title: Direct quantitative detection of Doc2b-induced hemifusion in optically trapped membranes.
abstract: Ca(2+)-sensor proteins control the secretion of many neuroendocrine substances. Calcium-secretion coupling may involve several mechanisms. First, Ca(2+)-dependent association of their tandem C2 domains with phosphatidylserine may induce membrane curvature and thereby enhance fusion. Second, their association with SNARE complexes may inhibit membrane fusion in the absence of a Ca(2+) trigger. Here we present a method using two optically trapped beads coated with SNARE-free synthetic membranes to elucidate the direct role of the C2AB domain of the soluble Ca(2+)-sensor Doc2b. Contacting membranes are often coupled by a Doc2b-coated membrane stalk that resists forces up to 600 pN upon bead separation. Stalk formation depends strictly on Ca(2+) and phosphatidylserine. Real-time fluorescence imaging shows phospholipid but not content mixing, indicating membrane hemifusion. Thus, Doc2b acts directly on membranes and stabilizes the hemifusion intermediate in this cell-free system. In living cells, this mechanism may co-occur with progressive SNARE complex assembly, together defining Ca(2+)-secretion coupling.
authors.affiliation: Department of Physics and Astronomy, LaserLab, VU University, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
authors.forename: Ineke
authors.initials: I
authors.lastname: Brouwer
authors.affiliation: Department of Functional Genomics and Clinical Genetics, CNCR, NCA, VU University and VU Medical Center, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
authors.forename: Asiya
authors.initials: A
authors.lastname: Giniatullina
authors.affiliation: Department of Physics and Astronomy, LaserLab, VU University, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
authors.forename: Niels
authors.initials: N
authors.lastname: Laurens
authors.affiliation: Department of Functional Genomics and Clinical Genetics, CNCR, NCA, VU University and VU Medical Center, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
authors.forename: Jan R T
authors.initials: JRT
authors.lastname: van Weering
authors.affiliation: Department of Molecular Cell Biology, VU University, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
authors.forename: Dirk
authors.initials: D
authors.lastname: Bald
authors.affiliation: Department of Physics and Astronomy, LaserLab, VU University, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
authors.forename: Gijs J L
authors.initials: GJL
authors.lastname: Wuite
authors.affiliation: Department of Functional Genomics and Clinical Genetics, CNCR, NCA, VU University and VU Medical Center, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Groffen
mesh_terms: D002118:Calcium / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D008567:Membranes, Artificial; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D010718:Phosphatidylserines / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry
chemical_list: D002135:Calcium-Binding Proteins; C490422:Doc2b protein, rat; D008567:Membranes, Artificial; D009419:Nerve Tissue Proteins; D010718:Phosphatidylserines; D010743:Phospholipids; D002118:Calcium