[
C.elegans Neuronal Development Meeting,
2008]
Neuropeptides are stored in and secreted from dense core vesicles (DCVs), which are found in many cell types, including neurons and neuroendocrine cells. While synaptic vesicles (SVs) are typically secreted only at an active zone in a neuron, DCVs can fuse almost anywhere in the cell, and our understanding of the molecular machinery required for DCV secretion and the ways in which secretion is regulated is incomplete. We have established a system recently developed by Zhou et al for direct electrophysiological assays of DCV secretion in primary cultured C.elegans neurons. This approach allows examination of vesicle docking, fusion, and release kinetics using total internal reflection fluorescence microscopy (TIRFM) and whole-cell patch clamp recordings. We have recorded from different neuronal cell types marked by GFP under cell-specific promoters, including cholinergic neurons, which release both SVs and DCVs, and neuroendocrine cells containing mostly DCVs. Analysis of the specific role that PKC-1, a protein kinase C ortholog, plays in DCV exocytosis is underway using these methods. PKC-1 regulates secretion of neuropeptides in cholinergic motor neurons. However, a lack of direct functional assays of vesicle secretion has until now prevented a mechanistic understanding of PKC-1''s role in the regulation of DCV exocytosis. We will look at the effects of PKC-1 null mutants and expression of a constitutively active mutant on DCV exocytotic burst kinetics in response to caged calcium photolysis. Additionally, we will employ TIRFM to detect changes in DCV docking and secretion by imaging fluorescently tagged neuropeptides in neuroendocrine cells. We also plan to use this approach to investigate other potential regulators of DCV exocytosis acting in the PKC-1 pathway that we identified in genetic screens for downstream effectors of PKC-1 signaling.