Abstract
In neuronal exocytosis, neurotransmitters are released via membrane fusion between synaptic vesicle and presynaptic plasma membrane. Assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) drive this membrane fusion in a millisecond within the Ca2+ trigger. While SNAREs constitutively catalyze relatively slow membrane fusion processes involved in intracellular vesicle trafficking, the ultrafast synaptic vesicle fusion process is accomplished through multiple protein-protein and protein-lipid interactions. Among the most extensively investigated interactions are those involving the lipid component phosphatidylinositol-4,5-bisphosphate (PIP2) and SNARE complex binding proteins complexin and synaptotagmin. Even though the regulatory function of these factors is indispensable for efficient membrane fusion and proper synaptic communication, the molecular mechanisms that underly their function have not been determined. Many previous study have focused on the interactions between these regulatory proteins and the SNARE complex; however, recent work indicates that the membrane interactions of these proteins may be critical to their function and are functionally indispensable. We speculate that part of the difficulty in understanding the molecular mechanisms of synaptic vesicle fusion originates from a lack of understanding regarding the membrane interactions made by complexin.
One of the main proposed functions of complexin-1 is to inhibit neurotransmitter release in the absence of Ca2+, while synaptotagmin-1 overcomes this inhibition in the presence of Ca2+ to facilitate synchronous neurotransmitter release. Previous work from our lab shows that complexin-1 simultaneously binds both acceptor SNAREs and membranes, where the membrane interactions occur via the terminal domains of complexin-1 in a highly membrane curvature-sensitive manner. In a fusion assay, where the target membrane contains an acceptor SNARE complex consisting of syntaxin-1a and SNAP25, these interactions inhibit fusion by lowering the affinity of the v-SNARE synaptobrevin-2 to the acceptor SNAREs. In other work, the membrane interactions of synaptotagmin-1 were shown to alter the order of the lipid acyl chains, thereby catalyzing a conformational change in the linker region of the SNARE complex and triggering fusion. As a follow-up to this work, this thesis is focused on characterizing the interaction between complexin-1 membranes in lipids of varied composition and in the presence or absence of the closely related players including synaptotagmin-1, PIP2, and the acceptor SNARE complex.
First, titration experiments using fluorescence anisotropy and electron paramagnetic resonance (EPR) demonstrate that complexin-1 not only senses but also modifies membrane curvature. This is evidenced by a self-competitive complexin-1/membrane interaction even at very low protein to lipid ratio. We propose that, in addition to preventing SNARE assembly, complexin-1 might inhibit spontaneous vesicle fusion via the introduction of curvature strain into the membrane bilayer.
Next, complexin-1 is shown to associate with PIP2 in bilayers. This has been observed using both vesicle binding assays that employ EPR or fluorescence anisotropy and using planar supported lipid bilayers that employ total internal reflection microscopy. This is the first time that a preferential association of complexin-1 for PIP2 containing bilayers has been characterized. We have observed that complexin-1 is recruited to the docking sites of dense core vesicles on planar supported bilayers reconstituted with the acceptor SNARE complex, which may be related to the curvature of the dense core vesicles or locally high concentrations of PIP2. We propose that the locally high concentration of complexin-1 at the site of vesicle docking might regulate fusion by locally modifying membrane curvature.
We also tested the idea that synaptotagmin-1 might compete with complexin-1 at the membrane interface and might modulate fusion through its membrane interaction. In a binding assay using total internal reflection microscopy, we find that complexin-1 competes with synaptotagmin-1 in either the presence or absence of the acceptor SNARE complex, but does so only when PIP2 is present in the membrane. Both complexin-1 and synaptotagmin-1 exhibit higher membrane affinity in the presence of PIP2, and we speculate that the competition for the membrane interface may in part be due to the ability of these proteins to sense and modulate membrane curvature.
