Model of SNARE-Mediated Membrane Adhesion Kinetics

SNARE proteins are conserved components of the core fusion machinery driving diverse membrane adhesion and fusion processes in the cell. In many cases micron-sized membranes adhere over large areas before fusion. Reconstituted in vitro assays have helped isolate SNARE mechanisms in small membrane adhesion-fusion and are emerging as powerful tools to study large membrane systems by use of giant unilamellar vesicles (GUVs). Here we model SNARE-mediated adhesion kinetics in SNARE-reconstituted GUV-GUV or GUV-supported bilayer experiments. Adhesion involves many SNAREs whose complexation pulls apposing membranes into contact. The contact region is a tightly bound rapidly expanding patch whose growth velocity increases with SNARE density . We find three patch expansion regimes: slow, intermediate, fast. Typical experiments belong to the fast regime where depends on SNARE diffusivities and complexation binding constant. The model predicts growth velocities s. The patch may provide a close contact region where SNAREs can trigger fusion. Extending the model to a simple description of fusion, a broad distribution of fusion times is predicted. Increasing SNARE density accelerates fusion by boosting the patch growth velocity, thereby providing more complexes to participate in fusion. This quantifies the notion of SNAREs as dual adhesion-fusion agents

Membrane Bridging and Hemifusion by Denaturated Munc18

Neuronal Munc18-1 and members of the Sec1/Munc18 (SM) protein family play a critical function(s) in intracellular membrane fusion together with SNARE proteins, but the mechanism of action of SM proteins remains highly enigmatic. During experiments designed to address this question employing a 7-nitrobenz-2-oxa-1,3-diazole (NBD) fluorescence de-quenching assay that is widely used to study lipid mixing between reconstituted proteoliposomes, we observed that Munc18-1 from squid (sMunc18-1) was able to increase the apparent NBD fluorescence emission intensity even in the absence of SNARE proteins. Fluorescence emission scans and dynamic light scattering experiments show that this phenomenon arises at least in part from increased light scattering due to sMunc18-1-induced liposome clustering. Nuclear magnetic resonance and circular dichroism data suggest that, although native sMunc18-1 does not bind significantly to lipids, sMunc18-1 denaturation at 37°C leads to insertion into membranes. The liposome clustering activity of sMunc18-1 can thus be attributed to its ability to bridge two membranes upon (perhaps partial) denaturation; correspondingly, this activity is hindered by addition of glycerol. Cryo-electron microscopy shows that liposome clusters induced by sMunc18-1 include extended interfaces where the bilayers of two liposomes come into very close proximity, and clear hemifusion diaphragms. Although the physiological relevance of our results is uncertain, they emphasize the necessity of complementing fluorescence de-quenching assays with alternative experiments in studies of membrane fusion, as well as the importance of considering the potential effects of protein denaturation. In addition, our data suggest a novel mechanism of membrane hemifusion induced by amphipathic macromolecules that does not involve formation of a stalk intermediate

Structure and function of longin SNAREs

International audienceSoluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins constitute the core membrane fusion machinery of intracellular transport and intercellular communication. A little more than ten years ago, it was proposed that the long N-terminal domain of a subset of SNAREs, henceforth called the longin domain, could be a crucial regulator with multiple functions in membrane trafficking. Structural, biochemical and cell biology studies have now produced a large set of data that support this hypothesis and indicate a role for the longin domain in regulating the sorting and activity of SNAREs. Here, we review the first decade of structure-function data on the three prototypical longin SNAREs: Ykt6, VAMP7 and Sec22b. We will, in particular, highlight the conserved molecular mechanisms that allow longin domains to fold back onto the fusion-inducing SNARE coiled-coil domain, thereby inhibiting membrane fusion, and describe the interactions of longin SNAREs with proteins that regulate their intracellular sorting. This dual function of the longin domain in regulating both the membrane localization and membrane fusion activity of SNAREs points to its role as a key regulatory module of intracellular trafficking

The SNARE Sec22b has a non-fusogenic function in plasma membrane expansion.

Development of the nervous system requires extensive axonal and dendritic growth during which neurons massively increase their surface area. Here we report that the endoplasmic reticulum (ER)-resident SNARE Sec22b has a conserved non-fusogenic function in plasma membrane expansion. Sec22b is closely apposed to the plasma membrane SNARE syntaxin1. Sec22b forms a trans-SNARE complex with syntaxin1 that does not include SNAP23/25/29, and does not mediate fusion. Insertion of a long rigid linker between the SNARE and transmembrane domains of Sec22b extends the distance between the ER and plasma membrane, and impairs neurite growth but not the secretion of VSV-G. In yeast, Sec22 interacts with lipid transfer proteins, and inhibition of Sec22 leads to defects in lipid metabolism at contact sites between the ER and plasma membrane. These results suggest that close apposition of the ER and plasma membrane mediated by Sec22 and plasma membrane syntaxins generates a non-fusogenic SNARE bridge contributing to plasma membrane expansion, probably through non-vesicular lipid transfer

SNAREpin/Munc18 promotes adhesion and fusion of large vesicles to giant membranes

Exocytic vesicle fusion requires both the SNARE family of fusion proteins and a closely associated regulatory subunit of the Sec1/Munc18 (SM) family. In principle, SM proteins could act at an early SNARE assembly step to promote vesicle–plasma membrane adhesion or at a late step to overcome the energetic barrier for fusion. Here, we use the neuronal cognates of each of these protein families to recapitulate, and distinguish, membrane adhesion and fusion on a novel lipidic platform suitable for imaging by fluorescence microscopy. Vesicle SNARE (v-SNARE) proteins reconstituted into giant vesicles (≈10 μm) are fully mobile and functional. Through confocal microscopy, we observe that large vesicles (≈100 nm) carrying target membrane SNAREs (t-SNAREs) both adhere to and freely move on the surface of the v-SNARE giant vesicle. Under conditions where the intrinsic ability of SNAREs to drive fusion is minimized, Munc18 stimulates both SNARE-dependent stable adhesion and fusion. Furthermore, mutation of a critical Munc18-binding residue on the N terminus of the t-SNARE syntaxin uncouples Munc18-stimulated vesicle adhesion from membrane fusion. We expect that the study of SNARE-mediated fusion with giant membranes will find wide applicability in distinguishing adhesion- and fusion-directed SNARE regulatory factors

Similarities between heterophilic and homophilic cadherin adhesion

The mechanism that drives the segregation of cells into tissue-specific subpopulations during development is largely attributed to differences in intercellular adhesion. This process requires the cadherin family of calcium-dependent glycoproteins. A widely held view is that protein-level discrimination between different cadherins on cell surfaces drives this sorting process. Despite this postulated molecular selectivity, adhesion selectivity has not been quantitatively verified at the protein level. In this work, molecular force measurements and bead aggregation assays tested whether differences in cadherin bond strengths could account for cell sorting in vivo and in vitro. Studies were conducted with chicken N-cadherin, canine E-cadherin, and Xenopus C-cadherin. Both qualitative bead aggregation and quantitative force measurements show that the cadherins cross-react. Furthermore, heterophilic adhesion is not substantially weaker than homophilic adhesion, and the measured differences in adhesion do not correlate with cell sorting behavior. These results suggest that the basis for cell segregation during morphogenesis does not map exclusively to protein-level differences in cadherin adhesion

Energetics and dynamics of SNAREpin folding across lipid bilayers

International audienceMembrane fusion occurs when SNAREpins fold up between lipid bilayers. How much energy is generated during SNAREpin folding and how this energy is coupled to the fusion of apposing membranes is unknown. We have used a surface forces apparatus to determine the energetics and dynamics of SNAREpin formation and characterize the different intermediate structures sampled by cognate SNAREs in the course of their assembly. The interaction energy-versus-distance profiles of assembling SNAREpins reveal that SNARE motifs begin to interact when the membranes are 8 nm apart. Even after very close approach of the bilayers (B2-4 nm), the SNAREpins remain partly unstructured in their membrane-proximal region. The energy stabilizing a single SNAREpin in this configuration (35 k B T) corresponds closely with the energy needed to fuse outer but not inner leaflets (hemifusion) of pure lipid bilayers (40-50 k B T). Intercellular communication and intracellular protein transport rely upon the fusion of cargo-containing vesicles with target membranes. As lipid bilayers are inherently stable, such fusion events are energetically costly and require specialized fusion proteins that harvest the energy made available during their own binding and folding to drive membrane disruption and merging 1-5. In neuronal synapses, the core of the fusion machinery consists of three proteins from the SNARE family: the synaptic vesicle (v)-SNARE protein VAMP-2 and the two target plasma membrane (t)-SNARE proteins syntaxin-1A and SNAP-25 (refs. 6-8). When separately reconstituted into synthetic liposomes or ectopically expressed on the surfaces of cells, neuronal v-and t-SNARE proteins are sufficient to drive membrane fusion through their assembly in the form of SNAREpins 2,9. The interacting domains of SNARE proteins (SNARE motifs) contain 60-70 amino acid residues; they are mostly unstruc-tured as monomers 10-12 and assemble in solution into a highly stable heterotrimer consisting of four a-helices aligned in parallel, with VAMP-2 and syntaxin-1A each contributing one helix and SNAP-25 contributing two helices 13,14. In the context of lipid bilayers, the assembly of SNAREs starts at their membrane-distal N termini and proceeds toward their membrane-proximal C termini (zipper model), a process that also includes passage through a stable intermediate binding state 15-21. This zipper-like assembly progressively brings the membranes into close apposition and creates a tight bridge between them that triggers lipid bilayer fusion. Progressive assembly of SNAREs may culminate in a release of energy sufficient to drive membrane merging. Alternatively, the assembling SNAREs may pass through a series of intermediates, each of which contributes enough energy for advancement through the successive stages of membrane fusion. Characterization of these inter-mediates requires the capacity to measure the interactions between membrane-associated proteins at nanometer distance resolutions. Thermodynamic and atomic force microscopy (AFM) measurements have successfully described the kinetics of SNARE assembly and disassembly in solution 22 and the rupture forces of SNARE complexes affixed to solid supports 23,24. However, none of these studies has been able to offer information about the dynamics and energetics of SNAREpin folding, including conformational changes and distance-energy correlations during SNARE assembly. Furthermore, the previous experiments were not performed in the context of lipid bilayers, preventing any investigation of the interplay of lipids and SNARE proteins in membrane interaction and fusion. Here, we have investigated these questions using the surface forces apparatus (SFA), which directly measures the interaction energy between two facing functionalized membranes as a function of their separation distance and makes it possible to identify molecular rearrangements of interacting species during their association 25. Direct measurements of force versus distance between membrane-embedded neuronal SNARE proteins (derived from mouse and rat) allow us to explore in real time the molecular details of SNAREpin formation across two lipid bilayers, including conformational changes, kinetics of association, binding energy and extent of assembly. RESULTS Interactions between SNAREs in apposing bilayers Force was measured between two mica-supported lipid bilayers reconstituted with the neuronal cognate t-and v-SNARE proteins (Fig. 1). The surfaces were approached toward each other and the