Kim, Jae-Yeol Choi, Bong-Kyu Choi, Mal-Gi Kim, Sun-Ae Lai, Ying Shin, Yeon-Kyun Lee, Nam Ki 2024-01-20T15:00:50Z 2024-01-20T15:00:50Z 2021-09-05 2012-05 0261-4189 https://pubs.kist.re.kr/handle/201004/129279 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 10(3) 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. The EMBO Journal (2012) 31, 2144-2155. doi:10.1038/emboj.2012.57; Published online 9 March 2012 English Nature Publishing Group Solution single-vesicle assay reveals PIP2-mediated sequential actions of synaptotagmin-1 on SNAREs Article 10.1038/emboj.2012.57 1 The EMBO Journal, v.31, no.9, pp.2144 - 2155 The EMBO Journal 31 9 2144 2155 N scie scopus 000303596900009 2-s2.0-84862778677 Biochemistry & Molecular Biology Cell Biology Biochemistry & Molecular Biology Cell Biology Article ALTERNATING-LASER EXCITATION DEPENDENT MEMBRANE-FUSION NEUROTRANSMITTER RELEASE TRANSMITTER RELEASE IN-VITRO REGULATED EXOCYTOSIS HIPPOCAMPAL-NEURONS SECRETORY VESICLES PLASMA-MEMBRANE CENTRAL SYNAPSE alternating-laser excitation (ALEX) exocytosis single-molecule FRET SNARE synaptotagmin-1