Bilayer Fusion Research Articles

Lipid Vesicle Aggregation Induced by Cooling

Lipid bilayer fusion is a complex process requiring several intermediate steps. Initially, the two bilayers are brought into close contact following removal of intervening water layers and overcoming electrostatic repulsions between opposing bilayer head groups. In this study we monitor by light scattering the reversible aggregation of phosphatidylcholine single shell vesicles during which adhesion occurs but stops prior to a fusion process. Light scattering measurements of dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in water show that lowering the temperature of about 0.14 micron single shell vesicles of DPPC (from 20 °C to 5 °C) and about 2 micron vesicles of DSPC (from 20 °C to 15 °C), but not of 1 micron vesicles of DMPC, results in extensive aggregation within 24 hours that is reversible by an increase in temperature. Aggregation of DSPC vesicles was confirmed by direct visual observation. Orientation of lipid head groups parallel to the plane of the bilayer and consequent reduction of the negative surface charge can account for the ability of DPPC and DSPC vesicles to aggregate. Retention of negatively charged phosphates on the surface and the burial of positively charged cholines within the bilayer offer an explanation for the failure of DMPC vesicles to aggregate. Lowering the temperature of 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS) vesicles from 20 °C to 5 °C failed to increase aggregation within 24 hours at Mg++/DPPS ratios that begin to initiate aggregation and fusion.

Direct Observation of Intermediate States in Membrane Fusion by Photonic Force Microscopy

Protein-free lipid bilayer fusion is an important model system for studying fundamental properties of biological membrane fusion. Fusion intermediates, as observed in viral or synaptic fusion, have been shown to be similar to those found with phospholipid membranes (Chernomordik and Kozlov, (2008), Nat. Struct. Mol. Biol., 15(7):675-683) To determine what role proteins play in the fusion process, one must understand the intermediate steps of protein-free fusion first. However, the characterization of all fusion intermediates in a single fusion event is difficult because some intermediates are expected to occur only on fast time scale.Fusion of a vesicle to a target membrane can be modeled by bringing an optically trapped and lipid bilayer-coated silica bead onto a membrane-coated glass coverslide. We use a Photonic Force Microscope to measure the position of the trapped bead in three-dimensions with microsecond temporal and nanometer spatial resolution (Bartsch et al., (2009), ChemPhysChem, 10(9-10):1541-1547). These position traces contain a wealth of information about the fusion event.In our assay, fusion can be induced with thermal energy alone; no additional force is needed. Fusion intermediates (transient fusion, stalk formation, hemifusion and full fusion) are clearly distinguishable. They are stable over periods of time and transitions between them are shorter than the lifetime of the intermediates by orders of magnitude. Each intermediate shows characteristic features in the bead's thermal fluctuation amplitude and position distribution. For instance, the confinement of the particle by the membrane in a hemifused stage shows typical features expected for a two-dimensional fluid.Our assay is general, as it allows one to study the influence of lipid composition, protein content, or buffer conditions on the intermediates of membrane fusion. The assay can be combined with other established methods for monitoring membrane fusion.

Fast Single Vesicle SNARE-Mediated Membrane Fusion Assay in Planar Supported Bilayers Reveals Details About Fusion Mechanism

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane fusion in neuronal exocytosis and intracellular membrane trafficking. It is widely accepted that the zippering interaction between syntaxin1a, SNAP25 and synaptobrevin2 brings plasma- and vesicle membrane together and leads to their merger. Membrane fusion has been extensively studied by liposome and single vesicle-planar membrane fusion assays. However, both approaches have been criticized because of slow fusion rates incompatible with cellular rates or lack of physiological specificity, respectively. Here we present a fluorescence-basedsingle vesicle-planar bilayer fusion assay with millisecond time resolution and an improved reconstitution procedure that preserves the native topology and mobility of the SNAREs. The acceptor-SNARE complex, composed of syntaxin1a (SyxH3), SNAP25 and a short synaptobrevin2 peptide (Syb49-96), was reconstituted into planar bilayers by a combined Langmuir-Blodgett - vesicle fusion technique. Docking and fusion of single Rh-DOPE labeled Syb vesicles to the supported acceptor-SNARE membranes were observed by total internal reflection fluorescence microscopy. No docking or fusion was observed in protein-free control membranes. Vesicle SNARE docking was dependent on the acceptor-SNARE complex density in the membranes. Moreover, docking was SNAP25-dependent, and subsequent fusion did not require Ca2+ and was efficient at ambient temperature. A detailed kinetic analysis of >1000 single fusion events revealed that each fusion reaction consists of 6 to 9 activating steps with 8 steps fitting the data best. This could be interpreted by fusion sites consisting of 8 SNARE complexes that each activate in a single rate-limiting step in 8 ms. We find that different lipid compositions of the supported and the vesicle membrane have a relatively minor influence on docking, but modulate the fusion efficiency and fusion kinetics more dramatically. Supported by NIH grant GM072694

Single Molecule Measurements of Interaction Free Energies Between the Proteins Within Binary and Ternary SNARE Complexes

We use an Atomic Force Microscope based single molecule measurements to evaluate the activation free energy in the interaction of SNARE proteins syntaxin 1A, SNAP25B and synaptobrevin 2 which regulate intracellular fusion of vesicles with target membranes. The dissociation rate of the binary syntaxin-synaptobrevin and the ternary syntaxin-SNAP25B-synaptobrevin complex was measured from the rupture force distribution as a function of the rate of applied force. The temperature dependence of the spontaneous dissociation rate was used to obtain the activation energy to the transition state of 19.8 +/- 3.5 kcal/mol = 33 +/- 6 k(B)T and 25.7 +/- 3.0 kcal/mol = 43 +/- 5 k(B)T for the binary and ternary complex, respectively. They are consistent with those measured previously for the ternary complex in lipid membranes and are of order expected for bilayer fusion and pore formation. The DeltaG was 12.4-16.6 kcal/mol = 21-28 k(B)T and 13.8-18.0 kcal/mol = 23-30 k(B)T for the binary and ternary complex, respectively. The ternary complex was more stable by 1.4 kcal/mol = 2.3 k(B)T, consistent with the spontaneous dissociation rates. The higher adhesion energies and smaller molecular extensions measured with SNAP25B point to its possible unique and important physiological role in tethering/docking the vesicle in closer proximity to the plasma membrane and increasing the probability for fusion completion.

Minimal membrane docking requirements revealed by reconstitution of Rab GTPase-dependent membrane fusion from purified components

Rab GTPases and their effectors mediate docking, the initial contact of intracellular membranes preceding bilayer fusion. However, it has been unclear whether Rab proteins and effectors are sufficient for intermembrane interactions. We have recently reported reconstituted membrane fusion that requires yeast vacuolar SNAREs, lipids, and the homotypic fusion and vacuole protein sorting (HOPS)/class C Vps complex, an effector and guanine nucleotide exchange factor for the yeast vacuolar Rab GTPase Ypt7p. We now report reconstitution of lysis-free membrane fusion that requires purified GTP-bound Ypt7p, HOPS complex, vacuolar SNAREs, ATP hydrolysis, and the SNARE disassembly catalysts Sec17p and Sec18p. We use this reconstituted system to show that SNAREs and Sec17p/Sec18p, and Ypt7p and the HOPS complex, are required for stable intermembrane interactions and that the three vacuolar Q-SNAREs are sufficient for these interactions.

Magnesium-induced lipid bilayer microdomain reorganizations: implications for membrane fusion.

Interactions between dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS), combined both as binary lipid bilayer assemblies and separately, under the influence of divalent Mg2+, a membrane bilayer fusogenic agent, are reported. Infrared vibrational spectroscopic analyses of the lipid acyl chain methylene symmetric stretching modes indicate that aggregates of the two phospholipid components exist as domains heterogeneously distributed throughout the binary bilayer system. In the presence of Mg2+, DPPS maintains an ordered orthorhombic subcell gel phase structure through the phase transition temperature, while the DPPC component is only minimally perturbed with respect to the gel to liquid crystalline phase change. The addition of Mg2+ induces a reorganization of the lipid domains in which the gel phase acyl chain planes rearrange from a hexagonal configuration toward a triclinic, parallel chain subcell. Examination of the acyl chain methylene deformation modes at low temperatures allows a determination of DPPS microdomain sizes, which decrease upon the addition of DPPC-d62 in the absence of Mg2+. On adding Mg2+, a uniform DPPS domain size is observed in the binary mixtures. In either the presence or absence of Mg2+, DPPC-d62 aggregates remain in a configuration for which microdomain sizes are not spectroscopically measurable. Analysis of the acyl chain methylene deformation modes for DPPC-d62 in the binary system suggests that clusters of the deuterated lipids are distributed throughout the DPPS matrix. Light scattering and fluorescence measurements indicate that Mg2+ induces both the aggregation and the fusion of the lipid assemblies as a function of the ratio of DPPS to DPPC. The structural reorganizations of the lipid microdomains within the DPPS-DPPC bilayer are interpreted in the context of current concepts regarding lipid bilayer fusion.

PTP1B Dephosphorylates N-Ethylmaleimide-sensitive Factor and Elicits SNARE Complex Disassembly during Human Sperm Exocytosis

The reversible phosphorylation of tyrosyl residues in proteins is a cornerstone of the signaling pathways that regulate numerous cellular responses. Protein tyrosine phosphorylation is controlled through the concerted actions of protein-tyrosine kinases and phosphatases. The goal of the present study was to unveil the mechanisms by which protein tyrosine dephosphorylation modulates secretion. The acrosome reaction, a specialized type of regulated exocytosis undergone by sperm, is initiated by calcium and carried out by a number of players, including tyrosine kinases and phosphatases, and fusion-related proteins such as Rab3A, alpha-SNAP, N-ethylmaleimide-sensitive factor (NSF), SNAREs, complexin, and synaptotagmin VI. We report here that inducers were unable to elicit the acrosome reaction when permeabilized human sperm were loaded with anti-PTP1B antibodies or with the dominant-negative mutant PTP1B D181A; subsequent introduction of wild type PTP1B or NSF rescued exocytosis. Wild type PTP1B, but not PTP1B D181A, caused cis SNARE complex dissociation during the acrosome reaction through a mechanism involving NSF. Unlike its non-phosphorylated counterpart, recombinant phospho-NSF failed to dissociate SNARE complexes from rat brain membranes. These results strengthen our previous observation that NSF activity is regulated rather than constitutive during sperm exocytosis and indicate that NSF must be dephosphorylated by PTP1B to disassemble SNARE complexes. Interestingly, phospho-NSF served as a substrate for PTP1B in an in vitro assay. Our findings demonstrate that phosphorylation of NSF on tyrosine residues prevents its SNARE complex dissociation activity and establish for the first time a role for PTP1B in the modulation of the membrane fusion machinery.

The Fusion of Membranes and Vesicles: Pathway and Energy Barriers from Dissipative Particle Dynamics

The fusion of lipid bilayers is studied with dissipative particle dynamics simulations. First, to achieve control over membrane properties, the effects of individual simulation parameters are studied and optimized. Then, a large number of fusion events for a vesicle and a planar bilayer are simulated using the optimized parameter set. In the observed fusion pathway, configurations of individual lipids play an important role. Fusion starts with individual lipids assuming a splayed tail configuration with one tail inserted in each membrane. To determine the corresponding energy barrier, we measure the average work for interbilayer flips of a lipid tail, i.e., the average work to displace one lipid tail from one bilayer to the other. This energy barrier is found to depend strongly on a certain dissipative particle dynamics parameter, and, thus, can be adjusted in the simulations. Overall, three subprocesses have been identified in the fusion pathway. Their energy barriers are estimated to lie in the range 8–15 k B T. The fusion probability is found to possess a maximum at intermediate tension values. As one decreases the tension, the fusion probability seems to vanish before the tensionless membrane state is attained. This would imply that the tension has to exceed a certain threshold value to induce fusion.

Negative Regulation of Syntaxin4/SNAP-23/VAMP2-Mediated Membrane Fusion by Munc18c In Vitro

BackgroundTranslocation of the facilitative glucose transporter GLUT4 from an intracellular store to the plasma membrane is responsible for the increased rate of glucose transport into fat and muscle cells in response to insulin. This represents a specialised form of regulated membrane trafficking. Intracellular membrane traffic is subject to multiple levels of regulation by conserved families of proteins in all eukaryotic cells. Notably, all intracellular fusion events require SNARE proteins and Sec1p/Munc18 family members. Fusion of GLUT4-containing vesicles with the plasma membrane of insulin-sensitive cells involves the SM protein Munc18c, and is regulated by the formation of syntaxin 4/SNAP23/VAMP2 SNARE complexes.Methodology/Principal FindingsHere we have used biochemical approaches to characterise the interaction(s) of Munc18c with its cognate SNARE proteins and to examine the role of Munc18c in regulating liposome fusion catalysed by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. We demonstrate that Munc18c makes contacts with both t- and v-SNARE proteins of this complex, and directly inhibits bilayer fusion mediated by the syntaxin 4/SNAP23/VAMP2 SNARE complex.Conclusion/SignificanceOur reductionist approach has enabled us to ascertain a direct inhibitory role for Munc18c in regulating membrane fusion mediated by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. It is important to note that two different SM proteins have recently been shown to stimulate liposome fusion mediated by their cognate SNARE complexes. Given the structural similarities between SM proteins, it seems unlikely that different members of this family perform opposing regulatory functions. Hence, our findings indicate that Munc18c requires a further level of regulation in order to stimulate SNARE-mediated membrane fusion.

Synaptotagmin arrests the SNARE complex before triggering fast, efficient membrane fusion in response to Ca2+

Neuronal communication is mediated by Ca2+-triggered fusion of transmitter-filled synaptic vesicles with the presynaptic plasma membrane. Synaptotagmin I functions as a Ca2+ sensor that regulates exocytosis, whereas soluble N-ethylmaleimide–sensitive factor attachment protein (SNAP) receptor (SNARE) proteins in the vesicle and target membrane assemble into complexes that directly catalyze bilayer fusion. Here we report that, before the Ca2+ trigger, synaptotagmin interacts with SNARE proteins in the target membrane to halt SNARE complex assembly at a step after donor vesicles attach, or dock, to target membranes. This results in fusion complexes that, when subsequently triggered by Ca2+, drive rapid, highly efficient lipid mixing. Ca2+-independent interactions with SNAREs also predispose synaptotagmin to selectively penetrate the target membrane in response to Ca2+; we demonstrate that Ca2+–synaptotagmin must insert into the target membrane to accelerate SNARE-catalyzed fusion. These findings demonstrate that Ca2+ converts synaptotagmin from a clamp to a trigger for exocytosis.

Membrane processes and biophysical characterization of living cells decorated with chromatic polydiacetylene vesicles

The structural complexity of the cell membrane makes analysis of membrane processes in living cells, as compared to model membrane systems, highly challenging. Living cells decorated with surface-attached colorimetric/fluorescent polydiacetylene patches might constitute an effective platform for analysis and visualization of membrane processes in situ. This work examines the biological and chemical consequences of plasma membrane labeling of promyelocytic leukemia cells with polydiacetylene. We show that the extent of fusion between incubated lipid/diacetylene vesicles and the plasma membrane is closely dependent upon the lipid composition of both vesicles and cell membrane. In particular, we find that cholesterol presence increased bilayer fusion between the chromatic vesicles and the plasma membrane, suggesting that membrane organization plays a significant role in the fusion process. Spectroscopic data and physiological assays show that decorating the cell membrane with the lipid/diacetylene patches reduces the overall lateral diffusion within the membrane bilayer, however polydiacetylene labeling does not adversely affect important cellular metabolic pathways. Overall, the experimental data indicate that the viability and physiological integrity of the surface-engineered cells are retained, making possible utilization of the platform for studying membrane processes in living cells. We demonstrate the use of the polydiacetylene-labeled cells for visualizing and discriminating among different membrane interaction mechanisms of pharmaceutical compounds.

Effect of Freeze−Thawing on Lipid Bilayer-Protected Gold Nanoparticles

In this study, varieties of lipid bilayer-protected gold nanoparticles (AuNPs) were synthesized through a simple wet chemical method, and then the effect of freeze-thawing on the as-prepared AuNPs was investigated. The freeze-thawing process induced fusion or fission of lipid bilayers tethered on the AuNPs. The UV-vis spectra and transmission electron microscopy experiments revealed that the disruption of lipid bilayer structures on the nanoparticles led to the fusion or aggregation of AuNPs. The role of freeze-thawing in the evolution of lipid bilayer-protected AuNPs was studied. The addition of adequate sucrose, a well-known cryoprotectant, effectively prevented the fusion or aggregation of lipid bilayer-protected AuNPs undergoing the freeze-thawing process. The possible mechanism of sucrose preserving the integrity of the lipid bilayer-protected AuNPs was also discussed.

Membranes: a meeting point for lipids, proteins and therapies

Membranes constitute a meeting point for lipids and proteins. Not only do they define the entity of cells and cytosolic organelles but they also display a wide variety of important functions previously ascribed to the activity of proteins alone. Indeed, lipids have commonly been considered a mere support for the transient or permanent association of membrane proteins, while acting as a selective cell/organelle barrier. However, mounting evidence demonstrates that lipids themselves regulate the location and activity of many membrane proteins, as well as defining membrane microdomains that serve as spatio-temporal platforms for interacting signalling proteins. Membrane lipids are crucial in the fission and fusion of lipid bilayers and they also act as sensors to control environmental or physiological conditions. Lipids and lipid structures participate directly as messengers or regulators of signal transduction. Moreover, their alteration has been associated with the development of numerous diseases. Proteins can interact with membranes through lipid co-/post-translational modifications, and electrostatic and hydrophobic interactions, van der Waals forces and hydrogen bonding are all involved in the associations among membrane proteins and lipids. The present study reviews these interactions from the molecular and biomedical point of view, and the effects of their modulation on the physiological activity of cells, the aetiology of human diseases and the design of clinical drugs. In fact, the influence of lipids on protein function is reflected in the possibility to use these molecular species as targets for therapies against cancer, obesity, neurodegenerative disorders, cardiovascular pathologies and other diseases, using a new approach called membrane-lipid therapy.

Field Theoretic Study of Bilayer Membrane Fusion III: Membranes with Leaves of Different Composition

We extend previous work on homogeneous bilayers to calculate the barriers to fusion of planar bilayers that contain two different amphiphiles, a lamellar former and a hexagonal former, with different compositions of the two in each leaf. Self-consistent field theory is employed, and both standard and alternative pathways are explored. We first calculate these barriers as the amount of hexagonal former is increased equally in both leaves to levels appropriate to the plasma membrane of human red blood cells. We follow these barriers as the composition of hexagonal formers is then increased in the cis layer and decreased in the trans layer, again to an extent comparable to the biological system. We find that, while the fusion pathway exhibits two barriers in both the standard and alternative pathways, in both cases the magnitudes of these barriers are comparable to one another, and small, on the order of 13 k B T. As a consequence, one expects that once the bilayers are brought sufficiently close to one another to initiate the process, fusion should occur rapidly.

Simultaneous Synthesis of Au and Cu Nanoparticles in Pseudo-Core−Shell Type Arrangement Facilitated by DMPG and 12-6-12 Capping Agents

A seed-growth method has been applied to synthesize the gold (Au) nanoparticles (NP) in the presence of 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DMPG) and hexamethylene-1,6-bis(dodecyldimethylammonium bromide)(12-6-12) as capping agents at ambient conditions. The systematic addition of CuSO4 leads to anisotropic growth of Au NP at low CuSO4 concentration in the presence of DMPG while this was not observed in the case of 12-6-12 where mostly spherical Au NP were obtained. At high CuSO4 concentration, small Cu NP appeared which arranged themselves around large Au NP in a typical pseudo-core−shell type arrangement. This was achieved by the fusion of lipid bilayers of lipid-capped Au and Cu NP. The cause of anisotropic growth of Au NP in the presence of DMPG has been discussed on the basis of its poor capping ability in comparison to 12-6-12.

Virus membrane fusion

Membrane fusion of enveloped viruses with cellular membranes is mediated by viral glycoproteins (GP). Interaction of GP with cellular receptors alone or coupled to exposure to the acidic environment of endosomes induces extensive conformational changes in the fusion protein which pull two membranes into close enough proximity to trigger bilayer fusion. The refolding process provides the energy for fusion and repositions both membrane anchors, the transmembrane and the fusion peptide regions, at the same end of an elongated hairpin structure in all fusion protein structures known to date. The fusion process follows several lipidic intermediate states, which are generated by the refolding process. Although the major principles of viral fusion are understood, the structures of fusion protein intermediates and their mode of lipid bilayer interaction, the structures and functions of the membrane anchors and the number of fusion proteins required for fusion, necessitate further investigations.

The Plasma Membrane Proteins Prm1 and Fig1 Ascertain Fidelity of Membrane Fusion during Yeast Mating

As for most cell-cell fusion events, the molecular details of membrane fusion during yeast mating are poorly understood. The multipass membrane protein Prm1 is the only known component that acts at the step of bilayer fusion. In its absence, mutant mating pairs lyse or arrest in the mating reaction with tightly apposed plasma membranes. We show that deletion of FIG 1, which controls pheromone-induced Ca(2+) influx, yields similar cell fusion defects. Although extracellular Ca(2+) is not required for efficient cell fusion of wild-type cells, cell fusion in prm1 mutant mating pairs is dramatically reduced when Ca(2+) is removed. This enhanced fusion defect is due to lysis. Time-lapse microscopy reveals that fusion and lysis events initiate with identical kinetics, suggesting that both outcomes result from engagement of the fusion machinery. The yeast synaptotagmin orthologue and Ca(2+) binding protein Tcb3 has a role in reducing lysis of prm1 mutants, which opens the possibility that the observed role of Ca(2+) is to engage a wound repair mechanism. Thus, our results suggest that Prm1 and Fig1 have a role in enhancing membrane fusion and maintaining its fidelity. Their absence results in frequent mating pair lysis, which is counteracted by Ca(2+)-dependent membrane repair.

Membrane Activation: Selective Vesicle Fusion via Small Molecule Recognition

We report herein the induction of selective vesicle fusion with biological recognition motifs not natively associated with lipid bilayer fusion, thus broadening the scope of recognition-guided membrane activation. Our system employs vancomycin glycopeptide, coupled to the antimicrobial peptide magainin, and D-Ala-D-Ala-OH dipeptide coupled to a phospholipid derivative, as surface-bound fusogens. Fusion was characterized by dynamic light scattering and FRET experiments with lipid bound fluorophores. We have demonstrated here that appropriately designed membrane anchored molecular recognition motifs have the biomimetic ability to activate specific membrane mergers; this principle has resonance with goals in targeted chemical delivery and nanoscale compartmentalized chemistry.

Membranes of the world unite!

Despite diverse origins, cellular fusion mechanisms converge at a pathway of phospholipid bilayer fusion. In this mini-review, we discuss how proteins can mediate each of the three major stages in the fusion pathway: contact, hemifusion, and the opening of an expanding fusion pore.

Lipidic Antagonists to SNARE-mediated Fusion

SNARE proteins mediate the fusion of lipid bilayers by the directed assembly of coiled-coil domains arising from apposing membranes. We have utilized inverted cone-shaped lipids, antagonists of the necessary membrane deformation during fusion to characterize the extent and range of SNARE assembly up to the moment of stalk formation between bilayers. The inverted cone-shaped lipid family of acyl-CoAs specifically inhibits the completion of fusion in an acyl-chain length-dependent manner. Removal of acyl-CoA from the membrane relieves the inhibition and initiates a burst of membrane fusion with rates exceeding any point in the control curves lacking acyl-CoA. This burst indicates the accumulation of semi-assembled fusion complexes. These preformed complexes are resistant to cleavage by botulinum toxin B and thus appear to have progressed beyond the "loosely zippered" state of docked synaptic vesicles. Surprisingly, application of the soluble domain of VAMP2, which blocks SNARE assembly by competing for binding on the available t-SNAREs, blocks recovery from the acyl-CoA inhibition. Thus, complexes formed in the presence of a lipidic antagonist to fusion are incompletely assembled, suggesting that the formation of tightly assembled SNARE pairs requires progression all the way through to membrane fusion. In this regard, physiologically docked exocytic vesicles may be anchored by a highly dynamic and potentially even reversible SNAREpin.