Exocytosis in neuronal cells requires the SNARE protein syntaxin-1A, which is clustered at sites where synaptic vesicles are poised to undergo exocytosis. Reinhard Jahn and colleagues use super-resolution stimulated-emission depletion (STED) microscopy to show that syntaxin clusters in the membrane through electrostatic interactions with the strongly anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2) into 70-nanometre microdomains. The results demonstrate that electrostatic protein–lipid interactions can result in the formation of microdomains independent of cholesterol or lipid phases and have important implications for the organization of the plasma membrane. Neuronal exocytosis is catalysed by the SNAP receptor protein syntaxin-1A1, which is clustered in the plasma membrane at sites where synaptic vesicles undergo exocytosis2,3. However, how syntaxin-1A is sequestered is unknown. Here we show that syntaxin clustering is mediated by electrostatic interactions with the strongly anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Using super-resolution stimulated-emission depletion microscopy on the plasma membranes of PC12 cells, we found that PIP2 is the dominant inner-leaflet lipid in microdomains about 73 nanometres in size. This high accumulation of PIP2 was required for syntaxin-1A sequestering, as destruction of PIP2 by the phosphatase synaptojanin-1 reduced syntaxin-1A clustering. Furthermore, co-reconstitution of PIP2 and the carboxy-terminal part of syntaxin-1A in artificial giant unilamellar vesicles resulted in segregation of PIP2 and syntaxin-1A into distinct domains even when cholesterol was absent. Our results demonstrate that electrostatic protein–lipid interactions can result in the formation of microdomains independently of cholesterol or lipid phases.

Interactions between synaptotagmin-1 and the SNARE syntaxin-1 are known to mediate synaptic-vesicle exocytosis. Fusion experiments with artificial lipid membranes combined with the crystal structure of synaptotagmin's C2B domain bound to phosphoserine indicate that PIP2 clusters, organized by syntaxin, act as molecular beacons for vesicle docking and direct Ca2+-dependent membrane fusion. Synaptic-vesicle exocytosis is mediated by the vesicular Ca2+ sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using PC12 cells from Rattus norvegicus and artificial supported bilayers, we show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca2+ through PIP2. This interaction allows both Ca2+-binding sites of synaptotagmin-1 to bind to phosphatidylserine in the vesicle membrane upon Ca2+ triggering. We determined the crystal structure of the C2B domain of synaptotagmin-1 bound to phosphoserine, allowing development of a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca2+ influx bringing the vesicle membrane close enough for membrane fusion.

The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein–protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes.

Synaptotagmin-1 is known to accelerate membrane fusion during neuronal exocytosis in response to Ca2+, but how it does this is unclear. By probing the activity of synaptotagmin-1 under conditions of low ionic strength, it is now shown that SNARE-mediated fusion is dependent on synaptotagmin-1, which tethers liposomes together but at distances too far for fusion. Ca2+ then induces synaptotagmin-1 to bring the liposomes closer together, allowing fusion to proceed. Synaptotagmin-1 triggers Ca2+-sensitive, rapid neurotransmitter release by promoting interactions between SNARE proteins on synaptic vesicles and the plasma membrane. How synaptotagmin-1 promotes this interaction is unclear, and the massive increase in membrane fusion efficiency of Ca2+-bound synaptotagmin-1 has not been reproduced in vitro. However, previous experiments have been performed at relatively high salt concentrations, screening potentially important electrostatic interactions. Using functional reconstitution in liposomes, we show here that at low ionic strength SNARE-mediated membrane fusion becomes strictly dependent on both Ca2+ and synaptotagmin-1. Under these conditions, synaptotagmin-1 functions as a distance regulator that tethers the liposomes too far from the plasma membrane for SNARE nucleation in the absence of Ca2+, but while bringing the liposomes close enough for membrane fusion in the presence of Ca2+. These results may explain how the relatively weak electrostatic interactions between synaptotagmin-1 and membranes substantially accelerate fusion.

Synaptotagmin-1 is the main Ca(2+) sensor of neuronal exocytosis. It binds to both Ca(2+) and the anionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)), but the precise cooperativity of this binding is still poorly understood. Here, we used microscale thermophoresis to quantify the cooperative binding of PIP(2) and Ca(2+) to synaptotagmin-1. We found that PIP(2) bound to the well conserved polybasic patch of the C2B domain with an apparent dissociation constant of ∼20 μM. PIP(2) binding reduced the apparent dissociation constant for Ca(2+) from ∼250 to 40-fold more sensitive to Ca(2+). This interplay between Ca(2+), synaptotagmin-1, and PIP(2) is crucial for neurotransmitter release.