Biochem. J. (2009) 423, 1–14 (Printed in Great Britain)
1
doi:10.1042/BJ20090969
REVIEW ARTICLE
Cholesterol, regulated exocytosis and the physiological fusion machine
Matthew A. CHURCHWARD*† and Jens R. COORSSEN‡§*†1
*Department of Physiology and Pharmacology, Faculty of Medicine, University of Calgary, Calgary, AB, Canada T2N 4N1, †Hotchkiss Brain Institute, Faculty of Medicine, University of
Calgary, Calgary, AB, Canada T2N 4N1, ‡Department of Molecular Physiology, School of Medicine, University of Western Sydney, Penrith South DC, NSW 1797, Australia, §Molecular
Medicine Research Group, University of Western Sydney, Penrith South DC, NSW 1797, Australia, and Nanoscale Organization and Dynamics Group, University of Western Sydney,
Penrith South DC, NSW 1797, Australia
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
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 endosomeindependent 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.
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
systems-level view of the as yet poorly understood process of
Ca2+ -triggered membrane mergers.
Key words: cholesterol, exocytosis, lipid, liposome, microdomain, soluble N-ethylmaleimide sensitive factor-attachment
protein receptor (SNARE).
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
Abbreviations used: 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.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2009 Biochemical Society
2
Figure 1
M. A. Churchward and J. R. Coorssen
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
c 2004 by Annual Reviews (www.annualreviews.org).
permission, from the Annual Review of Neuroscience , Volume 27 specific lipids according to the stalk-pore hypothesis [5–8]), 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 [11–14]. The PM Q-SNAREs syntaxin 1 and SNAP25 (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 [14–16]. 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 [3,17–21], 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 inter
c The Authors Journal compilation c 2009 Biochemical Society
actions, 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 proteoliposomes 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 proteoliposomes 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
Regulated exocytosis
Table 1
3
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
[15]
[16]
[25]
[246]
[247]
[248]
[249]
[59]
[64]
[58]
[115]
[116]
[250]
[251]
[22]
co
co
co
co
co
co
co
co
co
co
co
co
co
co
co
co
dir
dir
dir
dir
dir
dir
dir
dir
dir
dir
dir
dir
dir
dir
dir
dir
dir
11–18:1
11–18:1
11–18:1
22:1
11–18:1
22:1
100:1
75–100:1
25–35:1
17–300:1
27–270:1
280–450:1
600:1
10000–2000:1
20:1
160:1
300:1
420–950:1
N.R.f
100:1
300:1
300:1
150:1
50:1
100:1
200:1
100–200:1
100:1
50–400:1
200:1
200:1
200:1
240:1
First demonstration of SNARE-induced lipid mixing
Rapid lipid mixing
Content mixing assessed with oligonucleotides
Specificity of yeast SNAREs in lipid mixing
Zippering of SNAREs
Golgi inhibitory SNAREs
Mixing inhibited by positive-curvature lipids
Ca2+ -independent lipid mixing with synaptotagmin
Ca2+ -dependent lipid mixing with SV
Ca2+ -dependent lipid mixing with synaptotagmin
Synaptotagmin specificity for neuronal SNAREs
Direct effect of synaptotagmin in mixing
Mast cell SNAREs
Reconstituted endosome fusion with Rabs and cytosolic factors
Slow mixing
Little mixing
No detectable mixing
SNAREs affect only PEG-mediated fusion
Ca2+ -dependent liposome fusion without synaptotagmin
Mixing inhibited by botulinum toxin
Ca2+ -dependent mixing with full-length synaptotagmin
SV ‘fusion’ with liposomes, Ca2+ -independent
Kinetics of SNARE assembly and lipid mixing
Liposome fusion transits through hemifusion
Monitor single fusion events
Specificity of synaptotagmin for neuronal SNAREs
Synaptotagmin/Ca2+ relieve complexin block to mixing
Yeast SNAREs
SNARE assembly precedes hemifusion
Complexin and Ca2+ stimulate fusion without synaptotagmin
Cholesterol stimulates hemifusion
Cholesterol effects on SNAREs in lipid mixing
Fast mixing with planar bilayers - SNAP25, Ca2+ -independent
Lipid
Lipid
Content
Lipid
Lipid
Lipid
SNARE assemblyc
Lipid
Lipid
Lipid
Lipid
Lipid
Lipid
Content
Lipidd
Lipidd
Lipidd
Bothd
Lipid
Lipid
Lipid
Lipid
Lipid
Lipidd,e
Lipide
Lipide
Lipid e
Lipid
Lipid e
Lipid
Lipid e
Lipid e
Lipid
Söllner and co-workers
Davletov and co-workers
Chapman and co-workers
Hirashima and co-workers
Zerial and co-workers
Rizo and co-workers
Lentz and co-workers
Jena and co-workers
Jahn and co-workers
Shin and co-workers
Weisshaar and co-workers
[23]
[56]
[28]
[54]
[55]
[63]
[252]
[61]
[24]
[50]
[62]
[49]
[51]
[52]
[53]
[57]
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.
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 ∼29 000
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 [3,4,17–20,32], 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.
c The Authors Journal compilation c 2009 Biochemical Society
4
Figure 2
M. A. Churchward and J. R. Coorssen
Lipidic fusion according to the stalk-pore hypothesis
c 2008 Macmillan Magazines Ltd. (http://www.nature.com/).
This Figure is adapted from [254] and is reprinted with permission from Nature Structural Biology 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 [38–40]. 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} [33,41–43], or specific proteins [33,44–
48]. 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 proteoliposome 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 proteoliposomes 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
[24,49–55] (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
[24,49–53,60–63]. In the presence of PEG, VAMP proteo
c The Authors Journal compilation c 2009 Biochemical Society
liposomes 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 PMassociated 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 [65–68]. 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
[76–78] or directly in fusion [79–81], possibly modulating fusion
pore expansion [82].
An alternative to the stalk-pore model is the hypothesis that the
initial fusion pore itself is proteinaceous or ‘channel-like’ in nature
[83–85]. 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 [86–88].
Regulated exocytosis
Yeast vacuoles undergo a conserved, constitutive fusion reaction
that appears to be Ca2+ -regulated [89–91]. 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) [92–95]. 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 [40,99–103]. 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 vesicleassociated SNARE-binding protein with two Ca2+ -binding C2
domains [104,105] and a defined Ca2+ -dependent phospholipidbinding 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 C2 B 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 [60,109–114] 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 [24,54,114–116], 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.
Figure 3
5
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
c 2005 Elsevier Ltd.
[244] with permission 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 [5–8,119–124].
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 nonplanar (or non-bilayer) structures in hydrated assemblies. By
convention, the tendency of a lipid to form a convex micellelike 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
c The Authors Journal compilation c 2009 Biochemical Society
6
M. A. Churchward and J. R. Coorssen
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 [132–
134]. Lipids with little or no net curvature (such as diacylphosphatidylcholine) have hydrophilic and hydrophobic regions that
are of approximately equal proportions (Figure 3B).
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 [137–139]. 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 [26,27,139,147–
152]. 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].
c The Authors Journal compilation c 2009 Biochemical Society
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) [158–161], as were sphingolipids; however, virus
entry did not depend on intact cholesterol- and sphingomyelinenriched 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 haemagglutininmediated 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 cholesterolenriched 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 [168–170]. Disruption of cholesterolenriched 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 [176–178]).
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 sphingolipids; 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
[179–182]); 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]).
Regulated exocytosis
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 cholesteroldepleting 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 [190–193]. 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 cholesterolenriched 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-glutarylCoA) 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 antibodycoated colloidal gold particles have measured the lateral mobility
of putative cholesterol-enriched microdomain components
in native membranes [201–203]. 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.
7
Similarly, retention within TCZs was both glycosphingolipid- and
cholesterol-dependent, consistent with model and DRM analyses
[201–204]. Further studies using FRET in native membranes
showed that GPI-anchored proteins were found in cholesteroldependent 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 crosslinking 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 GPIanchored 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 cholesteroland 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 cholesterolenriched 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
c The Authors Journal compilation c 2009 Biochemical Society
8
M. A. Churchward and J. R. Coorssen
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 sphingomyelinenriched 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
c The Authors Journal compilation c 2009 Biochemical Society
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 [238–242], 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
Regulated exocytosis
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.
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 sphingomyelinenriched 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).
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
9
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 lo –ld 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 cholesterolenriched 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.
c The Authors Journal compilation c 2009 Biochemical Society
10
M. A. Churchward and J. R. Coorssen
ACKNOWLEDGEMENTS
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.
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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