Fast Vesicle Fusion in Living Cells Requires at Least Three SNARE Complexes

Mohrmann, Ralf; Wit, Heidi de; Verhage, Matthijs; Neher, Erwin; Sørensen, Jakob B.

doi:10.1126/science.1193134

Cited by 300 publications

(264 citation statements)

References 24 publications

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“…It is then reasonable to compare the hydration barrier with the energy release by SNARE formation. The ratio of 2-3 deduced from the above numbers is well compatible with recent observations that 1-3 SNARE complexes are sufficient to achieve membrane fusion (66,74). In line with the present results, the inhibition of fusion observed after depletion of cholesterol, both in SNARE-mediated (75) and viral fusion (76,77), as well an increase of the docking efficiency in SNARE-mediated vesicle fusion upon higher PE content (78), may result from the effects of these lipids on the hydration barrier W hyd .…”

Section: Discussionsupporting

confidence: 90%

Energetics of stalk intermediates in membrane fusion are controlled by lipid composition

Aeffner

Reusch²,

Weinhausen³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

162

246

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We have used X-ray diffraction on the rhombohedral phospholipid phase to reconstruct stalk structures in different pure lipids and lipid mixtures with unprecedented resolution, enabling a quantitative analysis of geometry, as well as curvature and hydration energies. Electron density isosurfaces are used to study shape and curvature properties of the bent lipid monolayers. We observe that the stalk structure is highly universal in different lipid systems. The associated curvatures change in a subtle, but systematic fashion upon changes in lipid composition. In addition, we have studied the hydration interaction prior to the transition from the lamellar to the stalk phase. The results indicate that facilitating dehydration is the key to promote stalk formation, which becomes favorable at an approximately constant interbilayer separation of 9.0 AE 0.5 Å for the investigated lipid compositions.curvature energy | hydration force | lipid bilayer | E xocytosis, intracellular transport, neurotransmission, fertilization, or viral entry, require that two membranes merge into one. This event, membrane fusion, involves a complex interplay of different membrane lipids, proteins, and water molecules on length scales of few nm. Following the "lipidic pore hypothesis", it is now well accepted that membrane fusion involves a sequence of lipidic nonbilayer intermediates, whose formation is catalyzed and guided by a specialized protein machinery (1, 2). A stage termed "hemifusion" in which the lipids of the outer leaflets of two membrane-enclosed compartments about to fuse can mix, whereas the inner leaflets and the enclosed content remain unaltered (3), has been confirmed by a variety of methods including conical electron tomography (4) and, more indirectly; e.g., by electron spin resonance (5) and fluorescence recovery after photobleaching (6). Studying the fusion of protein-free bilayers of well defined lipid composition can contribute useful insights into the physical principles governing the merger of their more complex biological counterparts and clarify the effect of individual lipid species.The first connection between two lipid bilayers is the so-called stalk sketched in Fig. 1A (7). The proximal lipid monolayers have merged into one strongly curved monolayer, whereas the distal ones are still separated and intact. A persistent problem in membrane biophysics is to determine the precise structure of stalks and the free energy barrier for stalk formation. In a long series of papers (8-16), this determination has been attempted within the framework of the continuum theory of membrane elasticity (17, 18). More recently, with the advent of sufficient computational power, simulations including molecular details have become feasible [e.g. (19, 20) and references therein]. While stalk formation between lipid bilayers in close contact is generally accepted as the initial step in possibly all membrane fusion reactions (7), the subsequent stages from stalk to complete fusion are still debated and different pathways may exist (21)....

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Section: Discussionsupporting

confidence: 90%

Energetics of stalk intermediates in membrane fusion are controlled by lipid composition

Aeffner

Reusch²,

Weinhausen³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

162

246

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“…However, at 23-30 sybs this assumption is not straightforward to apply owing to steric factors. Furthermore, estimates of the number of SNAREs involved in the homotypic fusion of early endosomes and the exocytosis of chromaffin granules are much lower than the total number of SNAREs available, suggesting that only local subsets of SNAREs take part in fusion (12,34).…”

Section: Discussionmentioning

confidence: 99%

“…Although some studies have left open the possibility that the number of SNARE complexes that cooperate during fusion is variable (11,12), much attention has been given to the notion of a preferred number of SNARE complexes, with estimates varying from a single SNARE complex (13) to 15 (14), although more recent estimates vary between two and eight (12,(15)(16)(17)(18). Unfortunately, this large disparity in results has not been appropriately explained, and it remains unclear whether the differences are a result of inherent properties of the particular set of SNAREs involved or rather originate from the biophysical characteristics of the fusing vesicles.…”

Section: Exocytosis | Fusion Intermediate | Liposome Dockingmentioning

confidence: 99%

Variable cooperativity in SNARE-mediated membrane fusion

Hernandez

Kreutzberger

Kiessling

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

101

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The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex drives the majority of intracellular and exocytic membrane fusion events. Whether and how SNAREs cooperate to mediate fusion has been a subject of intense study, with estimates ranging from a single SNARE complex to 15. Here we show that there is no universally conserved number of SNARE complexes involved as revealed by our observation that this varies greatly depending on membrane curvature. When docking rates of small (∼40 nm) and large (∼100 nm) liposomes reconstituted with different synaptobrevin (the SNARE present in synaptic vesicles) densities are taken into account, the lipid mixing efficiency was maximal with small liposomes with only one synaptobrevin, whereas 23-30 synaptobrevins were necessary for efficient lipid mixing in large liposomes. Our results can be rationalized in terms of strong and weak cooperative coupling of SNARE complex assembly where each mode implicates different intermediate states of fusion that have been recently identified by electron microscopy. We predict that even higher variability in cooperativity is present in different physiological scenarios of fusion, and we further hypothesize that plasticity of SNAREs to engage in different coupling modes is an important feature of the biologically ubiquitous SNARE-mediated fusion reactions.M embrane fusion is an essential reaction common to intracellular trafficking and exocytosis in eukaryotic cells. Although the process involves an intricate interplay of several proteins, the fusion of membranes is dependent on the conserved family of proteins known as soluble N-ethylmaleimide-sensitive factor attachment protein receptors, or SNAREs (1, 2). In the important case of the fusion of synaptic vesicles (SVs), the SNAREs responsible are vesicular synaptobrevin 2 (syb) and plasma membrane proteins SNAP-25A (SN25) and syntaxin-1A (syx). A critical intermediate seems to be an acceptor complex consisting of a threehelix bundle formed by a 1:1 syx:SN25 complex, which serves as a binding site for syb (3,4). According to the zipper hypothesis, the N termini of syb and the 1:1 syx:SN25 complex nucleate to form a parallel four-helix bundle called the SNARE complex. The directional assembly then proceeds toward the C termini, resulting in a pulling force between the membranes that leads to their fusion (4, 5). There is some consensus that the highly exergonic nature of the assembly of the SNARE complex provides the energy for overcoming the barrier for fusion (6, 7), although identification of putative fusion intermediates at molecular resolution as well as force measurement experiments suggest multiple energy barriers are present (7-10).The question of whether and how SNAREs cooperate to mediate fusion has received substantial attention. Although some studies have left open the possibility that the number of SNARE complexes that cooperate during fusion is variable (11, 12), much attention has been given to the notion of a preferred number of SNARE complexes, ...

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“…The four-helical trans-SnARE bundle between synaptobrevin (a synaptic vesicle transmembrane protein) and the plasma membrane proteins synaptosome-associated protein (relative molecular mass 25K) (SnAP25) and syntaxin is thought to provide the driving force for the fusion reaction. Recent data suggest that one to three SnARE complexes 24,25 may be sufficient to overcome the activation energy for the merger of lipid bilayers. The activity of SnAREs is tightly controlled by Munc13 and Munc18, two proteins that are considered to be involved in the priming of vesicles for release 23 (FIG.…”

Section: -2000 Neurotransmitter Moleculesmentioning

confidence: 99%

Protein scaffolds in the coupling of synaptic exocytosis and endocytosis

2011

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Communication between nerve cells largely occurs at chemical synapses -specialized sites of cell-cell contact where electrical signals trigger the exocytic release of neurotransmitter, which in turn activates postsynaptic receptor channels. The efficacy with which such chemical signals are transmitted is crucial for the functioning of the nervous system. Modulation of neurotransmission enables nerve cells to respond to a vast range of stimulus patterns. Synaptic activity can also be tremendously diverse; from the fast synapses of the hippocampus, to slow neuropeptide-containing synapses. Indeed, some signalling pathways are active in the absence of stimulation, such as those involved in the processing of sensory information, which transmit tonically at high rates of up to 100 Hz or more 1,2 .Intriguing questions arise from these facts, including how high rates of neurotransmission can be maintained over long periods of time, and what limits the ability of a given synapse to release neurotransmitter during sustained periods of activity. Recent data indicate that in many synapses, exocytosis of neurotransmitter is coupled to endocytosis, and that synapses have evolved a specialized apparatus of scaffolding proteins to comply with these demands. Such scaffolds aid the temporal and spatial coupling of exocytosis and endocytosis, and are crucial for maintaining rapid neurotransmission during sustained activity 3 .Exocytosis of neurotransmitter is triggered by stimulusinduced calcium influx into the nerve terminal 4,5 and the subsequent fusion of synaptic vesicles with the presynaptic plasma membrane at specialized regions called active zones (BOX 1). Exocytosed synaptic vesicle membrane proteins and lipids in turn are recycled at the endocytic or periactive zone (BOX 1) that surrounds the release site, to restore functional synaptic vesicle pools for reuse and to ensure long-term functionality of the synapse 6-8 (FIG. 1). Depending on the type of synapse and the stimulation frequency, several modes of endocytosis with different time constants -for example, fast and slow endocytosisseem to operate 7,9,10 . Clathrin-mediated endocytosis arguably represents the main pathway of synaptic vesicle endocytosis 6,8,11 , although other modes -for example, fast kiss-and-run exocytosis and endocytosis -could operate in parallel.Although the machineries for exocytic membrane fusion 12,13 and for endocytic retrieval of synaptic vesicle membranes have been studied separately in some detail 6,14 , comparatively little is known about the coupling between exocytosis and endocytosis. Here we synthesize recent data from physiological, morphological and biochemical studies in several model systems into hypothetical models for scaffold-based mechanisms underlying exocytic-endocytic coupling.The synaptic vesicle cycle Synaptic vesicle pools. Some defining features of chemical synapses are the presence of one or several clusters of synaptic vesicles in the presynaptic nerve terminal, and ultrastructural specializations at the pre-and postsy...

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Fast Vesicle Fusion in Living Cells Requires at Least Three SNARE Complexes

Cited by 300 publications

References 24 publications

Energetics of stalk intermediates in membrane fusion are controlled by lipid composition

Energetics of stalk intermediates in membrane fusion are controlled by lipid composition

Variable cooperativity in SNARE-mediated membrane fusion

Protein scaffolds in the coupling of synaptic exocytosis and endocytosis

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