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Sci Adv. 2017 Jul 19;3(7):e1603208. doi: 10.1126/sciadv.1603208. eCollection 2017 Jul.

Reconstitution of calcium-mediated exocytosis of dense-core vesicles.

Author information

1
Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA 22908, USA.
2
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908, USA.
3
Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
4
Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA.

Abstract

Regulated exocytosis is a process by which neurotransmitters, hormones, and secretory proteins are released from the cell in response to elevated levels of calcium. In cells, secretory vesicles are targeted to the plasma membrane, where they dock, undergo priming, and then fuse with the plasma membrane in response to calcium. The specific roles of essential proteins and how calcium regulates progression through these sequential steps are currently incompletely resolved. We have used purified neuroendocrine dense-core vesicles and artificial membranes to reconstruct in vitro the serial events that mimic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent membrane docking and fusion during exocytosis. Calcium recruits these vesicles to the target membrane aided by the protein CAPS (calcium-dependent activator protein for secretion), whereas synaptotagmin catalyzes calcium-dependent fusion; both processes are dependent on phosphatidylinositol 4,5-bisphosphate. The soluble proteins Munc18 and complexin-1 are necessary to arrest vesicles in a docked state in the absence of calcium, whereas CAPS and/or Munc13 are involved in priming the system for an efficient fusion reaction.

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