Membrane fusion is one of the most important cellular processes by which two initially distinct lipid bilayers merge their hydrophobic cores, resulting in one interconnected structure. Proteins, called SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor), play a central role in the fusion process that is also regulated by several accessory proteins. In order to study the SNARE-mediated membrane fusion, the in vitro protein reconstitution assay involving ensemble FRET (fluorescence resonance energy transfer) has been used over a decade. In this mini-review, we describe several single-molecule-based FRET approaches that have been applied to this field to overcome the shortage of the bulk assay in terms of protein and fusion dynamics.

INTRODUCTION

SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor)-mediated membrane fusion is a fundamental cellular process involved in many important life activities, such as egg fertilization, protein trafficking and neurotransmitter release [1]. In order to communicate with each other, molecules called neurotransmitters are used for messages across the synaptic cleft between two neurons at the synapse. Synaptic vesicles in the axon bulb containing neurotransmitters are released in the submillisecond timescale by exocytosis into the synaptic cleft. As summarized in Table 1, this calcium-triggered fast neurotransmitter release is mediated by SNARE proteins and is tightly regulated by a number of accessory proteins, such as synaptotagmin, complexin and Munc18 [2].

Table 1
Key proteins involved in SNARE-mediated membrane fusion for fast neurotransmitter release
NameFamilyLocationFunction
Synaptobrevin SNARE Synaptic vesicle To form SNARE complex, which is known as the minimum fusion machinery 
SNAP-25 SNARE Target membrane To form SNARE complex, which is known as the minimum fusion machinery 
Syntaxin SNARE Target membrane To form SNARE complex, which is known as the minimum fusion machinery 
Complexin  Free Dual-function regulator 
Synaptotagmin  Synaptic vesicle Calcium sensor 
Munc18 Sec1/Munc18 Free Docking and priming regulator 
NameFamilyLocationFunction
Synaptobrevin SNARE Synaptic vesicle To form SNARE complex, which is known as the minimum fusion machinery 
SNAP-25 SNARE Target membrane To form SNARE complex, which is known as the minimum fusion machinery 
Syntaxin SNARE Target membrane To form SNARE complex, which is known as the minimum fusion machinery 
Complexin  Free Dual-function regulator 
Synaptotagmin  Synaptic vesicle Calcium sensor 
Munc18 Sec1/Munc18 Free Docking and priming regulator 

Although many proteins involved in membrane fusion have been identified, the detailed mechanism still remains ambiguous. Traditionally, most studies rely on two major techniques, in vivo knockout and in vitro reconstitution methods [2,3]. For the scope of this review, we will only focus on the advancement of in vitro assays.

The in vitro protein reconstitution assay involving ensemble FRET (fluorescence resonance energy transfer) has been used to study SNARE-mediated membrane fusion for a decade. Despite the great success of ensemble FRET for studying SNARE-mediated membrane fusion, it falls short in revealing the fast and transient fusion dynamics. In order to overcome the limitations of conventional methods, more advanced assays are expected to study membrane fusion. Since single-molecule techniques have many advantages over ensemble or bulk measurements, it naturally becomes a good candidate of the substantial technique [4]. By watching single molecules, people can see many details such as different fusion states that would have been averaged out in an ensemble measurement.

FRET is a popular biological technique because of its sensitive distance range and for its ability to observe real-time reactions in biologically relevant conditions. The FRET signal is sensitive to 2–10 nm-scale distances between donor and acceptor fluorophores [5]. This range helps us understand molecular interactions, such as protein–protein, protein–DNA and antibody–antigen. Fluorophores commonly used for FRET measurements are small organic dyes, and with control, they do not purturb the biological function and are stable under most biologically relevant conditions. Two major ways of utilizing the smFRET (single-molecule FRET) technique have been applied to SNARE-mediated membrane fusion through monitoring SNARE protein interactions [610] or fusion of lipid molecules [1116]. The most recent application of the smFRET approach has been the use of labelled content [17] and the combination of the lipid marker and the labelled content.

MONITORING PROTEINS

Many biologically relevant protein conformation and protein–protein interactions occur within the sensitivity range of FRET measurement. SNARE and its accessory proteins that are site-specifically conjugated with fluorescent dyes may be used to provide unique structural information [19]. There are a number of conventional techniques to study the structure and conformation of SNARE proteins: X-ray crystallography, a variety of NMR techniques, electron paramagnetic resonance, electron microscopy and CD to name a few. However, there are many instances where structural determination is challenging using these conventional methods for various reasons. Some examples include instability of protein complex at a high concentration, fluctuation of the structure between multiple conformations and the averaging out of the active complex signal by being only a fraction of the large ensemble.

The smFRET approach provides an alternative to conventional methodologies. High sensitivity of the state-of-the-art camera allows detection down to a single fluorophore. Because signals from individual molecules or complexes are independently recorded, synchronization is not necessary and transient processes (such as domain conformational change) may be studied without being averaged out. Here, we discuss the overview of smFRET measurement applications on fluorescent-dye-labelled SNARE proteins by categorizing them into three groups depending on the type of data obtained: stoichiometry, intramolecular conformation and intermolecular orientation.

Stoichiometry

One of the fundamental information obtained from single molecule detection is the localization of the molecule on the surface. Although FRET signal is not required, precise localization (within diffraction limited spot of ~200 nm without fitting or within several nanometres with fitting) allows identification of the target protein and co-localization of different proteins of interest. By measuring the resident on and off times from the fluorescence intensity time trace, an accurate binding constant of the protein may be obtained [7]. However, perhaps the most useful information that may be obtained from localizing individual molecules is the stoichiometry of the complex. Each individual fluorophore photobleaches with a sharp drop of intensity. The number of drops within the fluorescence intensity time trace reveals the number of molecules. The cytoplasmic domain of syntaxin forms a fusion inactive dimer complex with micromolar affinity and also an inactive tetramer with tens of micromolar to millimolar affinity [20]. Photobleaching analysis of individual fluorescence intensity time profile was used to ensure that the dye-labelled-syntaxin molecules are in the active monomeric state within the surface-deposited lipid bilayer [8].

The minimum number of SNARE complexes necessary for membrane fusion is of fundamental interest. Van den Bogaart et al. [21] applied a similar photobleaching analysis to quantify the number of synaptobrevin and acceptor SNARE complexes [syntaxin and SNAP-25 (25 kDa synaptosome-associated protein)] reconstituted in the vesicle embedded in an agarose gel matrix. Using vesicle samples containing, on an average, one synaptobrevin in one and one acceptor complex in another set of vesicles, they have concluded that a single SNARE complex is sufficient to induce lipid mixing.

Intramolecular conformation

The neurotransmitter release process takes place within several milliseconds, and it is important to study the protein conformation dynamic in a similar timescale. In one of the earliest works to apply smFRET to SNARE proteins, syntaxin 1a was dually labelled with Alexa Fluor® 488 (donor) and Alexa Fluor® 594 (acceptor) at several combinations of protein sites and the multiple physical parameters (intramolecular distance from FRET, fluorescence lifetime and anisotropy) were simultaneously quantified in the presence and absence of interacting partners [10]. While contradictory of an NMR study, dynamic switching between opening and closing of the N-terminal three helix bundle of syntaxin 1a with a relaxation time of 0.8 ms was observed from free diffusing syntaxin molecules [22]. Once the four-helix bundle of the SNARE complex is formed, its unusual stability is well known [23,24]. However, the conformational dynamic of two helices of SNAP-25 in the context of a 1:1 binary acceptor complex was not known until recently [8].

Weninger et al. [8] labelled SNAP-25 helices with Cy3 (indocarbocyanine; donor) and Cy5 (indodicarbocyanine; acceptor) and observed, to their surprise, a significant conformational flexibility of helices bound to syntaxin molecule embedded in the lipid bilayer represented by the mid-FRET efficiency signal. The real-time analysis of the FRET efficiency profile showed a transient motion of helices between two stable parallel and anti-parallel conformations. Completion of a four-helix bundle by addition of synaptobrevin stabilizes the binding SNAP-25 helices. Interestingly, addition of a cytosolic fragment of synaptotagmin 1 to the binary acceptor complex also stabilized the conformational dynamic to a similar extent as synaptobrevin, which authors suggest its role in ‘setting the stage’ for efficient trans-SNARE formation. Binding to the SNARE binary complex also stabilized the conformational dynamic of synaptotagmin itself even in the absence of Ca2+, but the effect is more pronounced in the presence of Ca2+ [25,26].

In a recent study, a similarly dual-labelled neuronal SNAP-25 and widely expressed SNAP-29 were microinjected into live cells to investigate the conformational dynamic of the protein by combining smFRET and single-particle tracking [27]. From this proof of concept study, they verified the promiscuous nature of SNAP-25 binding, which results in folding, to non-neuronal origin syntaxin molecules.

Intermolecular interaction and orientation

Structural determination of multicomplex systems that may bind to one another in more than one conformation or that are weakly interacting is challenging. To obtain accurate structural information on such system, FRET pair fluorophores may be conjugated to different interacting partner proteins. Using this strategy, the relative orientations and stabilities of syntaxin, synaptobrevin and SNAP-25 have been investigated through smFRET [6,8].

With careful calibration, FRET efficiency signal may be converted into distance information [2830]. Taking full advantage of this, Choi et al. obtained 34 smFRET-derived distances to elucidate the relative arrangement of synaptotagmin 1 with respect to the SNARE complex [26]. In conjunction with previously known crystal structures, they have fitted these measured distances to show that synaptotagmin interacts with the central region of the SNARE complex without obstructing the complexin binding site.

Caveats

The smFRET measurements from labelled SNARE protein provides dynamic structural information unobtainable by conventional ensemble methods. A typical strategy to label proteins with fluorescent dyes is done through forming covalent linkage to naturally existing or cloned cysteine residue(s). However, special care must be taken in selecting the site of dye conjugation. First, the labelling site of the dye needs to be chosen such that the native function of the protein is not perturbed [9]. High-resolution crystal structure will greatly assist in this process, but with or without such information, a series of control biochemical assays must be done to ensure the functionality. Secondly, the labelled fluorescent dye should be free of interactions with nearby amino acid residues, otherwise it may result in unpredictable photoemission. Both enhancement and quenching of dye are known to happen. This will potentially skew the conversion of FRET efficiencies into distances. A further review on this topic may be found elsewhere [31].

MONITORING FUSION

The application of smFRET has a great advantage in monitoring different stages of membrane fusion events. Compared with the ensemble fusion assay, where averaged FRET signal from the entire population is obtained, instead, the FRET efficiency value from each pair of vesicles may be collected to identify different stages of fusion such as docking, hemifusion and full fusion. The importance of such detailed information may be exemplified in the experiment of fast vesicle aggregation induced by C2AB/Ca2+ [14]. Using the ensemble lipid mixing assay (Figure 1A), upon the addition of Ca2+ into the system, a sudden increase of the FRET signal has been observed. This indicates a high degree of lipid mixing, which may be interpreted as an efficient fusion. However, on testing the same system using the single vesicle–vesicle lipid mixing assay (Figure 1B), the FRET efficiency distribution peaks at ~0.3, which signifies the aggregation of vesicles [11]. The ensemble assay is incapable of distinguishing between the aggregation and the fused membranes, and therefore additional supplemental experiments may be necessary for an accurate interpretation of the data. In order to watch the fusion process, labelled lipid or content molecules are mainly used as fusion reporters for smFRET.

Schematic illustrations of the bulk fusion assay (A) and the single-vesicle lipid-mixing assay (B) for a mixture of protein-free vesicles (35% PS and 65% PC, DiD or DiI labelled), C2AB (cytoplasmic domain of synaptotagmin) and 1 mM calcium

Figure 1
Schematic illustrations of the bulk fusion assay (A) and the single-vesicle lipid-mixing assay (B) for a mixture of protein-free vesicles (35% PS and 65% PC, DiD or DiI labelled), C2AB (cytoplasmic domain of synaptotagmin) and 1 mM calcium

The single-vesicle FRET histogram is plotted by compiling FRET signals from over 1000 vesicles. The y-axis is normalized population, where we divided the distribution by the total number of vesicles measured and the x-axis is FRET efficiency value.

Figure 1
Schematic illustrations of the bulk fusion assay (A) and the single-vesicle lipid-mixing assay (B) for a mixture of protein-free vesicles (35% PS and 65% PC, DiD or DiI labelled), C2AB (cytoplasmic domain of synaptotagmin) and 1 mM calcium

The single-vesicle FRET histogram is plotted by compiling FRET signals from over 1000 vesicles. The y-axis is normalized population, where we divided the distribution by the total number of vesicles measured and the x-axis is FRET efficiency value.

Labelling lipid

When membrane fusion occurs between two membranes, lipid molecules in these membranes undergo a two-dimensional diffusion and completely get mixed together. This process called lipid mixing can be used as a marker for membrane fusion [32]. Actually lipid mixing is a good marker to observe membrane fusion because labelling membrane is relatively easy with in vitro systems and the lipid mixing process is fast enough and so it does not hamper time resolution of many microscopy systems [33].

First, an in vitro lipid mixing experiment on SNARE-driven membrane fusion used a FRET pair consisting of NBD (7-nitrobenz-2-oxa-1,3-diazole) and rhodamine [34]. Two groups of vesicles were prepared containing v-SNARE proteins (SNARE proteins located on synaptic vesicle, v-vesicle) and t-SNARE proteins (SNARE proteins on target membrane, t-vesicle) respectively. V-vesicles were labelled with NBD and rhodamine so that NBD fluorescence is quenched by nearby rhodamine via FRET. When t-vesicles were added to v-vesicles, vesicle fusion and substantial lipid mixing caused dilution of labelled v-vesicle lipids with unlabelled t-vesicle lipids. As the average inter-dye distance between NBD and rhodamine increased, fluorescence signal from NBD recovered. This NBD fluorescence recovery was recorded in bulk solution, which worked as the evidence that SNARE proteins can work as the minimal fusion machinery in vitro.

The ensemble lipid mixing assay is a convenient tool for recording overall fusion kinetics in the solution. However, it records the total signal from a large number of vesicles, and therefore, does not give information about the kinetics of each fusion step, which is an important component to understand the detailed fusion mechanism. To observe vesicle fusion with single vesicle resolution, several methods including single vesicle–bilayer fusion [3537] and single vesicle–vesicle fusion systems were invented [11].

The single vesicle–bilayer fusion assay with geometry similar to the real synaptic fusion has been developed by several groups [3537]. They were able to record single vesicles fusing to the planar lipid bilayer. However, these studies based on planar bilayers suffered from results contradictory to the known SNARE mechanism. For example, SNAP-25 was not required in the acceptor t-SNARE complex [36,37] or the calcium-dependent fusion was observed in the absence of the calcium sensor protein, synaptotagmin [35], which might be induced by the substrate surface. Recently, several groups recovered SNAP-25 dependence of single–vesicle fusion assays in planar bilayers by directly incorporating PEG [poly(ethylene glycol)], a synthetic polymer to mimic surface effect, into the fusion system [38,39].

Single vesicle–vesicle fusion system utilizes specifically attached acceptor dye (DiD) labelled v-vesicles on the polymer passivated and functionalized imaging surface. After immobilization is complete, donor dye (DiI)-labelled t-vesicles are added and membrane fusion between t- and v-vesicles is measured using a TIR (total internal reflection) microscope. What is notable in this approach is, from calculated FRET efficiency values from single v- and t-vesicle complexes, docked (FRET<0.25) and fused (FRET>0.6) vesicle complexes are clearly distinguishable. Compared with the bulk fusion method, this technique can also distinguish intermediate states between docked and fully fused vesicles (e.g. hemifusion), quantify the kinetics of transitions between individual intermediate states and detect post-fusion pathways such as the kiss-and-run event. Such information may be obtained at a certain time point as well as from real-time fluorescence time traces from hundreds of vesicle–vesicle interactions in parallel. Through this assay, accessory proteins, including complexin [13], Munc18 [15] and membrane-anchored synaptotagmin 1 [16] have been shown to work with SNARE proteins to accelerate the lipid mixing process, but in unique manners.

Labelling content

The fundamental assumption of using lipid mixing to study membrane fusion is that there is a direct correlation between lipid and content mixing. However, through simultaneous detection of lipid and small content indicators of DNA-mediated membrane fusion, Boxer and co-workers revealed that efficient lipid mixing (>90%) of both outer and inner leaflets can occur without content mixing (<2%) [40]. The present study calls into question liposome ‘fusion’ assays that rely on lipid-mixing indicators alone to assess the fusion. The present study strongly suggests that future vesicle fusion experiments should employ content-mixing indicators in addition to lipid-mixing indicators.

Even prior to the wide spread of ensemble lipid-mixing assays, Rothman and co-workers utilized radioactive probe-labelled DNA to report that SNARE proteins constitute the minimal machinery for membrane fusion [41]. However, in this assay, the content mixing signal was measured after vesicles were lysed by detergent, leaving a possibility that docked vesicles without fusion could still contribute to the final readout. To observe the SNARE-mediated membrane fusion, fluorescent-dye-based small indicators have previously been substituted for lipid-dye through the ensemble approach. However, when a large number of transmembrane domain-containing SNARE proteins are reconstituted into vesicles, membranes are destabilized, causing the indicator molecules to leak out of the vesicle over time [42]. Unless a very low protein-to-lipid ratio (<1:1000) was used [21,43], this leakage problem makes the data analysis challenging.

Approaches involving smFRET have also been used to detect the content mixing. For single-vesicle fusion assays utilizing planar bilayers, small content indicators showed that SNAP-25 was not required in the acceptor t-SNARE complex for fusion, which is contradictory of known physiology and biochemistry [36,44].

A more promising result comes from a vesicle–vesicle content mixing assay [17]. In this assay, a large Cy3/Cy5 dual-labelled DNA probe was encapsulated inside a surface-immobilized v-vesicle. The soluble t-vesicles contain complementary DNA, and when two cavities of vesicles interconnect through fusion the two DNA strands may hybridize to induce a conformational change. Through smFRET detection of this conformational change, authors are able to detect the fusion pore expansion, a very late step of the membrane fusion process, for the first time.

OUTLOOK

The single-molecule FRET methodologies are still evolving outside of the SNARE applications. By using an increased number of fluorophores, 3-colour, 4-colour and other variations of FRET measurements are possible [4548]. This enables monitoring of multiple relative distances simultaneously. Although previously done, multiparameter measurements to include parameters such as anisotropy and fluorescence lifetime may benefit from observation of assembly of complexes and domain flexibility [10]. In order to increase the number of fluorophores, novel labelling schemes, such as incorporation of unnatural amino acids and short amino acid tags, to allow orthogonal site-specific labelling are necessary [49]. In order to achieve physiologically relevant micromolar concentrations of labelled protein, the use of nanofabricated device such as zero-mode waveguide or the tightly focused excitation beam of STED (stimulated emission depletion) microscopy may broaden the concentration range of the study [50,51].

Studying membrane fusion based on protein-reconstituted vesicles has achieved tremendous successes, but certainly has areas of further development. In order to understand how lipid-mixing steps are related to the pore expansion step, simultaneous lipid- and content-mixing detection assays are desirable. With such assays, the exact roles of multiple accessory proteins that all seemingly promote (or inhibit) lipid-mixing may be identified. The single-vesicle lipid- and content-mixing assays described above are blind to the conformations of SNARE protein. Understanding how SNARE proteins and its accessory proteins co-ordinate themselves during these observed fusion intermediate steps will not only deepen our understanding of the system, but also lead to the discovery of new drug targets. Because the size of common vesicles (50–100 nm in diameter) is smaller than the diffraction limit, direct optical observation is impossible. In order to overcome this issue, utilization of new super-resolution imaging techniques are demanded, such as STORM (stochastic optical reconstruction microscopy) [52], and may become an ultimate tool to study protein–protein interactions during the SNARE-mediated membrane fusion process.

Abbreviations

     
  • Cy3

    indocarbocyanine

  •  
  • Cy5

    indodicarbocyanine

  •  
  • FRET

    fluorescence resonance energy transfer

  •  
  • NBD

    7-nitrobenz-2-oxa-1,3-diazole

  •  
  • smFRET

    single-molecule FRET

  •  
  • SNAP-25

    25 kDa synaptosome-associated protein

  •  
  • SNARE

    soluble N-ethylmaleimide-sensitive factor-attachment protein receptor

  •  
  • t-SNARE

    target SNARE

We thank Dr Taekjip Ha, Dr Yeon-Kyun Shin and Dr Tae-Young Yoon for their continuous support, and Dr Zengliu Su for illustration preparation.

FUNDING

Our work on single-vesicle FRET assay in the present review was supported by National Institutes of Health [grant number R21 GM074526] and the Howard Hughes Medical Institute through Dr Taekjip Ha at the University of Illinois.

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Author notes

1

Present address: Howard Hughes Medical Institute and School of Medicine, Stanford University, Stanford, CA 94305, U.S.A.