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

Author

Listed:
  • Ineke Brouwer

    (LaserLab, VU University)

  • Asiya Giniatullina

    (CNCR, NCA, VU University and VU Medical Center)

  • Niels Laurens

    (LaserLab, VU University)

  • Jan R. T. van Weering

    (CNCR, NCA, VU University and VU Medical Center)

  • Dirk Bald

    (VU University)

  • Gijs J. L. Wuite

    (LaserLab, VU University)

  • Alexander J. Groffen

    (CNCR, NCA, VU University and VU Medical Center)

Abstract

Ca2+-sensor proteins control the secretion of many neuroendocrine substances. Calcium-secretion coupling may involve several mechanisms. First, Ca2+-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 Ca2+ 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 Ca2+-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 Ca2+ 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 Ca2+-secretion coupling.

Suggested Citation

  • Ineke Brouwer & Asiya Giniatullina & Niels Laurens & Jan R. T. van Weering & Dirk Bald & Gijs J. L. Wuite & Alexander J. Groffen, 2015. "Direct quantitative detection of Doc2b-induced hemifusion in optically trapped membranes," Nature Communications, Nature, vol. 6(1), pages 1-8, December.
    • Handle: RePEc:nat:natcom:v:6:y:2015:i:1:d:10.1038_ncomms9387
    DOI: 10.1038/ncomms9387
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    Cited by:

    1. Thomas Litschel & Charlotte F. Kelley & Xiaohang Cheng & Leon Babl & Naoko Mizuno & Lindsay B. Case & Petra Schwille, 2024. "Membrane-induced 2D phase separation of the focal adhesion protein talin," Nature Communications, Nature, vol. 15(1), pages 1-13, December.
    2. Bar Manori & Alisa Vaknin & Pavla Vaňková & Anat Nitzan & Ronen Zaidel-Bar & Petr Man & Moshe Giladi & Yoni Haitin, 2024. "Chloride intracellular channel (CLIC) proteins function as fusogens," Nature Communications, Nature, vol. 15(1), pages 1-14, December.

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