP-SNAP-251-206 (acceptor) and munc18-1 showedclusters at the base of the cell. The colour scale [1500 (red) – 2000 pseconds (blue)] in the FLIM map represents the donor fluorescencelifetime in the presence of ionomycin and Ca2+. The donor fluorescence lifetime was significantly shortened, indicative of FRET between thet-SNAREs. Bar, 5 �m. The boxed region of interest is shown in the lower panels and illustrates clusters at the plasma membrane. Pixelscontaining fluorescence lifetimes below the 99% confidence interval of the non-FRET distribution in panel A are shown in red. SNAREclusters where no FRET could be detected are highlighted with a dashed circle. Bar, 1 �m. (D) Similar results were obtained using KCldepolarization. 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 plottedas a frequency distribution histogram. The fluorescence lifetimes in the KCl-treated samples (open circles, grey fit line) were significantlyreduced compared with those from resting cells (filled circles, black fit line).

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The role of munc18-1 has been difficult to define andcontroversial. Conflicting data regarding its role in traffickingsyntaxin and its ability to interact with the assembled binary-and ternary-SNARE complexes have hampered progress. Wepresent a model here based on the simplest interpretation ofmunc18-1 function. The role of munc18-1 in traffickingsyntaxin through the secretory pathway relies on it beingpresent in appropriate concentrations at the correct time and inthe correct place. If the t-SNAREs encounter one another earlyin their biogenesis, before munc18-1 inactivation of syntaxin,they can form a misplaced SNARE complex. This complexcannot traffic out of the Golgi complex, perhaps because afourth SNARE helix is provided by an intracellular SNARE,so trapping the ectopic complex. This hypothesis is supportedby including the observations that syntaxin can interactpromiscuously with intracellular SNAREs (Fasshauer et al.,1999). SNAP-25 is palmitoylated by Golgi-resident enzymesand requires a functional Golgi complex for membraneassociation (Gonzalo et al., 1999). Once through intracellularmembrane systems, syntaxin remains inactive, while SNAP-25can proceed efficiently and independently to the plasmamembrane and associate with the inner leaflet (Loranger andLinder, 2002). Newly delivered SNAREs form clusters butmight remain nonreactive until required. As munc18-1 canremain associated with syntaxin throughout the formation ofthe binary and ternary SNARE complex, it is possible thatmunc18-1 can be bound to syntaxin for its entire life cycle, indifferent conformational forms.

Data from inside-out membrane preparations showedthat exogenously added SNARE proteins could interactspontaneously with pre-existing SNARE clusters (Lang et al.,2002). Interestingly, this observation relied on the ‘ageing’ ofthe membrane preparations: freshly prepared sheets did notallow the exogenous SNAREs to interact. In addition,exogenously added recombinant SNAP-25 could not enterinto a complex with the endogenous SNAREs in clusters. Theauthors concluded that the SNAREs are constitutively reactiveand that no additional factor was required to prevent SNAREprotein interactions (Lang et al., 2002). Our data are incomplete agreement with these findings, but support adifferent conclusion. We argue that syntaxin is held in aninactive closed form by munc18-1 and that it is feasibletherefore that the run-down of the membrane sheet preparationseen during ‘ageing’ is due to loss of soluble munc18-1, alongwith other soluble cytoplasmic material. In addition, theobserved failure of exogenous SNAP-25 to enter intocomplexes (Lang et al., 2002) also supports our model, wheresyntaxin is held in an inactive state until required. It haspreviously been observed in vitro that syntaxin and SNAP-25can form a stable dimeric complex (Fasshauer and Margittai,2004; Rickman et al., 2004) and that the formation of thisbinary intermediate is the rate-limiting step to formation ofternary SNARE complexes (Fasshauer and Margittai, 2004).It is common in biological pathways for the rate-limiting stepto form the natural point of regulation (Fersht, 2003). It ishighly likely, therefore, that formation of the t-SNARE binaryintermediate is a crucial point of regulation in living cells.Indeed, the slow fusion kinetics observed for in vitroreconstituted fusion assays are dramatically accelerated by thepresence of a stabilised binary t-SNARE intermediate(Pobbati et al., 2006).

The approaches used in this study permit the determinationof protein interactions in situ, meaning that individual SNAREclusters can be examined without prior cell lysis and proteininteraction disruption and re-formation in vitro. Thus, thesedata provide important insights into the spatial mechanismsregulating the biogenesis and function of SNARE proteins.Further work and development of the technologies will berequired to quantify protein interactions in living cells todetermine protein dynamics immediately before, during andafter exocytosis.

Materials and MethodsVectors and cell cultureThe vector pEGFP-C2 was obtained from Clontech (Basingstoke, UK). An EYFP-SNAP-25 fusion was generated by ligation of SNAP-251-206 into BamHI/EcoRI sitesof pEGFP-C, followed by the replacement of EGFP with EYFP. SNAP-25–EGFPin pEGFP-N1 was a gift from M. Linder. This construct was used in control FRETexperiments. A SNAP-25 mutation, SNAP-251-121, was generated using site-directed mutagenesis with a Quickchange site-directed mutagenesis kit (StratageneEurope). The plasmids pmCerulean-Syntaxin1a1-288, open (L165A, E166A)mutation and N-terminal truncations of syntaxin1a were described previously(Rickman et al., 2007). A native munc18-1 expression vector was constructed byamplifying the open reading frame of rat munc18-1 and ligating the PCR productto pTarget (Promega). The Golgi complex marker GRASP65-mCherry was obtainedfrom Jon Lane (University of Bristol, UK). Neuroblastoma 2a (N2a) cells andhuman embryonic kidney (HEK293) cells were cultured in Dulbecco’s modifiedEagle’s medium supplemented with 10% fetal bovine serum, 50 units of penicillin,50 �g/ml streptomycin and maintained at 37°C in 5% (v/v) CO2, 95% (v/v) air. Allcells were cultured on glass coverslips and transfections were performed usingExGen500 (Fermentas). In stimulation experiments, the medium was replaced withKrebs-Ringer bicarbonate buffer (115 mM NaCl, 5 mM KCl, 24 mM NaHCO3, 2.5mM CaCl2, 1 mM MgCl2, 10 mM HEPES pH 7.4 and 0.1% BSA) for imaging.Cells were stimulated with Krebs-Ringer bicarbonate buffer adjusted to 55 mM KClor by addition of 100 nM ionomycin and incubated at 37°C for 20 minutes beforeimaging. At the concentration and time scales used, we saw no morphologicalevidence of apoptosis.

Confocal laser scanning microscopy and image analysis All imaging experiments were performed using a Zeiss LSM510 Axiovert confocallaser scanning microscope, equipped with a pulsed excitation source [MIRA 900Ti:Sapphire femto-second pulsed laser, coupled with a VERDI 10 W pump laser(Coherent)]. Data acquisition was performed using a 1024�1024 pixel image size,using a Zeiss Plan NeoFLUAR 1.4 NA 63� oil-immersion objective lens. Separatefluorescence emission channels were collected simultaneously using multi-track ordual-laser excitation. All imaging was performed using live cells maintained at 37°Cin 5% (v/v) CO2, 95% (v/v) air. Image data acquired at Nyquist sampling rates weredeconvolved using Huygens software (Scientific Volume Imaging), and theresulting images analysed using NIH ImageJ software (http://rsb.info.nih.gov/ij/).Residual maps were generated by calculating the residual of each voxel from thelinear regression fit to the intensities of each channel within each voxel. Theresulting residuals are displayed on a colour scale from –1 to 1 (with zero residualcoloured cyan), with brightness corresponding to the combined intensity of the twochannels. Cell peripheries were determined using transmitted light imagingcombined with CLSM data.

TCSPC-FLIM acquisition and analysisTime-correlated single-photon counting (TCSPC) measurements were made under800-820 nm two-photon excitation (TPE), which efficiently excited cerulean,without any detectable direct excitation or emission from EYFP, using a fastphotomultiplier tube (H7422; Hamamatsu Photonics UK) coupled directly to therear port of the Axiovert microscope. TCSPC recordings were acquired for between10 seconds and 60 seconds, mean photon counts were between 105-106 counts persecond. Images were recorded at 256�256 pixels from a 1024�1024 image scanwith 256 time bins over a 12 nsecond period. Off-line FLIM data analysis usedpixel-based fitting software (SPCImage, Becker & Hickl). The fluorescence wasassumed to follow a multi-exponential decay for the purposes of data fitting. Inaddition, an adaptive offset-correction was performed. This constant offset takesinto consideration the time-independent baseline due to dark noise of the detectorand the background caused by room light, calculated from the average number ofphotons per channel preceding the rising part of the fluorescence trace. To fit theparameters of the multi-exponential decay to the fluorescence decay trace measuredby the system, a convolution with the instrumental response function was carriedout. The optimisation of the fit parameters was performed by using the Levenberg-Marquardt algorithm, minimising the weighted chi-square quantity. This approachcan be used to separate the interacting from the non-interacting donor fraction in

Journal of Cell Science 120 (24)

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our FRET systems. The long lifetime component �2 was determined by controlassays with cerulean alone, or expressed with (non-interacting) EYFP, as describedabove. This value was subsequently used as a fixed �2 lifetime for all otherexperiments. As controls for non-specific FRET, or FRET between GFPs that mightform dimers spontaneously when overexpressed in cells, we determined thefluorescence lifetimes of cerulean-Syx1-288 alone, cerulean alone, or cerulean-Syx1-288 co-transfected with EYFP (data not shown). No FRET was detected in anyof these experiments.

We thank Jon Lane, University of Bristol, UK, for providing avector encoding mCherry-GRASP65. SNAP-25 EGFP in pEGFP-N1was a gift from Maurine Linder, Washington University in St Louis,USA. This work was supported by a Wellcome Trust Research CareerDevelopment Fellowship to R.R.D. and a Project Grant to R.R.D. andL.H.C.

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