Forces, Kinetics, and Fusion Efficiency Altered by the Full-Length Synaptotagmin-1 -PI(4,5)P2 Interaction in Constrained Geometries.

A mechanism for full-length synaptotagmin-1 (syt-1) to interact with anionic bilayers and to promote fusion in the presence of SNAREs is proposed. Colloidal probe force spectroscopy in conjunction with tethered particle motion monitoring showed that in the absence of Ca2+ the binding of syt-1 to membranes depends on the presence and content of PI(4,5)P2. Addition of Ca2+ switches the interaction forces from weak to strong, eventually exceeding the cohesion of the C2A domain of syt-1 leading to partial unfolding of the protein. Fusion of single unilamellar vesicles equipped with syt-1 and synaptobrevin 2 with planar pore-spanning target membranes containing PS and PI(4,5)P2 shows an almost complete suppression of stalled intermediate fusion states and an accelerated fusion kinetics in the presence of Ca2+, which is further enhanced upon addition of ATP.

Membrane fusion mediated by peptidic SNARE protein analogues: Evaluation of FRET-based bulk leaflet mixing assays.

Peptide-mediated membrane fusion is frequently studied with in vitro bulk leaflet mixing assays based on Förster resonance energy transfer (FRET). In these, customized liposomes with fusogenic peptides are equipped with lipids which are labeled with fluorophores that form a FRET pair. Since FRET is dependent on distance and membrane fusion comes along with lipid mixing, the assays allow for conclusions on the membrane fusion process. The experimental outcome of these assays, however, greatly depends on the applied parameters. In the present study, the influence of the peptides, the size of liposomes, their lipid composition and the liposome stoichiometry on the fusogenicity of liposomes are evaluated. As fusogenic peptides, soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) protein analogues featuring artificial recognition units attached to the native SNARE transmembrane domains are used. The work shows that it is important to control these parameters in order to be able to properly investigate the fusion process and to prevent undesired effects of aggregation.

SNARE-Mediated Fusion of Single Chromaffin Granules with Pore-Spanning Membranes.

Pore-spanning membranes (PSMs) composed of supported membrane parts as well as freestanding membrane parts are shown to be very versatile to investigate SNARE-mediated fusion on the single-particle level. They provide a planar geometry readily accessible by confocal fluorescence microscopy, which enabled us for the first time, to our knowledge, to investigate the fusion of individual natural secretory granules (i.e., chromaffin granules (CGs)) on the single-particle level by two-color fluorescence microscopy in a time-resolved manner. The t-SNARE acceptor complex ΔN49 was reconstituted into PSMs containing 2 mol % 1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol-4,5-bisphosphate and Atto488-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, and CGs were fluorescently labeled with 2-((1E,3E)-5-((Z)-3,3-dimethyl-1-octadecylindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-1-octadecyl-3H-indol-1-ium perchlorate. We compared the dynamics of docked and hemifused CGs as well as their fusion efficacy and kinetics with the results obtained for synthetic synaptobrevin 2-doped vesicles fusing with PSMs of the same composition. Whereas the synthetic vesicles were fully immobile on supported PSMs, docked as well as hemifused CGs were mobile on both PSM parts, which suggests that this system resembles more closely the natural situation. The fusion process of CGs proceeded through three-dimensional post-lipid-mixing structures, which were readily resolved on the gold-covered pore rims of the PSMs and which are discussed in the context of intermediate states observed in live cells.

Resolving single membrane fusion events on planar pore-spanning membranes.

Even though a number of different in vitro fusion assays have been developed to analyze protein mediated fusion, they still only partially capture the essential features of the in vivo situation. Here we established an in vitro fusion assay that mimics the fluidity and planar geometry of the cellular plasma membrane to be able to monitor fusion of single protein-containing vesicles. As a proof of concept, planar pore-spanning membranes harboring SNARE-proteins were generated on highly ordered functionalized 1.2 µm-sized pore arrays in Si3N4. Full mobility of the membrane components was demonstrated by fluorescence correlation spectroscopy. Fusion was analyzed by two color confocal laser scanning fluorescence microscopy in a time resolved manner allowing to readily distinguish between vesicle docking, intermediate states such as hemifusion and full fusion. The importance of the membrane geometry on the fusion process was highlighted by comparing SNARE-mediated fusion with that of a minimal SNARE fusion mimetic.

Phosphorylation of C-terminal polycystin-2 influences the interaction with PIGEA14: a QCM study based on solid supported membranes.

Polycystin-2 (PC2) trafficking has been proposed to be a result of the interaction of PIGEA14 with PC2 as a function of the phosphorylation state of PC2. Here, we investigated the interaction of PIGEA14 with the C-terminal part of polycystin-2 wild type (cPC2wt) and the pseudophosphorylated mutant (cPC2S812D) to first, quantify the binding affinity between cPC2 and PIGEA14 and second, to elucidate the influence of PC2 phosphorylation on PIGEA14 binding. Solid supported membranes composed of octanethiol/1,2-dioleoyl-sn-glycero-3-phosphocholine doped with the receptor lipid DOGS-NTA-Ni were used to attach PIGEA14 to the membrane via its hexahistidine tag. By means of the quartz crystal microbalance technique, binding affinities as well as kinetic constants of the interaction were extracted in a label-free manner by applying the scaled particle theory. The results show that the dissociation constant of cPC2 to PIGEA14 is in the 10 nM regime providing strong evidence of a very specific interaction of cPC2 with PIGEA14. The interaction of cPC2wt is twofold larger than that of cPC2S812D. The moderate higher binding affinity of cPC2wt to PIGEA14 is discussed in light of PC2 trafficking to the plasma membrane.