Cellular membrane fusion is thought to proceed through intermediates including docking of apposed lipid bilayers, merging of proximal leaflets to form a hemifusion diaphragm, and fusion pore opening. A membrane-bridging four-helix complex of soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) mediates fusion. However, how assembly of the SNARE complex generates docking and other fusion intermediates is unknown. Using a cell-free reaction we identified intermediates visually and then arrested the SNARE fusion machinery when fusion was about to begin. Partial and directional assembly of SNAREs tightly docked bilayers, but efficient fusion and an extended form of hemifusion required assembly beyond the core complex to the membrane-connecting linkers. We propose that straining of lipids at the edges of an extended docking zone initiates fusion.

Summary Exocytosis of neurosecretory vesicles is mediated bythe SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins syntaxin-1, synaptobrevin, and SNAP-25, with synaptotagmin functioning as the major Ca2+-sensor for triggering membrane fusion. Here we show that bovine chromaffin granules readily fuse with large unilamellar liposomes in a SNARE-dependent manner. Fusion is enhanced by Ca2+ but only if the target liposomes contain PI(4,5)P2 and if polyphosphate anions such as nucleotides or pyrophosphate are present. Ca2+-dependent enhancement is mediated by endogenous synaptotagmin-1. Polyphosphates operate by an electrostatic mechanism that reverses an inactivating cis-association of synaptotagmin-1 with its own membrane whereas trans-binding is not affected. Hence, balancing trans- and cis-membrane interactions of synaptotagmin may be a crucial element in the pathway of Ca2+-dependent exocytosis.

Cell-cell fusion is essential for fertilization and organ development. Dedicated proteins known as fusogens are responsible for mediating membrane fusion. However, until recently, these proteins either remained unidentified or were poorly understood at the mechanistic level. Here, we review how fusogens surmount multiple energy barriers to mediate cell-cell fusion. We describe how early preparatory steps bring membranes to a distance of ∼10 nm, while fusogens act in the final approach between membranes. The mechanical force exerted by cell fusogens and the accompanying lipidic rearrangements constitute the hallmarks of cell-cell fusion. Finally, we discuss the relationship between viral and eukaryotic fusogens, highlight a classification scheme regrouping a superfamily of fusogens called Fusexins, and propose new questions and avenues of enquiry.

SEC separates complex branched polymers by hydrodynamic volume, rather than by molecular weight or branching characteristics. Equations relating the response of different types of detectors are derived including band broadening, by defining a distribution function N′(M,Vh), the number of chains with molecular weight M and hydrodynamic volume Vh. While the true molecular weight distribution of complex polymers cannot be determined by SEC, irrespective of the detector used, the formalism enables multiple detection SEC data to be processed to both analyze the polymer sample and reveal mechanistic information about polymer synthesis. The formalism also shows how the true weight‐ and number‐average molecular weight, $\overline M _{\rm w}$ and $\overline M _{\rm n}$, can be obtained from correct processing of the hydrodynamic volume distributions.

Neuronal exocytosis is mediated by the SNARE proteins synaptobrevin 2/VAMP, syntaxin 1A, and SNAP-25A. While it is wellestablished that these proteins mediate membrane fusion after reconstitution in artificial membranes, it has so far been difficult to monitor intermediate stages of the reaction. Using a confocal two-photon setup, we applied fluorescence cross-correlation spectroscopy (FCCS) and fluorescence lifetime analysis to discriminate between docking and fusion of liposomes. We show that liposome populations that are either non-interacting, or are undergoing docking and fusion, as well as multiple interactions can be quantitatively discriminated without the need for immobilizing the lipid bilayers. When liposomes containing a stabilized syntaxin 1A/ SNAP-25A complex were mixed with liposomes containing synaptobrevin 2, we observed that rapid docking precedes fusion. Accordingly, docked intermediates accumulated in the initial phase of the reaction. Furthermore, rapid formation of multiple docked states was observed with on average four liposomes interacting with each other. When liposomes of different sizes were compared, only the rate of lipid mixing depended on the liposome size but not the rate of docking. Our results show that under appropriate conditions a docked state, mediated by trans-SNARE interactions, can be isolated that constitutes an intermediate in the fusion pathway.fluorescence cross-correlation spectroscopy ͉ fluorescence lifetime analysis ͉ fusion intermediate ͉ single-particle detection ͉ SNAREs

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, ...

Background: Soluble N-ethylmaleimide-sensitive factor attachment protein ␣ (␣-SNAP) regulates the pre-fusion step as well as SNARE disassembly. Results: ␣-SNAP on its own interferes with SNARE zippering and inhibits chromaffin granule fusion, but not synaptic vesicle fusion. Conclusion: Retardation of SNARE zippering by ␣-SNAP results in the partial SNARE zippering. Significance: This is the first direct evidence showing the partial SNARE zippering in the physiological context.

Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion in the secretory pathway. They contain conserved regions, termed SNARE motifs, that assemble between opposing membranes directionally from their N termini to their membrane-proximal C termini in a highly exergonic reaction. However, how this energy is utilized to overcome the energy barriers along the fusion pathway is still under debate. Here, we have used mutants of the SNARE synaptobrevin to arrest trans-SNARE zippering at defined stages. We have uncovered two distinct vesicle docking intermediates where the membranes are loosely and tightly connected, respectively. The tightly connected state is irreversible and independent of maintaining assembled SNARE complexes. Together, our results shed new light on the intermediate stages along the pathway of membrane fusion.