title: Phosphomimetic mutation of cysteine string protein-α increases the rate of regulated exocytosis by modulating fusion pore dynamics in PC12 cells.
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.
  
  
  RESULTS
  Using amperometry, we investigated how phosphorylation at Ser10 of CSPα (CSPα-Ser10) 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
  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.
authors: Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan; Department of Life Science, National Taiwan University, Taipei, Taiwan; Neurobiology and Cognitive Science Center, National Taiwan University, Taipei, Taiwan.
authors: Ning
authors: N
authors: Chiang
authors: Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan.
authors: Yu-Tien
authors: YT
authors: Hsiao
authors: Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan.
authors: Hui-Ju
authors: HJ
authors: Yang
authors: Department of Physiology and Pharmacology, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
authors: Yu-Chun
authors: YC
authors: Lin
authors: Department of Physiology and Pharmacology, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
authors: Juu-Chin
authors: JC
authors: Lu
authors: Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan; Department of Life Science, National Taiwan University, Taipei, Taiwan; Neurobiology and Cognitive Science Center, National Taiwan University, Taipei, Taiwan; Genome and Systems Biology Degree Program, National Taiwan University, Taipei, Taiwan.
authors: Chih-Tien
authors: CT
authors: Wang
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D005089:Exocytosis; D050956:HSP40 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D016716:PC12 Cells; D010766:Phosphorylation; D051381:Rats
chemical_list: D050956:HSP40 Heat-Shock Proteins; D008565:Membrane Proteins; D050505:Mutant Proteins; C088460:cysteine string protein