Direct quantitative detection of Doc2b-induced hemifusion in optically trapped membranes

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 as...

Full description

Saved in:
Bibliographic Details
Published in:Nature communications 2015-09, Vol.6 (1), p.8387-8387, Article 8387
Main Authors: Brouwer, Ineke, Giniatullina, Asiya, Laurens, Niels, van Weering, Jan R. T., Bald, Dirk, Wuite, Gijs J. L., Groffen, Alexander J.
Format: Article
Language:English
Subjects:
14/19
631/45/56
631/57/2270
631/80/313/2104
82/62
82/80
82/83
Calcium - chemistry
Calcium-Binding Proteins - chemistry
Humanities and Social Sciences
Membranes, Artificial
multidisciplinary
Nerve Tissue Proteins - chemistry
Phosphatidylserines - chemistry
Phospholipids - chemistry
Science
Science (multidisciplinary)
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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. Membrane fusion in cells is triggered by an increase in Ca 2+ and involves SNARE complexes and calcium-sensing proteins, but the mechanism underlying the Ca 2+ -sensors’ role in fusion remains unclear. Here the authors show in vitro that the Ca 2+ -sensor Doc2b acts directly on membranes and induces a hemifusion intermediate in the presence of calcium.
ISSN:2041-1723
2041-1723
DOI:10.1038/ncomms9387