title: A Chemical Controller of SNARE-Driven Membrane Fusion That Primes Vesicles for Ca(2+)-Triggered Millisecond Exocytosis.
abstract: Membrane fusion is mediated by the SNARE complex which is formed through a zippering process. Here, we developed a chemical controller for the progress of membrane fusion. A hemifusion state was arrested by a polyphenol myricetin which binds to the SNARE complex. The arrest of membrane fusion was rescued by an enzyme laccase that removes myricetin from the SNARE complex. The rescued hemifusion state was metastable and long-lived with a decay constant of 39 min. This membrane fusion controller was applied to delineate how Ca(2+) stimulates fusion-pore formation in a millisecond time scale. We found, using a single-vesicle fusion assay, that such myricetin-primed vesicles with synaptotagmin 1 respond synchronously to physiological concentrations of Ca(2+). When 10 μM Ca(2+) was added to the hemifused vesicles, the majority of vesicles rapidly advanced to fusion pores with a time constant of 16.2 ms. Thus, the results demonstrate that a minimal exocytotic membrane fusion machinery composed of SNAREs and synaptotagmin 1 is capable of driving membrane fusion in a millisecond time scale when a proper vesicle priming is established. The chemical controller of SNARE-driven membrane fusion should serve as a versatile tool for investigating the differential roles of various synaptic proteins in discrete fusion steps.
authors.affiliation: Department of Genetic Engineering, College of Biotechnology and Bioengineering, and Center for Human Interface Nano Technology, Sungkyunkwan University , Suwon, Gyeonggi-do 440-746, South Korea.
authors.forename: Paul
authors.initials: P
authors.lastname: Heo
authors.affiliation: Department of Genetic Engineering, College of Biotechnology and Bioengineering, and Center for Human Interface Nano Technology, Sungkyunkwan University , Suwon, Gyeonggi-do 440-746, South Korea.|Biomedical Research Institute, Korea Institute of Science and Technology , Seoul 136-791, South Korea.
authors.forename: Yoosoo
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Howard Hughes Medical Institute , Baltimore, Maryland 21205, United States.|CREOL, The College of Optics & Photonics, University of Central Florida , Orlando, Florida 32816, United States.
authors.forename: Kyu Young
authors.initials: KY
authors.lastname: Han
authors.affiliation: Department of Genetic Engineering, College of Biotechnology and Bioengineering, and Center for Human Interface Nano Technology, Sungkyunkwan University , Suwon, Gyeonggi-do 440-746, South Korea.
authors.forename: Byoungjae
authors.initials: B
authors.lastname: Kong
authors.affiliation: Department of Genetic Engineering, College of Biotechnology and Bioengineering, and Center for Human Interface Nano Technology, Sungkyunkwan University , Suwon, Gyeonggi-do 440-746, South Korea.
authors.forename: Jong-Hyeok
authors.initials: JH
authors.lastname: Shin
authors.affiliation: Department of Genetic Engineering, College of Biotechnology and Bioengineering, and Center for Human Interface Nano Technology, Sungkyunkwan University , Suwon, Gyeonggi-do 440-746, South Korea.
authors.forename: Younghoon
authors.initials: Y
authors.lastname: Jung
authors.affiliation: Biomedical Research Institute, Korea Institute of Science and Technology , Seoul 136-791, South Korea.
authors.forename: Cherlhyun
authors.initials: C
authors.lastname: Jeong
authors.affiliation: Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University , Ames, Iowa 50011, United States.
authors.forename: Jaeil
authors.initials: J
authors.lastname: Shin
authors.affiliation: Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University , Ames, Iowa 50011, United States.
authors.forename: Yeon-Kyun
authors.initials: YK
authors.lastname: Shin
authors.affiliation: Howard Hughes Medical Institute , Baltimore, Maryland 21205, United States.|Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine , Baltimore, Maryland 21205, United States.|Department of Biophysics, Johns Hopkins University , Baltimore, Maryland 21218, United States.|Department of Biomedical Engineering, Johns Hopkins University , Baltimore, Maryland 21205, United States.
authors.forename: Taekjip
authors.initials: T
authors.lastname: Ha
authors.affiliation: Department of Genetic Engineering, College of Biotechnology and Bioengineering, and Center for Human Interface Nano Technology, Sungkyunkwan University , Suwon, Gyeonggi-do 440-746, South Korea.
authors.forename: Dae-Hyuk
authors.initials: DH
authors.lastname: Kweon
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005089:Exocytosis; D005419:Flavonoids / Q000378:metabolism; D042845:Laccase / Q000378:metabolism; D008561:Membrane Fusion; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D050600:SNARE Proteins / Q000378:metabolism*; D050863:Synaptotagmin I / Q000378:metabolism
chemical_list: D005419:Flavonoids; D009419:Nerve Tissue Proteins; D050600:SNARE Proteins; D050863:Synaptotagmin I; C040015:myricetin; D042845:Laccase; D002118:Calcium