Mal-Gi Choi

Large α-synuclein oligomers inhibit neuronal SNARE-mediated vesicle docking.

Parkinson disease and dementia with Lewy bodies are featured with the formation of Lewy bodies composed mostly of α-synuclein (α-Syn) in the brain. Although evidence indicates that the large oligomeric or protofibril forms of α-Syn are neurotoxic agents, the detailed mechanisms of the toxic functions of the oligomers remain unclear. Here, we show that large α-Syn oligomers efficiently inhibit neuronal SNARE-mediated vesicle lipid mixing. Large α-Syn oligomers preferentially bind to the N-terminal domain of a vesicular SNARE protein, synaptobrevin-2, which blocks SNARE-mediated lipid mixing by preventing SNARE complex formation. In sharp contrast, the α-Syn monomer has a negligible effect on lipid mixing even with a 30-fold excess compared with the case of large α-Syn oligomers. Thus, the results suggest that large α-Syn oligomers function as inhibitors of dopamine release, which thus provides a clue, at the molecular level, to their neurotoxicity.

Solution single-vesicle assay reveals PIP2-mediated sequential actions of synaptotagmin-1 on SNAREs.

Synaptotagmin‐1 (Syt1) is a major Ca2+ sensor for synchronous neurotransmitter release, which requires vesicle fusion mediated by SNAREs (soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors). Syt1 utilizes its diverse interactions with target membrane (t‐) SNARE, SNAREpin, and phospholipids, to regulate vesicle fusion. To dissect the functions of Syt1, we apply a single‐molecule technique, alternating‐laser excitation (ALEX), which is capable of sorting out subpopulations of fusion intermediates and measuring their kinetics in solution. The results show that Syt1 undergoes at least three distinct steps prior to lipid mixing. First, without Ca2+, Syt1 mediates vesicle docking by directly binding to t‐SNARE/phosphatidylinositol 4,5‐biphosphate (PIP2) complex and increases the docking rate by 103 times. Second, synaptobrevin‐2 binding to t‐SNARE displaces Syt1 from SNAREpin. Third, with Ca2+, Syt1 rebinds to SNAREpin, which again requires PIP2. Thus without Ca2+, Syt1 may bring vesicles to the plasma membrane in proximity via binding to t‐SNARE/PIP2 to help SNAREpin formation and then, upon Ca2+ influx, it may rebind to SNAREpin, which may trigger synchronous fusion. The results show that ALEX is a powerful method to dissect multiple kinetic steps in the vesicle fusion pathway.

Real-Time Observation of Multiple-Protein Complex Formation with Single-Molecule FRET

Current single-molecule techniques do not permit the real-time observation of multiple proteins interacting closely with each other. We here report an approach enabling us to determine the single-molecule fluorescence resonance energy transfer (FRET) kinetics of multiple protein-protein interactions occurring far below the diffraction limit. We observe a strongly cooperative formation of multimeric soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, which suggests that formation of the first SNARE complex triggers a cascade of SNARE complex formation.

Mechanical Unzipping and Rezipping of a Single SNARE Complex Reveals Large Hysteresis as the Force Generating Mechanism

Formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex provides mechanical thrust for membrane fusion, but its molecular mechanism is still unclear. Here using magnetic tweezers, we observe mechanical responses of a single neuronal SNARE complex under constant pulling force. Single SNARE complexes may be unzipped with 34 pN force. When rezipping is induced by lowering the force to 11 pN, only a partially assembled state results, with the C-terminal half of the SNARE complex remaining disassembled. Reassembly of the C-terminal half occurs only when the force is further lowered below 11 pN. Thus, mechanical hysteresis, characterized by the unzipping and rezipping cycle of a single SNARE complex, produces the partially assembled state. In this metastable state, unzipping toward the N-terminus is suppressed while zippering toward the C-terminus is initiated as a steep function of force. This ensures the directionality of SNARE-complex formation, making the SNARE complex a robust force-generating machine.