TY - JOUR
T1 - Direct quantitative detection of Doc2b-induced hemifusion in optically trapped membranes
AU - Brouwer, Ineke
AU - Giniatullina, Asiya
AU - Laurens, Niels
AU - Van Weering, Jan R.T.
AU - Bald, Dirk
AU - Wuite, Gijs J.L.
AU - Groffen, Alexander J.
N1 - Funding Information: We thank Dr Maarten Noom for his help with preliminary experiments and Rien Dekker for technical assistance. This study was supported by the Netherlands Organization for Scientific Research (grant numbers 91111009, 021002077 and a VICI grant to G.J.L.W.), Funding Information: the Netherlands Organization for Health Research and Development (ZonMW, 91113022), an ERC starting grant and a FOM program grant to G.J.L.W. Publisher Copyright: © 2015 Macmillan Publishers Limited. All rights reserved.
PY - 2015/9/23
Y1 - 2015/9/23
N2 - 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 600a €‰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.
AB - 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 600a €‰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.
UR - http://www.scopus.com/inward/record.url?scp=84942163114&partnerID=8YFLogxK
U2 - https://doi.org/10.1038/ncomms9387
DO - https://doi.org/10.1038/ncomms9387
M3 - Article
C2 - 26395669
SN - 2041-1723
VL - 6
SP - 520A-520A
JO - Nature communications
JF - Nature communications
IS - 3, Supplement 1
M1 - 8387
ER -