Exocytosis is a highly conserved and essential process. Although numerous proteins are involved throughout the exocytotic process, the defining membrane fusion step appears to occur through a lipid-dominated mechanism. Here we review and integrate the current literature on protein and lipid roles in exocytosis, with emphasis on the multiple roles of cholesterol in exocytosis and membrane fusion, in an effort to promote a more molecular systems-level view of the as yet poorly understood process of Ca2+-triggered membrane mergers.

EXOCYTOSIS AND MEMBRANE MERGER

Regulated exocytosis

Exocytosis is a highly conserved process essential to the function of numerous cellular activities. At the neuronal synapse, the rapid, triggered release of neurotransmitter following a depolarizing action potential is perhaps one of the most widely studied exocytotic events. In this case, SV (synaptic vesicles) are trafficked to the active zone where they are docked, tethered and undergo final ATP-dependent priming reactions (Figure 1). These SV are now defined as release-ready and will undergo a regulated fusion reaction with the PM (plasma membrane) dependent on only an increase in intracellular [Ca2+]free to trigger release of neurotransmitter into the synaptic cleft; at any time there are a limited number of such readily releasable SV (e.g. ∼7–12) at an active zone [1,2]. Subsequent endocytosis and refilling of vesicles retrieved from the PM (via endosomal fusion or endosome-independent refilling) serves to replenish the pool of SV. As the entire exo- and endo-cytotic machinery is intact in healthy synapses this, in theory, allows for the study and identification of protein and lipid components integral to the mechanism of exocytosis; however, the cyclic nature of this pathway can confound the study of the Ca2+-triggered fusion steps as blockade of any step will result in the eventual inhibition of exocytosis and thus neurotransmitter release. To some extent, this has resulted in the tendency to equate the exocytotic process directly with the membrane fusion event.

Schematic diagram of exocytosis in the presynaptic terminal

Figure 1
Schematic diagram of exocytosis in the presynaptic terminal

Note that although shown as a strict sequence, the timing of the steps of the priming reaction may occur before, during or after the docking process. NT, neurotransmitter. Adapted from [253], with permission, from the Annual Review of Neuroscience, Volume 27 © 2004 by Annual Reviews (www.annualreviews.org).

Figure 1
Schematic diagram of exocytosis in the presynaptic terminal

Note that although shown as a strict sequence, the timing of the steps of the priming reaction may occur before, during or after the docking process. NT, neurotransmitter. Adapted from [253], with permission, from the Annual Review of Neuroscience, Volume 27 © 2004 by Annual Reviews (www.annualreviews.org).

Membrane fusion is strictly defined as the intermixing of apposed bilayer membranes (e.g. the vesicle membrane and the PM) to enable release of vesicular contents, or content mixing in the case of homotypic vesicle fusion as occurs in compound exocytosis. Although exocytosis includes the release of vesicle contents, it is a more general descriptor for the entire trafficking pathway, and includes both essential and regulatory steps upstream of membrane fusion (targeting, tethering, docking and priming). This interchangeable use of terms has led to the description of a number of exocytotic proteins as ‘fusion proteins’ without rigorous evidence of a direct role for the protein in native membrane fusion. Rather than a simple issue of semantics, the distinction between a fusion protein and an exocytotic protein is one of molecular mechanisms. Although a fusion protein would be expected to transduce some or all of the local energy in vivo to fully drive the membrane merger steps, contributions upstream of membrane merger that ensure the efficiency of the triggered fusion event – e.g. promoting close membrane apposition by helping to dissipate the hydration layer, ensuring stable docking and the effective binding of Ca2+ required for the characterized physiological response of the system – may not equate with a fusion protein. Although a range of different cell types exhibit physiologically different secretory events (e.g. differences in Ca2+ sensitivity and fusion pores of different sizes and stabilities), evidence suggests that this is accomplished with a single fundamentally conserved fusion mechanism that is co-ordinated by any number of accessory molecules to promote the efficiency of the fusion process, thus yielding the physiology characteristic of the cell type in question [3,4]. In this light, the entire ensemble of interacting molecules could be more globally considered to be a fusion machine: a multicomponent system composed of varied constituents, including those necessary and sufficient to enable fusion (i.e. membrane merger), as well as those serving modulatory roles critical to the physiology of the system.

Thus, whereas only specific components may be necessary and sufficient for membrane merger, based on purely (bio)physical considerations, many additional components contribute to the overall physiology of any given native membrane fusion event. In terms of the most basic components required to actually merge apposed native bilayer membranes (perhaps only cholesterol and specific lipids according to the stalk-pore hypothesis [58]), we consider this to be the FFM (fundamental fusion mechanism). Superimposed on this inherently inefficient mechanism [3] are those various components that provide for the regulation, sensitivity, triggering, speed and overall efficiency of the native fusion reaction – the characteristic physiology – we consider this to be the PFM (physiological fusion machine) [9,10].

Proteins in regulated exocytosis

Studies of exocytotic pathways in a range of secretory cell types have led to the clear identification of a number of proteins involved in exo- and endo-cytosis. Among the first identified and best studied exocytotic proteins are NSF (N-ethylmaleimide-sensitive factor), the soluble NSF-attachment protein, α/β SNAP, and the SNAP receptor, SNARE, family of proteins [1114]. The PM Q-SNAREs syntaxin 1 and SNAP-25 (synaptosome-associated protein of 25 kDa, no relation to α/β SNAP) as well as the vesicular R-SNARE synaptobrevin (or VAMP; vesicle-associated membrane protein) are targets of the clostridial toxins. A number of studies subsequently identified the SNAREs as fusion proteins, and they are sometimes described as the minimal machinery required for membrane fusion [1416]. Quantitative analyses have demonstrated that the SNARE protein cytosolic domains, although essential for efficient exocytosis, are not absolutely required for native Ca2+-triggered membrane fusion [3,1721], nor are these proteins capable of providing a minimal framework for the fusion of artificial membranes when reconstituted at native densities [22,23].

As such, although the importance of the SNARE proteins to eukaryotic exocytosis is undisputed, the specific role of the SNAREs as the membrane fusion proteins still remains highly controversial. Studies of the SNARE proteins in vitro have provided interesting information regarding their structural interactions, yet it is still unclear how or whether these results directly translate to the native membrane interactions or fusion. Early studies demonstrated that two discrete populations of proteo-liposomes containing Q- and R-SNAREs were able to undergo spontaneous lipid mixing when the populations were combined [15,16]. This was heralded as evidence that the assembly of the trans-SNARE complex was sufficient and necessary to drive the full fusion of two lipid membranes. Subsequent studies identified three unaccounted for experimental variables which may have influenced the results (summarized in Table 1). First, the comicellization method used to generate SNARE proteo-liposomes resulted in a heterogeneous distribution of vesicle sizes, which led to a comparably heterogeneous distribution (and thus density) of proteins within the vesicle population [22]. Second, the authors measured only lipid mixing as an indicator of vesicle fusion; as content mixing was not assessed, full vesicle fusion events could not be distinguished from transient localized disruption and re-annealing of contacting membranes. Indeed, the SNAREs (with synaptotagmin and Ca2+) have since been shown to promote hemifusion over fusion pore formation in these in vitro assays [24]. Recent studies, with protein incorporated directly into pre-formed liposomes, did not demonstrate significant content mixing in SNARE proteoliposomes at native protein densities, even in the presence of significant lipid mixing [23], in contrast with an original report of content mixing in similar SNARE proteoliposomes reconstituted using the comicellization approach [25]. Finally, the authors observed lipid mixing between the two populations of SNARE proteoliposomes at protein/lipid ratios that vastly exceed the native densities of the Q- and R-SNAREs. Recent reports indicate that the native density of VAMP in rat brain SV is approx. 180:1 (lipid/protein, mol/mol): this is equivalent to ∼3200 copies of VAMP/μm2 of membrane [26], which contrasts with an earlier estimate of ∼2800 copies/μm2 [14]. The density of VAMP on sea urchin CV (cortical vesicles), another well-studied, fast, Ca2+-triggered fusion system, is somewhat lower at ∼1700 copies/μm2 [3,19]. The initial studies of SNARE-induced lipid mixing in vitro used VAMP at an ∼20:1 lipid/protein ratio, equivalent to ∼29000 copies/μm2 of membrane (assuming uniform distribution and liposome size), almost 10- and 20-fold higher than reported for native SV and CV VAMP densities respectively [3,15,16,26]. In contrast, the sum total of the 15 most abundant SV membrane proteins only equates to a 72:1 lipid/protein ratio (mol/mol; 1:1.94 lipid/protein, w/w), ∼8100 proteins/μm2 [26]. Sea urchin CV have a comparable lipid/protein ratio of 1:2.44 (w/w) [27]. Thus in the original SNARE reconstitution experiments VAMP was present at ∼3.5-fold higher densities than the total protein composition of native SV. When incorporated into pre-formed liposomes at approximately native densities (160–185:1), these SNARE proteoliposomes were unable to undergo efficient lipid-mixing, and content mixing (when assessed) was negligible [22,23]. Furthermore, as the comicellization method originally used to generate SNARE-loaded proteoliposomes results in a widely heterogeneous population in terms of both liposome size and protein density, it is probable that the subpopulation of liposomes involved in the lipid mixing events detected had much higher protein densities than originally estimated [22,28]. As high SNARE protein densities can cause increased leakage of trapped vesicle contents [23], SNARE interactions between such proteoliposomes probably caused catastrophic local dehydration and membrane lysis, similar in effect to the interaction of Ca2+ with anionic lipids [29,30]. Additionally, SNARE overexpression in native vacuoles has also been shown to result in extensive lysis [31]. To our knowledge, there is no known biological membrane that equates to SNAREs reconstituted to densities equivalent to or greater than the total native protein density. Thus, although in vivo results are consistent with a role for the SNARE proteins in the process of exocytosis, there is insufficient in vitro evidence to support a direct role in the membrane merger steps of native fusion. Although it has been clearly demonstrated that the targets of the clostridial toxins (i.e. the SNARE proteins) are linked to the probability of fusion events, including the Ca2+ sensitivity of release [3,4,1720,32], they do not appear to be essential to the fusion process itself, but rather enable critical inter-membrane attachment and perhaps closer apposition. Thus there is likely some validity to the notion that SNAREs ‘catalyse’ fusion since, similar to a true catalyst, they are present in small amounts relative to the reactants (i.e. lipids) and they are neither consumed nor are they a part of the reaction (i.e. the FFM) itself.

Table 1
Summary of experimental systems utilized in some SNARE proteoliposome studies
Group Citation Method (co/dir)a Density (lipid/protein)b Findings Lipid/content mix 
Rothman and co-workers [15co 11–18:1 First demonstration of SNARE-induced lipid mixing Lipid 
 [16co 11–18:1 Rapid lipid mixing Lipid 
 [25co 11–18:1 Content mixing assessed with oligonucleotides Content 
 [246co 22:1 Specificity of yeast SNAREs in lipid mixing Lipid 
 [247co 11–18:1 Zippering of SNAREs Lipid 
 [248co 22:1 Golgi inhibitory SNAREs Lipid 
 [249co 100:1 Mixing inhibited by positive-curvature lipids SNARE assemblyc 
Söllner and co-workers [59co 75–100:1 Ca2+-independent lipid mixing with synaptotagmin Lipid 
Davletov and co-workers [64co 25–35:1 Ca2+-dependent lipid mixing with SV Lipid 
Chapman and co-workers [58co 17–300:1 Ca2+-dependent lipid mixing with synaptotagmin Lipid 
 [115co 27–270:1 Synaptotagmin specificity for neuronal SNAREs Lipid 
 [116co 280–450:1 Direct effect of synaptotagmin in mixing Lipid 
Hirashima and co-workers [250co 600:1 Mast cell SNAREs Lipid 
Zerial and co-workers [251co 10000–2000:1 Reconstituted endosome fusion with Rabs and cytosolic factors Content 
Rizo and co-workers [22co 20:1 Slow mixing Lipidd 
  co 160:1 Little mixing Lipidd 
  dir 300:1 No detectable mixing Lipidd 
Lentz and co-workers [23dir 420–950:1 SNAREs affect only PEG-mediated fusion Bothd 
Jena and co-workers [56dir N.R.f Ca2+-dependent liposome fusion without synaptotagmin Lipid 
Jahn and co-workers [28dir 100:1 Mixing inhibited by botulinum toxin Lipid 
 [54dir 300:1 Ca2+-dependent mixing with full-length synaptotagmin Lipid 
 [55dir 300:1 SV ‘fusion’ with liposomes, Ca2+-independent Lipid 
Shin and co-workers [63dir 150:1 Kinetics of SNARE assembly and lipid mixing Lipid 
 [252dir 50:1 Liposome fusion transits through hemifusion Lipidd,e 
 [61dir 100:1 Monitor single fusion events Lipide 
 [24dir 200:1 Specificity of synaptotagmin for neuronal SNAREs Lipide 
 [50dir 100–200:1 Synaptotagmin/Ca2+ relieve complexin block to mixing Lipid e 
 [62dir 100:1 Yeast SNAREs Lipid 
 [49dir 50–400:1 SNARE assembly precedes hemifusion Lipid e 
 [51dir 200:1 Complexin and Ca2+ stimulate fusion without synaptotagmin Lipid 
 [52dir 200:1 Cholesterol stimulates hemifusion Lipid e 
 [53dir 200:1 Cholesterol effects on SNAREs in lipid mixing Lipid e 
Weisshaar and co-workers [57dir 240:1 Fast mixing with planar bilayers - SNAP25, Ca2+-independent Lipid 
Group Citation Method (co/dir)a Density (lipid/protein)b Findings Lipid/content mix 
Rothman and co-workers [15co 11–18:1 First demonstration of SNARE-induced lipid mixing Lipid 
 [16co 11–18:1 Rapid lipid mixing Lipid 
 [25co 11–18:1 Content mixing assessed with oligonucleotides Content 
 [246co 22:1 Specificity of yeast SNAREs in lipid mixing Lipid 
 [247co 11–18:1 Zippering of SNAREs Lipid 
 [248co 22:1 Golgi inhibitory SNAREs Lipid 
 [249co 100:1 Mixing inhibited by positive-curvature lipids SNARE assemblyc 
Söllner and co-workers [59co 75–100:1 Ca2+-independent lipid mixing with synaptotagmin Lipid 
Davletov and co-workers [64co 25–35:1 Ca2+-dependent lipid mixing with SV Lipid 
Chapman and co-workers [58co 17–300:1 Ca2+-dependent lipid mixing with synaptotagmin Lipid 
 [115co 27–270:1 Synaptotagmin specificity for neuronal SNAREs Lipid 
 [116co 280–450:1 Direct effect of synaptotagmin in mixing Lipid 
Hirashima and co-workers [250co 600:1 Mast cell SNAREs Lipid 
Zerial and co-workers [251co 10000–2000:1 Reconstituted endosome fusion with Rabs and cytosolic factors Content 
Rizo and co-workers [22co 20:1 Slow mixing Lipidd 
  co 160:1 Little mixing Lipidd 
  dir 300:1 No detectable mixing Lipidd 
Lentz and co-workers [23dir 420–950:1 SNAREs affect only PEG-mediated fusion Bothd 
Jena and co-workers [56dir N.R.f Ca2+-dependent liposome fusion without synaptotagmin Lipid 
Jahn and co-workers [28dir 100:1 Mixing inhibited by botulinum toxin Lipid 
 [54dir 300:1 Ca2+-dependent mixing with full-length synaptotagmin Lipid 
 [55dir 300:1 SV ‘fusion’ with liposomes, Ca2+-independent Lipid 
Shin and co-workers [63dir 150:1 Kinetics of SNARE assembly and lipid mixing Lipid 
 [252dir 50:1 Liposome fusion transits through hemifusion Lipidd,e 
 [61dir 100:1 Monitor single fusion events Lipide 
 [24dir 200:1 Specificity of synaptotagmin for neuronal SNAREs Lipide 
 [50dir 100–200:1 Synaptotagmin/Ca2+ relieve complexin block to mixing Lipid e 
 [62dir 100:1 Yeast SNAREs Lipid 
 [49dir 50–400:1 SNARE assembly precedes hemifusion Lipid e 
 [51dir 200:1 Complexin and Ca2+ stimulate fusion without synaptotagmin Lipid 
 [52dir 200:1 Cholesterol stimulates hemifusion Lipid e 
 [53dir 200:1 Cholesterol effects on SNAREs in lipid mixing Lipid e 
Weisshaar and co-workers [57dir 240:1 Fast mixing with planar bilayers - SNAP25, Ca2+-independent Lipid 
a

co, reconstitution of protein during liposome formation by comicellization method, detergent dilution and dialysis; dir, direct reconstitution of protein into pre-formed ∼100 nm liposomes using detergents followed by dialysis.

b

Molar lipid/protein ratio – native ratio estimated to be 180:1 [26].

c

‘Lipid mixing’ was assessed based on FRET between fluorescent protein-labelled Q- and R-SNAREs upon SNARE complex formation.

d

Studies in which vesicle size was assessed.

e

Lipid mixing was assessed separately for both inner and outer leaflets - outer leaflet mixing occurs after hemifusion whereas inner leaflet mixing is more suggestive of fusion

f

Not reported.

Pure lipid vesicles can undergo fusion in the absence of proteins [33] and can exhibit properties that closely resemble those of native membrane fusion, including Ca2+ triggering [34,35]. Protein-free liposomes are capable of interacting with [36,37] and fusing to biological membranes [3840]. Furthermore, liposome fusion can be facilitated or promoted by a number of factors, including lipid composition [33,35], reagents that induce aggregation or dehydration {such as PEG [poly(ethylene glycol)] and DMSO} [33,4143], or specific proteins [33,4448]. Myelin basic protein can bring protein-free liposomes into close apposition [44] and induces measurable lipid mixing on timescales comparable with that seen with SNARE proteoliposomes (t½=0.5–5 min) [33,45,46]. Similarly, serum albumin has been observed to induce fusion of protein-free liposomes under specific conditions [47]. Perhaps most notably, biotin/avidin/antibody tethering of liposomes to cultured cells resulted in dramatically enhanced PEG-mediated membrane fusion, as assessed by liposome content delivery [48]. As none of the aforementioned proteins are found on SV (at least not at detectable levels [26]), it is safe to conclude that none are involved in neuronal exocytosis, nor would they necessarily be expected to participate in any exocytotic process. Such results complicate any interpretation of SNARE-mediated proteo-liposome mixing assays. Rather than a definitive demonstration of a minimal FFM, the SNARE proteins may be acting to tether two membranes into the close apposition required to increase the probability of a spontaneous and purely lipidic fusion event. This interpretation is also consistent with the observation that SNAREs promote PEG-mediated fusion [23]. This would clearly explain the dramatic differences in the speed of proteoliposome ‘fusion’ as compared with fast SV fusion or other release events (e.g. neuroendocrine, CV). Further complicating matters is the wide variability of assay results for SNARE proteoliposome fusion from different groups. Most notably, some groups have reported that the SNARE proteins, incorporated into proteo-liposomes at approximately native densities, were unable to drive or even promote lipid mixing [22,23], whereas subsequent studies at comparable protein densities showed efficient lipid mixing [24,4955] (summarized in Table 1). Further discrepancies include reports of Ca2+-dependent [56] and -independent [57] lipid mixing of proteoliposomes containing only the Q- and R-SNAREs, as well as Ca2+-dependent [54,58] and -independent [59] stimulation of lipid mixing by synaptotagmin. When complexin is included in the proteoliposome assays, it has been reported that synaptotagmin promoted the ‘unclamping’ of complexin in a Ca2+-dependent manner [50], consistent with the proposed role of synaptotagmin as a Ca2+ sensor in exocytosis; however, further studies from the same group found that Ca2+ also promoted lipid mixing of complexin-treated SNARE proteoliposomes even in the absence of synaptotagmin [51]. Efficient lipid mixing of SNARE proteoliposomes has even been reported in the absence of SNAP-25 [57], despite numerous reports to the contrary [24,4953,6063]. In the presence of PEG, VAMP proteoliposomes underwent homotypic (VAMP→VAMP) lipid mixing with efficiency very close to that of the fully assembled SNARE complex (VAMP→syntaxin/SNAP-25) [23]. Furthermore, in an assay of SV fusion to Q-SNARE proteoliposomes, two separate groups have reported conflicting observations concerning Ca2+ sensitivity: SV fusion in vitro has been reported to be both Ca2+-sensitive [64] and Ca2+-insensitive [55]. As a whole, these observations minimally suggest caution when interpreting the results of any such reconstituted ‘fusion’ assay, and indicate that better communication and standardized protocols across labs will be necessary in order to achieve the consistency and uniformity expected of a reproducible and quantitative assay format.

The SNARE proteins are proposed to play a role in exocytosis (i.e. the PFM) by forming a heterotrimeric trans-SNARE complex between the vesicle-bound R-SNARE VAMP and the PM-associated Q-SNAREs syntaxin and SNAP-25. This complex is defined by a coiled-coil of four α-helices (one each from VAMP and syntaxin, and two from SNAP-25) which serve to draw the two lipid bilayers into close proximity, to better enable the subsequent steps required for actual membrane merger (i.e. the FFM) (Figure 2). After membrane merger, all components of the intact SNARE complex are retained within one bilayer and this is referred to as the cis-SNARE complex. The SNARE proteins are themselves quite promiscuous, and this property has been used to identify a number of proteins that interact with the SNAREs, both in complexes or as isolated components [6568]. Downstream of the fusion steps, the cis-SNARE complex binds the accessory protein α/β SNAP and the ATPase NSF. The ATPase activity of NSF drives the dissolution of the cis-SNARE complex, to enable recycling of the individual protein components [69]. Complexins are similarly capable of binding to the assembled SNARE complex in a manner that competes with α/β SNAP [70,71]; however, their exact role in the PFM remains to be debated. In vivo evidence suggests that the complexins are responsible for binding to the assembled SNARE complex only in the context of regulated (i.e. Ca2+-dependent) exocytosis. Surprisingly, in many systems, both increasing and decreasing levels of cellular complexin cause an inhibitory effect on regulated exocytosis (reviewed in [72]). It has been suggested that this binding to the assembled SNARE complex acts as a fusion clamp that stabilizes the assembled SNARE complex to prevent fusion in advance of a Ca2+ stimulus [50,60,73]; other results suggest that complexin plays a critical role as a positive regulator of vesicle priming [74]. Upstream of membrane fusion, the SM (Sec1/Munc18) proteins bind to syntaxin1, and this is proposed to limit or modulate the formation of cis- or trans-SNARE complexes [75], although it has also been suggested that SM proteins may play roles in docking and priming [7678] or directly in fusion [7981], possibly modulating fusion pore expansion [82].

Lipidic fusion according to the stalk-pore hypothesis

Figure 2
Lipidic fusion according to the stalk-pore hypothesis

This Figure is adapted from [254] and is reprinted with permission from Nature Structural Biology © 2008 Macmillan Magazines Ltd. (http://www.nature.com/).

Figure 2
Lipidic fusion according to the stalk-pore hypothesis

This Figure is adapted from [254] and is reprinted with permission from Nature Structural Biology © 2008 Macmillan Magazines Ltd. (http://www.nature.com/).

An alternative to the stalk-pore model is the hypothesis that the initial fusion pore itself is proteinaceous or ‘channel-like’ in nature [8385]. Multiple proteins have been proposed to comprise such an initial fusion pore. Perhaps the best described pore-forming protein in this regard is the V0 subunit of the V-ATPase [8688]. Yeast vacuoles undergo a conserved, constitutive fusion reaction that appears to be Ca2+-regulated [8991]. As with the basic exocytotic pathway in other eukaryotes, yeast vacuolar contact, priming, docking and fusion utilizes specific proteins, including NSF (Sec18p), α-SNAP (Sec17p), the R-SNARE VAMP (Snc1p, Snc2p), and the Q-SNAREs SNAP-25 (Sec9p) and syntaxin (Sso1p, Sso2p) [9295]. The membrane integral subunit, V0, is capable of forming a stable inter-membrane dodecameric complex (i.e. two rings of six subunits each in two adjacent membranes), which would have an ion-channel-like pore functionality [86]. A role for at least one component of this model, actin, has been tested in fast, Ca2+-triggered membrane fusion and was found not to be essential to the FFM [96]. Substantiation of a central role for V0 has not been forthcoming in other secretory systems, suggesting that this may be a constitutive release pathway utilized largely by yeast. Other studies have implicated the transmembrane helices of syntaxin and VAMP as the pore-forming proteins in neuroendocrine cells [97], although there has been some discussion of this model [98]. In both cases the proteinaceous structure is proposed to form the initial aqueous fusion pore after triggering of fusion with Ca2+, whereas expansion of the structure by the invasion of lipids is thought to enable full membrane merger. A critical factor of any proteinaceous fusion pore model is that it requires specific sets of proteins in both fusing membranes, yet several secretory vesicle types have been shown to require proteins in only one fusing membrane [40,99103]. Ultimately, substantiation of any such protein pore model will require both identification of critical proteins and the in vitro reconstitution of fast, Ca2+-triggered membrane fusion.

Two proteins remain prominent contenders for roles as Ca2+ sensors during exocytosis: synaptotagmin is a vesicle-associated SNARE-binding protein with two Ca2+-binding C2 domains [104,105] and a defined Ca2+-dependent phospholipid-binding activity, whereas annexin-2 has a similar Ca2+-dependent binding of anionic phospholipids that is capable of inducing structural changes in membranes [106]. In part, due to its close association with the known exocytotic SNARE proteins [107,108], synaptotagmin has been better studied for a possible direct role in the Ca2+-triggered steps of membrane fusion. Binding of Ca2+ to the C2B domain of synaptotagmin co-ordinates binding of phospholipids in two adjacent bilayers [109] and is capable of bringing membranes into close proximity and inducing membrane deformations [60,109114] that may be consistent with the early steps of lipidic membrane fusion. Synaptotagmin strongly associates with the SNARE proteins and promotes SNARE-mediated lipid mixing in vitro in a Ca2+-dependent manner [24,54,114116], although as noted above there is at least one study that does not support this [59]. At approximately native lipid/protein ratios (1300:1 lipid/protein compared with 850:1 for SV) the C2 domains of synaptotagmin are proposed to insert short hydrophobic loops into the membrane in the presence of Ca2+ [114,117]. This insertion is thought to promote the formation of positive curvature structures in vitro, possibly providing some early membrane stress required to initiate the FFM via formation of initial lipidic fusion intermediates [113,114], and to subsequently promote fusion pore expansion [113]. Ongoing approaches promise an unbiased alternate route to independently identify critical Ca2+-sensors as well as other protein components of the PFM [118].

Underlying every proposed protein role in membrane fusion is the fundamental problem of merging two predominantly lipid bilayers. The lipidic process of membrane merger is itself best described by the stalk-pore model, which details the structural and energetic requirements of membrane fusion at the level of two lipid bilayers.

The stalk-pore model of membrane fusion

Based on an extensive body of work in viral and lipid model systems, the stalk-pore hypothesis is a mathematical and physical description of the merger of two pure lipid bilayers that has been vetted by extensive computational analysis [58,119124]. The model describes a series of transient intermediate structures that two fusing bilayers must progress through in order to facilitate content mixing (Figure 2). Each intermediate structure involves the formation of highly curved membrane structures, which present an energy barrier to the fusion process. Initial membrane deformation, in the form of a point-like protrusion [125], disrupts the hydration layer [126,127] and allows initial contact between the hydrophobic bilayer regions; such membrane dimpling has been observed in fusion-ready native membranes [128]. This initial contact enables lipid mixing and results in the fusion of the two contacting monolayers to form the initial stalk structure (Figure 2). The stalk is defined by the high curvature of the merged proximal monolayers. Expansion of the stalk into a hemifusion diaphragm brings the two distal monolayers into close proximity, enabling these to fuse, forming the initial fusion pore. Much as the stalk is defined by the curvature of the contacting monolayers, the fusion pore is defined by the curvature of the distal monolayers [122]. Molecular dynamics simulations are consistent with such a fusion pathway (i.e. FFM), as are studies of membrane merger in different artificial lipid assemblies. Notably, a FFM based on the stalk-pore pathway does not obviate more direct protein/proteolipid involvement [129], although how protein transmembrane domains might also contribute to this process requires substantial further investigation [97,130,131].

Individual lipid species can contribute curvature to the lipid monolayers, and the spontaneous (or intrinsic) curvature of a given lipid species is a measure of its tendency to form non-planar (or non-bilayer) structures in hydrated assemblies. By convention, the tendency of a lipid to form a convex micelle-like structure is defined as positive curvature (Figure 3A), whereas the tendency to form concave structures is defined as negative curvature (Figure 3C). At the simplest level, the relative curvature contribution can be related to the average effective molecular conformation (i.e. shape) of a lipid. Positive curvature lipids (such as lysophospholipids) have a hydrophilic headgroup that is large relative to the hydrophobic acyl chain, whereas negative curvature lipids (such as diacylglycerols) have small polar heads relative to the non-polar acyl chains [132134]. Lipids with little or no net curvature (such as diacylphosphatidylcholine) have hydrophilic and hydrophobic regions that are of approximately equal proportions (Figure 3B).

Membrane curvature and domain structure

Figure 3
Membrane curvature and domain structure

(A) Positive curvature lipids form convex micelle-like structures. (B) Neutral curvature lipids form planar structures. (C) Negative curvature lipids form concave structures. (D) Cholesterol-enriched lo microdomains (green) are characterized by a thickening of the membrane (relative to ld, blue) and highly ordered packing of acyl chains. (D) Adapted from [244] with permission © 2005 Elsevier Ltd.

Figure 3
Membrane curvature and domain structure

(A) Positive curvature lipids form convex micelle-like structures. (B) Neutral curvature lipids form planar structures. (C) Negative curvature lipids form concave structures. (D) Cholesterol-enriched lo microdomains (green) are characterized by a thickening of the membrane (relative to ld, blue) and highly ordered packing of acyl chains. (D) Adapted from [244] with permission © 2005 Elsevier Ltd.

The highly curved intermediates of the stalk-pore model are defined energetically by their net curvature. Formation of the stalk, having a net negative curvature, is supported by lipids having negative spontaneous curvature. The transition from hemifusion diaphragm to fusion pore is supported by lipids of positive spontaneous curvature in the distal monolayers [122,135,136]. The addition of negative curvature lipids in the contacting monolayers lowers the energy barrier to the formation of the stalk, whereas adding positive curvature lipids to the contacting monolayers arrests the fusion process [137139]. The formation of a fusion pore can be likewise manipulated by the select addition of positive and negative curvature lipids to the distal monolayers [135,140], including the products of snake phospholipase A2 neurotoxins [141,142]. Thus, although the PFM may be predominantly proteinaceous in nature, considering as well that pure lipid membranes can also support flickering fusion pores [143], it is clear that the FFM could itself be largely or purely lipidic (see also [144]).

CHOLESTEROL

Cholesterol in model membranes

Cholesterol is a molecule of particular interest in the field of membrane fusion; as a naturally occurring lipidic component of established negative curvature, it is capable of locally contributing directly to the FFM in order to facilitate efficient membrane merger [145,146]. Cholesterol is enriched in a wide range of secretory vesicles, and comprises up to ∼40% (mol/mol) of the total lipid in SV, CV, and other secretory vesicles [26,27,139,147152]. These observations, along with the tendency of cholesterol to promote formation of negative curvature structures in vitro, have led to widespread interest into the role of cholesterol in membrane fusion [145,146].

Various models have been used to study the in vitro fusion of pure lipid vesicles, synthetic bilayers or the lipidic fusion of biological membranes. The PEG-mediated fusion of lipid vesicles has been used extensively to study the lipid dependence of the fusion process. In this model system, cholesterol, although not strictly required, dramatically increases the efficiency of fusion [41,43]. Cholesterol, as well as diacylphosphatidylethanolamine and diacylglycerol, dramatically enhanced the PEG-mediated fusion of liposomes in a manner that is consistent with a direct contribution of negative curvature to the lipidic fusion process [41]. An optimally fusogenic membrane composition for PEG-mediated fusion contained ∼20 mole % cholesterol, closely mimicking that of native SV and CV [27,43]. In the absence of PEG, cholesterol enhanced the fusion of phosphatidylcholine vesicles [153] and was required for the aggregation and fusion of vesicles induced by myelin basic protein [46]. In model systems measuring the fusion of biological membranes, cholesterol was required for the fusion of small unilamellar vesicles with Mycoplasma capricolum [154], and enhanced both the homotypic fusion of hen erythrocytes and the fusion of the Sendai virus with erythrocytes [155]. Similarly, in studies of Ca2+-triggered CV–liposome fusion, cholesterol was a necessary component of the target membrane [40]. Cholesterol was similarly required for the fusion of Sendai virus particles with pure lipid vesicles [156,157].

Cholesterol in viral fusion

Cholesterol was required in the target membrane for the entry of a number of encapsulated viruses, such as SFV (Semliki Forest virus) [158161], as were sphingolipids; however, virus entry did not depend on intact cholesterol- and sphingomyelin-enriched microdomains, suggesting a direct role of cholesterol and sphingolipids in the fusion process [162,163]. The influenza virus does not require cholesterol in the target membrane [164]; however, depletion of envelope-associated cholesterol with mβcd (methyl-β-cyclodextrin) [165], but not filipin [166], was observed to inhibit viral infectivity. In studies of haemagglutinin-mediated cell fusion, both haemagglutinin-associated [163] and target membrane cholesterol [167] were found to promote fusion, with specific effects on pore formation. Many other encapsulated viruses have been found to depend on cholesterol-enriched microdomains to organize the site of virus entry on the target membrane, including the murine leukaemia virus, the human T-cell leukaemia virus and HIV-1. Fusion of the HIV-1 virion required cholesterol-enriched microdomains on the target membrane, where they organize the target proteins CD4 and chemokine receptors [168170]. Disruption of cholesterol-enriched microdomains with the reagent mβcd resulted in inhibition of membrane fusion and blockade of HIV-1 infectivity, and was recovered by restoration of cholesterol to native levels. HIV-1 particles are highly enriched in cholesterol, and the virion membrane-associated cholesterol was also required for fusion, internalization and infectivity of HIV-1 [171,172]; however, virion-associated cholesterol was not required for membrane binding, which suggests a direct role for cholesterol in the fusion of HIV-1 to target membranes. Virion-associated cholesterol and sphingomyelin were similarly required for the maturation and infectivity of the hepatitis C virus [173,174]. A role for sphingomyelin in the efficiency of secretory vesicle fusion has also been established [175].

In addition to its role as a structural membrane component, a number of proteins have specific binding sites for free (annular) cholesterol. In the best studied example, cholesterol binding is necessary for the ion-gating activity of the AChR (acetylcholine receptor), and numerous other proteins have since been demonstrated to bind cholesterol (for reviews, see [176178]).

Cholesterol as a membrane organizer

One remarkable role of cholesterol in biological membranes is its ability to spontaneously self-organize into discrete domains within the plane of the bilayer. At a critical concentration in model membranes, cholesterol self-organizes into lo (liquid ordered) microdomains within the ld (liquid disordered) phospholipid membrane. The cholesterol-rich lo phase is characterized by the specific inclusion of long-chain saturated phospho- and sphingo-lipids; the close association of cholesterol and saturated lipids yields a highly ordered packing of lipid acyl chains and a distinct thickening of the membrane similar to the ordered gel phase in a pure phospholipid bilayer (Figure 3D; reviewed extensively in [179182]); however, unlike the gel phase, the cholesterol-rich lo domain has a high rate of lateral diffusion, comparable with the ld phase [183,184]. In addition to the spontaneous segregation of lipids, cholesterol-enriched microdomains provide organization of proteins within the plane of model bilayers: both lipid-anchored proteins, most notably GPI (glycophosphatidylinositol)-anchored proteins [185], and transmembrane proteins (reviewed in [178]) can associate with cholesterol-enriched microdomains. Discrete microdomains can be readily visualized in model bilayers using various techniques of fluorescence microscopy (for a review, see [186]).

The existence of cholesterol-enriched microdomains in biological membranes remains somewhat contentious, largely due to the biochemical techniques that have been used to classically define such domains. The earliest biochemical evidence of cholesterol-enriched microdomains came from observations that cold Triton X-100 extraction of membranes yielded a low-density insoluble fraction that was enriched in cholesterol, sphingomyelin, glycolipids and specific proteins [187]. These DRMs (detergent-resistant microdomains) are sensitive to cholesterol-depleting reagents such as mβcd [188], and can be reconstituted into model lipid membranes [189]. Some controversy arose when it was observed that distinct DRMs could be isolated using different non-ionic detergents, such as those of the Lubrol or Brij families [190193]. These findings did, however, demonstrate that, unlike earlier studies using Triton X-100, the isolation of DRMs was possible at physiological temperatures (up to 37 °C, [194]). Recent observations that cold Triton X-100 induced the aggregation of cholesterol-enriched PM regions [195] suggests that DRMs do not in fact represent physiologically intact microdomains, but rather could be aggregates of cholesterol-enriched microdomains of different sizes and compositions.

Additional biochemical techniques, notably the manipulation of membrane cholesterol content, have been used to dissect the functional importance of cholesterol-enriched microdomains in native membranes. Traditional cholesterol modulating agents such as mβcd and the statin class of drugs are capable of inducing both cholesterol-dependent and -independent effects in vivo. In addition to the high affinity for reducing membrane cholesterol levels, at acute doses mβcd causes a significant disruption of glycerophospholipids and sphingolipids, and can interact with proteins (reviewed extensively in [196]). Statin drugs, in addition to potent inhibition of HMG CoA (3-hydroxy-3-methyl-glutaryl-CoA) reductase, have been observed to regulate the NOS (nitric oxide synthase) pathway in human aortic endothelial cells, leading to downstream effects including the modulation of exocytosis [197]. Longer term use also affects isoprenoids, and thus the prenylation of different proteins, notably small GTP-binding proteins (reviewed in [198]). Any studies looking to rigorously examine specific molecular roles of cholesterol must integrate a number of quantitative approaches in order to account for secondary effects of any such reagents.

Within model membranes, the cholesterol-enriched lo phase can be readily visualized using fluorescent lipids or selective binding of the CTB (cholera toxin B) to GM1 ganglioside, a classic marker for such microdomains (reviewed extensively in [181,182,186]). In biological membranes, cholesterol- and sphingomyelin-enriched microdomains are far more difficult to detect. Although some groups have reported direct visualization of cholesterol-enriched microdomains in living cells [199,200], the majority of evidence supporting the existence of biologically relevant cholesterol-enriched microdomains comes from alternate methods: FRET (fluorescence resonance energy transfer), SPT (single-particle tracking) and other advanced microscopy techniques provide evidence in favour of the cholesterol-enriched microdomain hypothesis. Studies using video-enhanced microscopy to track the motion of antibody-coated colloidal gold particles have measured the lateral mobility of putative cholesterol-enriched microdomain components in native membranes [201203]. These initial observations demonstrated that the lateral mobility of GPI-anchored proteins in the PM was restricted to TCZs (transient confinement zones), which are temporally and/or spatially discrete subdomains of the plasma membrane. Protein and lipid components isolated with DRMs showed high retention times within TCZs, whereas non-DRM components typically had very low retention times. Similarly, retention within TCZs was both glycosphingolipid- and cholesterol-dependent, consistent with model and DRM analyses [201204]. Further studies using FRET in native membranes showed that GPI-anchored proteins were found in cholesterol-dependent submicron domains [205] and were associated with specific lipids, such as GM1 ganglioside [206]. Domains occupied by GPI-anchored proteins specifically excluded PM proteins such as the transferrin receptor [206]. Chemical cross-linking studies demonstrated that GPI-anchored proteins were clustered in cholesterol-dependent domains within the PM, whereas detergent treatments, as for DRM isolation, caused an increase in the apparent size of GPI-anchored protein domains [207]. Similar results were observed when GPI-anchored proteins were cross-linked using antibodies [208]. Recent studies using fluorescence photoactivation localization microscopy to image protein dynamics at subdiffraction resolution (i.e. 40 nm) suggest that, although correct, our perception of membrane domains remains perhaps oversimplified [209].

Although it remains unclear if detergent-based isolation methods for cholesterol-enriched microdomains yield accurate protein maps of a single type of domain, it is clear that cholesterol plays a role in providing lateral organization in the plane of the membrane in a manner consistent with the cholesterol- and sphingomyelin-enriched microdomains described in model membranes. It is more likely that DRM isolation methods are generating a conglomerate of cholesterol-enriched microdomains that would otherwise be spatially and/or temporally discrete, and may in fact contain distinct lipid and protein components. Thus, as long as the limitations of the technique are taken into account, isolation of DRMs remains a valuable tool for the analysis of cholesterol-dependent protein organization.

CO-ORDINATION OF CHOLESTEROL AND PROTEINS IN EXOCYTOSIS

Cholesterol-enriched microdomains

Owing to the versatility of cholesterol in biological processes, there are three mechanisms by which cholesterol is capable of contributing to the process of native membrane fusion. First, as a component of cholesterol- and sphingomyelin-enriched microdomains, cholesterol can organize essential protein and lipid components at the fusion site. Second, as an abundant membrane component, cholesterol can contribute to the fusion process by modulating the physical properties of the membrane, such as fluidity and/or curvature [210]. Finally, as a functional ligand or cofactor, cholesterol can directly modulate the activity of proteins essential to the fusion process.

A number of proteins linked with or directly involved in exocytosis have been reported to associate with cholesterol-enriched DRMs The SNARE proteins have been well characterized in terms of their association with cholesterol-enriched microdomains in a range of cell types. The Q-SNARE proteins syntaxin and SNAP25 have been isolated in cholesterol-enriched DRMs from diverse secretory cell types, including PC12 cells [211,212], mast cells [75], pancreatic β- [213] and α-cells [214], endothelial cells [215] and rat brain synaptosomes [216]. Synaptotagmin was also found to associate with DRMs in rat brain synaptosomes [216] and rat brain synaptic vesicles [217]. Voltage-gated Ca2+ channels have been isolated in DRMs from pancreatic α- and β-cells [213,214,218], mouse cerebellum [219] and rat cerebrum [220]. The SNARE-regulatory SM protein Munc18/n-Sec1 is specifically excluded from cholesterol-rich microdomains as either a free protein or in complex with syntaxin1; indeed, the exclusion of SM from cholesterol-enriched microdomains may provide spatial control over syntaxin1 recycling and activity [75,211,213]. Taken together, the association of exocytotic proteins with and by cholesterol- and sphingomyelin-enriched microdomains strongly suggests that the site of membrane fusion is defined and organized by cholesterol. Global treatments of neurons with mβcd have linked cholesterol to aspects of effective neurotransmission, including possible effects on evoked release [221,222]. In a direct assay of native Ca2+-triggered CV fusion, cholesterol is required to enable fusion, and also contributes to the efficiency (Ca2+ sensitivity and rate) of the process [139]. Altering membrane cholesterol with specific reagents such as mβcd and the cholesterol-binding polyene antibiotics (e.g. filipin, amphotericin or pimaricin), as well as enzymatically altering cholesterol using cholesterol oxidase, resulted in the potent inhibition of membrane fusion. Treatments capable of disrupting cholesterol- and sphingomyelin-enriched microdomains (mβcd and cholesterol oxidase) potently inhibited the Ca2+ sensitivity and kinetics of membrane fusion, whereas treatment with the polyene antibiotics, which bind and sequester cholesterol without altering microdomain stability [223], resulted in inhibition of only the ability of vesicles to fuse [139]. Further studies demonstrated that treatments capable of disrupting cholesterol- and sphingomyelin-enriched microdomains without altering membrane cholesterol (e.g. treatment with exogenous sphingomyelinase) inhibited the Ca2+ sensitivity of fusion without altering the ability of CV to fuse [175]. Taken together, these results strongly suggest that the Ca2+-triggering steps of fusion depend on the integrity of cholesterol- and sphingomyelin-enriched microdomains in the fusing membranes, most likely through organization of proteins at the fusion site [224].

Direct contributions to membrane fusion

Careful experimental design has enabled us to separate the functional effects of cholesterol depletion on membrane fusion, allowing the identification of two discrete roles for cholesterol in the native fusion mechanism. In contrast to the indirect effects of cholesterol on the Ca2+ sensitivity and kinetics of fusion, the ability of vesicles to undergo Ca2+-triggered fusion depends on a direct role of cholesterol in the fusion process. Following cholesterol depletion, the resulting inhibition of fusion was fully reversed by delivery of exogenous cholesterol to recover the original native levels, consistent with recovery of microdomains [139]. The fundamental ability of CV to fuse, as measured by the extent of fusion, was also selectively recovered by delivery of exogenous structurally dissimilar lipids having intrinsic curvatures comparable with or greater than that of cholesterol [139,225]. This recovery was dose-dependent, and correlated not only to the molar amounts of these exogenous lipids, but to the respective curvature that each contributed to the membrane. For example, by replacing a proportion of membrane cholesterol with dioleoylglycerol, a neutral lipid with a measured negative curvature more than twice that of cholesterol, less than half of the molar quantity of this lipid (relative to cholesterol) was required to fully recover the extent of fusion [225]. We have thus effectively separated the fundamental ability to fuse (the FFM) from the efficiency of the native mechanism (as defined by the PFM). It is important to note that of all lipids tested, only cholesterol was able to recover both the efficiency (Ca2+ sensitivity and kinetics) of fusion and the fundamental ability of CV to fuse. This ability of negative curvature lipids to selectively replace cholesterol in the fusion mechanism (FFM) demonstrates that in biological membrane fusion cholesterol contributes a critical membrane curvature to lower the energy barriers and thus promote formation of fusion intermediates, consistent with the stalk-pore model. This work also identifies other common native membrane components of intrinsic negative curvature, including tocopherols, phosphatidylethanolamine and diacylglycerols, that could substitute for or work with cholesterol to locally facilitate the FFM. Thus different types of secretory cells and vesicles may well utilize different negative curvature lipids, or different local mixtures of these lipids, to facilitate the FFM, providing yet another layer of regulation to pore formation and modulation by the PFM. Indeed, in PC12 cells, exogenous phosphatidylethanolamine has been shown to increase the kinetics of release in a manner consistent with the stalk-pore hypothesis [226].

In addition to the defined roles of cholesterol as both a membrane organizer to co-ordinate proteins that are essential upstream of and during the triggered fusion process (the PFM), and as a negative curvature lipid contributing the local curvature required to enable fusion (the FFM), there remains a third possible, unexplored role for cholesterol in exocytosis. No study of the cholesterol dependence of exocytosis, including the specific analyses of the role of cholesterol in membrane fusion [139,175,225], has provided evidence against the existence of a cholesterol-binding protein in the fusion machinery. It thus remains a possibility that cholesterol directly regulates the activity of one or more proteins through direct binding.

STRUCTURAL ASPECTS OF EXOCYTOSIS

Modulation of exocytosis

Conceptually, the absolute minimal FFM of all biological fusion events may be a strictly defined pure-lipid site, which is enriched in cholesterol and other negative curvature lipids. The PFM, responsible for the regulation, timing, efficiency and frequency of fusion, includes numerous protein and lipid components and can vary greatly between different vesicle and cell types. At the synapse, the PFM includes numerous identified proteins upstream (e.g. S/M proteins, complexin, the SNAREs, Ca2+ channels, synaptotagmin and SNARE-interacting proteins such as snapin [227], to name but a few) and downstream (e.g. NSF, α/β and γ SNAP) of the fusion event. The defined role for cholesterol in organizing the fusion site [139,211,212,225,228] suggests that cholesterol- or, more generally, sterol-enriched microdomains are a key lipidic component of the PFM. In neuroendocrine cells, PLD1 (phospholipase D1) may also play a regulatory role in the fusion process (reviewed in [229]); in this system enzymatic generation of PA (phosphatidic acid) has been suggested to contribute to the requisite negative curvature for fusion. In light of the modulatory role of PLD in human platelets [230], PA might also contribute upstream, possibly as a charged protein binding site and/or activator of modulatory kinases [231]. Regulation of PA levels expands the PFM in this exocytotic event to include not only PLD1 [232,233], but its interacting partners the GTPases ARF6 (ADP-ribosylation factor 6) [234] and RalA [235] and the GTPase-activating protein GIT1 [236]. In this regard, a G-protein more directly involved in the triggered fusion process in some cells (i.e. the so-called ‘GE’) would also be a component of the PFM (reviewed in [237]). Cytoskeletal components can also be considered components of the PFM. In addition to trafficking vesicles to sites of fusion, specific cytoskeletal components have been shown to regulate fusion kinetics by promoting the dilation and stability of the fusion pore in neuroendocrine, exocrine and epithelial cells. F-actin and myosin II contribute to the duration of fusion pore opening [238242], possibly by contributing tension to increase pore dilation [238,240] or by assembly of a supporting scaffold to maintain the open fusion pore [239,241]. The latter case would result in a state intermediate to a kiss-and-run event and full fusion in which an F-actin scaffold prevents the vesicle from collapsing into the PM, possibly as a means to facilitate efficient endocytosis [243]. Experiments that rule out a role for actin in the fusion process itself confirm the assignment of cytoskeletal elements to the PFM [96].

The structure of a cholesterol-enriched fusion site

Given the evidence in favour of multiple roles for cholesterol not only in the process of exocytosis, but directly in the molecular mechanism of native Ca2+-triggered membrane fusion, we can speculate as to the nature and organization of the fusion site. As an organizer of proteins critical to exocytosis, we expect that the fusion site is defined by cholesterol- and sphingomyelin-enriched microdomains. As cholesterol acts directly in the fusion process by contributing negative curvature to the formation of high-curvature fusion intermediates (as well as to pore formation and expansion [52,163]), we expect that the lipidic fusion site will be enriched in cholesterol. Additionally, considering the enrichment of cholesterol in the vesicle membrane relative to the PM, and the ability of isolated native vesicles to fuse with pure lipid membranes, we note that the two apposed fusogenic membrane domains need not be of identical composition (but will be shown in Figure 4 as such for simplicity) [40,139,225]. On the basis of these considerations, we can explore three possible models for the fusion site (Figure 4).

Models for a cholesterol-enriched fusion site

Figure 4
Models for a cholesterol-enriched fusion site

(A) Cholesterol-enriched microdomains (green) surround and define the ld fusion site (blue); after triggering, microdomains remain intact. (B) A fusion site composed of a contiguous cholesterol-enriched microdomain which disperses into a highly fusogenic ld phase upon triggering. (C) A fusion site composed of a contiguous cholesterol-enriched microdomain as in (B), but with regions of varied fluidity such that the lipidic fusion site has a fusogenic composition; upon triggering the microdomain remains intact surrounding the fusion site.

Figure 4
Models for a cholesterol-enriched fusion site

(A) Cholesterol-enriched microdomains (green) surround and define the ld fusion site (blue); after triggering, microdomains remain intact. (B) A fusion site composed of a contiguous cholesterol-enriched microdomain which disperses into a highly fusogenic ld phase upon triggering. (C) A fusion site composed of a contiguous cholesterol-enriched microdomain as in (B), but with regions of varied fluidity such that the lipidic fusion site has a fusogenic composition; upon triggering the microdomain remains intact surrounding the fusion site.

The fusion site could exist as a ld phase constrained or bordered by lo microdomains (Figure 4A), effectively corralling the cholesterol-enriched fusion site with the appropriate modulatory proteins. In this model, proteins associated with the fusion mechanism, including the putative Ca2+ sensor synaptotagmin and upstream effectors such as the SNARE proteins would be carefully positioned around the site of fusion as per their demonstrated association with cholesterol-enriched microdomains. Triggering of fusion with Ca2+ would result in the formation of initial lipid fusion structures, such as the point-like protrusion and initial stalk within the ld membrane region corralled at the centre of the surrounding cholesterol-rich microdomains. This fusogenic lipid region may have a finely tuned lipid composition, enriched in negative curvature lipids (such as cholesterol, diacylglycerol, phosphatidylethanolamine or even a tocopherol) in the proximal membrane leaflets, with positive curvature contributing lipids (such as lysophosphatidylcholine and fatty acids) in the distal leaflets. Such a lipid composition would reduce the energy barriers required to form the high curvature stalk-pore fusion intermediates. The maintained presence of cholesterol-enriched microdomains at the periphery of the fusion site could contribute line tension to promote the formation and expansion of the fusion pore [244].

An alternative model would have the entire fusion site consist of a lo cholesterol-enriched microdomain (Figure 4B). Exocytotic proteins, including the SNARE proteins, would most likely be associated directly with the microdomain, possibly at the peripheral lold interface. In this model, the entire microdomain may have a finely tuned lipid composition such that, upon triggering with Ca2+, proteins induce a phase transition from a stable lo domain to a highly fusogenic ld state. This microdomain may similarly be enriched in negative curvature lipids (especially cholesterol) in the proximal leaflets; however, due to the reported trans-bilayer symmetry of most cholesterol-enriched microdomains, it seems less likely that the distal leaflets could be selectively enriched in positive curvature lipids to support the transition from hemifusion to fusion pore.

Finally, the fusion site may exist as a contiguous cholesterol-enriched microdomain with regions of varied fluidity (Figure 4C). As with the previous models, this allows for the close association of exocytotic proteins with the optimized lipid mixture of the fusion site; however, this model would not require a triggered transition from lo to ld. As with the first model, the persistence of the lo subdomains could contribute line tension to drive the expansion of the fusion pore, whereas the lipidic fusion site would be enriched in fusogenic negative curvature lipids. As one of the earliest and best studied cholesterol-rich lo membrane structures (i.e. caveolae) is defined functionally by its membrane curvature, it is reasonable to expect that a lipid composition similar to that of cholesterol-enriched microdomains could be potentially fusogenic, forming highly curved membrane structures (see also [245]). As the inherent size of lipidic fusion intermediates is much smaller than the curved structures formed by caveolae, such a model would probably require additional lipid optimization at the fusion site to reduce the structured nature of the domain and possibly increase the local membrane fluidity.

Novel physiological observations may allow us to predict which of these models will be more likely. Notably, electrophysiological and imaging analyses of ‘kiss-and-run’ phenomena suggest that the two membranes do not lose their identity following a series of transient fusion events. The simplest biophysical interpretation of these observations is that during a kiss-and-run event lipid mixing between the vesicle and PM is minimized, which suggests that some barrier exists to limit the intermixing of lipids and the diffusion of proteins. In both the first and third models described above (Figures 4A and 4C), cholesterol-enriched microdomains may remain intact following the initial fusion stalk formation, and if these domains are in fact retained through the formation of the fusion pore they may act as a physical barrier to further mixing of membrane contents; however, in the case of full fusion, microdomains may largely dissipate [225]. Thus, based on interpretation of these limited data, we can predict that the second model (Figure 4B) described above is less likely as may be the third (Figure 4C); if the latter does describe the prefusion state, then triggering may also yield either of the outcomes shown in Figures 4(A) and 4(B). Substantially more experimental evidence and the integration of a number of different technical approaches will be required to provide further insight into the nature of the fusion site and the integration of the FFM and PFM in native Ca2+-triggered exocytosis.

We thank Kendra Furber and Dr Tatiana Rogasevskaia and Dr R. Hussain Butt for helpful discussions. We also acknowledge the critical work of so many colleagues in the field that simply could not be cited due to space limitations.

Abbreviations

     
  • CV

    cortical vesicle(s)

  •  
  • DRM

    detergent-resistant microdomain

  •  
  • FFM

    fundamental fusion mechanism

  •  
  • FRET

    fluorescence resonance energy transfer

  •  
  • GPI

    glycophosphatidylinositol

  •  
  • ld

    liquid disordered

  •  
  • lo

    liquid ordered

  •  
  • mβcd

    methyl-β-cyclodextrin

  •  
  • NSF

    N-ethylmaleimide sensitive factor

  •  
  • PA

    phosphatidic acid

  •  
  • PEG

    poly(ethylene glycol)

  •  
  • PFM

    physiological fusion machine

  •  
  • PLD

    phospholipase D

  •  
  • PM

    plasma membrane

  •  
  • SM

    Sec1/Munc18

  •  
  • α/β SNAP

    soluble NSF-attachment protein

  •  
  • SNAP-25

    synaptosome-associated protein of 25 kDa

  •  
  • SNARE

    SNAP receptor

  •  
  • SV

    synaptic vesicle(s)

  •  
  • TCZ

    transient confinement zone

  •  
  • VAMP

    vesicle-associated membrane protein

FUNDING

J. R. C. acknowledges support from the Canadian Institutes of Health Research (CIHR), Alberta Heritage Foundation for Medical Research (AHFMR), Natural Sciences and Engineering Research Council of Canada (NSERC) and the University of Western Sydney. M. A. C. is the recipient of postgraduate studentship awards from NSERC and AHFMR.

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