Investigation of Neuronal Membrane Fusion Using Fluorescence Correlation Spectroscopy

Neurotransmission requires rapid fusion of synaptic vesicles with the presynaptic membrane. This fusion is triggered by calcium influx and promoted by SNARE assembly. The vesicular protein synaptotagmin-1 acts as a calcium sensor and its trans-tethering to the plasma membrane is an essential step in the membrane fusion process. In this study, several details in membrane fusion have been investigated and discussed. First, the molecular requirements of the trans- and cis-membrane interaction of synaptotagmin-1 have been analyzed. All of the three binding sites of synaptotagmin-1 (the C2A domain, the C2B domain and the polylysine patch) are involved in both of these processes. Cis-binding to the host membrane containing 20% phosphatidylserine seems to be a stable state and can be overcome by neither trans-binding between synaptotagmin-1 and the target membrane containing acidic phospholipids, nor by the interaction between synaptotagmin-1 and SNAREs inserted into the acceptor membrane. Similarly, with 12% phosphatidylserine in the host membrane-a situation comparable to that in wild-type vesicles-cis-binding is found nearly exclusively. With 5% phosphatidylserine, however, the presence of either phosphatidylinositol-4,5-bisphosphate in the target membrane or calcium in the solution can invoke conversion to trans-binding with great efficiency. Second, liposome cross-linking can be mediated by soluble C2AB fragments, when all the binding sites for C2AB are saturated on the liposome surface. To rationalize this result, it is assumed that binding with only the C2A- or the C2B domain might occur under these conditions. The other-unbound-domain of the same synaptotagmin molecule will eventually lead to cross-linking, if it attaches to a different liposome. This finding confirms the observation of cis-binding and explains some of the conflicts in the existing literature. Apparently, a subtle balance between cis- and trans-binding of synaptotagmin-1 may play an important role in the regulation of neuronal membrane fusion. Moreover, first data for the characterization of mouse synaptic vesicles, in terms of average protein mass per vesicle, was measured in this study. The results obtained for mouse synaptic vesicles are similar to those of rat synaptic vesicles and offer a single vesicle basis for further investigating mouse synaptic vesicles as well as membrane fusion in a quantitative fashion. Finally, details of the inhibiting effect of α-SNAP on the fusion of chromaffin granules with larger liposomes were also investigated in the present study. The experimental findings suggest that α-SNAP inhibits fusion of chromaffin granules and stops the SNARE assembly at the C-terminal site, so that partial SNARE zippering can still mediate docking of chromaffin granules.