Calcium-Mediated Docking and Fusion of Purified Dense Core Vesicles with Reconstituted Membranes

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. This is mediated by SNARE proteins which catalyze membrane fusion of the secretory vesicle to the plasma membrane. While SNAREs are sufficient to catalyze fusion in vitro, they do not convey calcium sensitivity to the process. The calcium sensor is thought to be the protein synaptotagmin, which has two calcium binding C2 domains and resides on the secretory vesicle. To investigate the calcium response of native secretory vesicles, we developed a novel iso-osmotic fractionation procedure to purify dense core vesicles (DCVs) from PC12 cells, an immortalized neuroendocrine cell line. Fusion assays based on bulk lipid mixing of DCVs with proteoliposomes containing a highly reactive SNARE acceptor complex were used to determine the overall calcium response of fusion. A fluorescently tagged secretory protein, neuropeptide Y (NPY), was used to specifically label the contents of the DCVs to monitor single vesicle docking and fusion events to a planar supported bilayer containing reconstituted acceptor SNARE complexes. The results show that docking and fusion both depend on calcium, but in different fashions, indicating that different molecular players contribute to each sub-reaction. Knock downs using shRNA were used to determine which resident DCV proteins are responsible for the calcium response of docking and fusion. Soluble accessory proteins known to modulate SNARE proteins were added to fusion assays to determine the molecular requirements to have a fully calcium dependent SNARE mediated fusion process.