Complexin arrests a pool of docked vesicles for fast Ca2+-dependent release

• Published in: The EMBO journal 2012-08, Vol.31 (15), p.3270-3281
• Main Authors: Malsam, Jörg, Parisotto, Daniel, Bharat, Tanmay A M, Scheutzow, Andrea, Krause, Jean Michel, Briggs, John A G, Söllner, Thomas H
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Vesicular Transport - chemistry; Adaptor Proteins, Vesicular Transport - metabolism; Adaptor Proteins, Vesicular Transport - physiology; Biochemistry; Biological Transport - drug effects; Biological Transport - physiology; Calcium; Calcium - pharmacology; Electron microscopy; EMBO20; EMBO27; exocytosis; Exocytosis - drug effects; Exocytosis - physiology; fusion; Humans; In Vitro Techniques; Membrane Fusion - drug effects; Membranes; Models, Biological; Molecular biology; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - metabolism; Nerve Tissue Proteins - physiology; Neurons; Physiology; Protein Binding - drug effects; reconstitution; SNARE; Synaptic Transmission - drug effects; synaptic vesicle; Synaptic Vesicles - drug effects; Synaptic Vesicles - metabolism; Synaptic Vesicles - secretion; Synaptotagmin I - chemistry; Synaptotagmin I - metabolism; Synaptotagmin I - physiology; Time Factors; Unilamellar Liposomes - metabolism
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1038/emboj.2012.164

Regulated exocytosis requires that the assembly of the basic membrane fusion machinery is temporarily arrested. Synchronized membrane fusion is then caused by a specific trigger—a local rise of the Ca 2+ concentration. Using reconstituted giant unilamellar vesicles (GUVs), we have analysed the role of complexin and membrane‐anchored synaptotagmin 1 in arresting and synchronizing fusion by lipid‐mixing and cryo‐electron microscopy. We find that they mediate the formation and consumption of docked small unilamellar vesicles (SUVs) via the following sequence of events: Synaptotagmin 1 mediates v‐SNARE‐SUV docking to t‐SNARE‐GUVs in a Ca 2+ ‐independent manner. Complexin blocks vesicle consumption, causing accumulation of docked vesicles. Together with synaptotagmin 1, complexin synchronizes and stimulates rapid fusion of accumulated docked vesicles in response to physiological Ca 2+ concentrations. Thus, the reconstituted assay resolves both the stimulatory and inhibitory function of complexin and mimics key aspects of synaptic vesicle fusion. Regulated exocytosis of synaptic vesicles is triggered by a local increase in calcium levels, relieving a block in the assembly of the membrane fusion machinery. Based on a reconstituted system, this study characterizes the roles of complexin and synaptotagmin 1 in arresting and synchronizing fusion.