Phosphomimetic mutation of cysteine string protein-α Increases the rate of regulated exocytosis by modulating fusion pore dynamics in PC12 cells
Journal
PLoS ONE
Journal Volume
9
Journal Issue
6
Date Issued
2014-06-23
Author(s)
Abstract
Background: Cysteine string protein-α (CSPα) is a chaperone to ensure protein folding. Loss of CSPα function associates with many neurological diseases. However, its function in modulating regulated exocytosis remains elusive. Although cspα-knockouts exhibit impaired synaptic transmission, overexpression of CSPα in neuroendocrine cells inhibits secretion. These seemingly conflicting results lead to a hypothesis that CSPα may undergo a modification that switches its function in regulating neurotransmitter and hormone secretion. Previous studies implied that CSPα undergoes phosphorylation at Ser10 that may influence exocytosis by altering fusion pore dynamics. However, direct evidence is missing up to date. Methodology/Principal Findings: Using amperometry, we investigated how phosphorylation at Ser 10 of CSPα (CSPα-Ser 10 ) modulates regulated exocytosis and if this modulation involves regulating a specific kinetic step of fusion pore dynamics. The real-time exocytosis of single vesicles was detected in PC12 cells overexpressing control vector, wild-type CSPα (WT), the CSPα phosphodeficient mutant (S10A), or the CSPα phosphomimetic mutants (S10D and S10E). The shapes of amperometric signals were used to distinguish the full-fusion events (i.e., prespike feet followed by spikes) and the kiss-and-run events (i.e., square-shaped flickers). We found that the secretion rate was significantly increased in cells overexpressing S10D or S10E compared to WT or S10A. Further analysis showed that overexpression of S10D or S10E prolonged fusion pore lifetime compared to WT or S10A. The fraction of kiss-and-run events was significantly lower but the frequency of full-fusion events was higher in cells overexpressing S10D or S10E compared to WT or S10A. Advanced kinetic analysis suggests that overexpression of S10D or S10E may stabilize open fusion pores mainly by inhibiting them from closing. Conclusions/Significance: CSPα may modulate fusion pore dynamics in a phosphorylation-dependent manner. Therefore, through changing its phosphorylated state influenced by diverse cellular signalings, CSPα may have a great capacity to modulate the rate of regulated exocytosis. © 2014 Chiang et al.
Other Subjects
cysteine string protein; cysteine string protein alpha; unclassified drug; cysteine string protein; heat shock protein 40; membrane protein; mutant protein; amperometry; article; cell activity; controlled study; exocytosis; fusion pore dynamics; mutational analysis; PC12 cell line; porosity; protein expression; protein function; protein phosphorylation; protein secretion; wild type; animal; cell membrane; genetics; kinetics; membrane fusion; metabolism; mutation; phosphorylation; rat; secretion (process); Animals; Cell Membrane; Exocytosis; HSP40 Heat-Shock Proteins; Kinetics; Membrane Fusion; Membrane Proteins; Mutant Proteins; Mutation; PC12 Cells; Phosphorylation; Rats
Publisher
PUBLIC LIBRARY SCIENCE
Type
journal article
File(s)![Thumbnail Image]()
Loading...
Name
10.1371_journal.pone.0099180.PDF
Size
987.24 KB
Format
Adobe PDF
Checksum
(MD5):e74d13e9b8647c71cb72be649673eb6e