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Phosphomimetic mutation of cysteine string protein-α increases the rate of regulated exocytosis by modulating fusion pore dynamics in PC12 cells.

Chiang N, Hsiao YT, Yang HJ, Lin YC, Lu JC, Wang CT - PLoS ONE (2014)

Bottom Line: These seemingly conflicting results lead to a hypothesis that CSPα may undergo a modification that switches its function in regulating neurotransmitter and hormone secretion.We found that the secretion rate was significantly increased in cells overexpressing S10D or S10E 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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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 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/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.

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Related in: MedlinePlus

KR events in cells overexpressing CSP and its phosphomutants.A, Mean amplitude histograms of KR events fitted by Gaussian distributions (red) for all groups. The mean values and R2 from Gaussian distributions were as indicated. B, Scatter plots of mean amplitudes vs. durations for KR events. The R2 from linear regression (red) were as indicated. For A and B, n = 797–1112 events. C, Scatter plots of PSF mean amplitudes vs. durations. The R2 from linear regression (red) were as indicated. Total 105–242 PSF events.
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pone-0099180-g004: KR events in cells overexpressing CSP and its phosphomutants.A, Mean amplitude histograms of KR events fitted by Gaussian distributions (red) for all groups. The mean values and R2 from Gaussian distributions were as indicated. B, Scatter plots of mean amplitudes vs. durations for KR events. The R2 from linear regression (red) were as indicated. For A and B, n = 797–1112 events. C, Scatter plots of PSF mean amplitudes vs. durations. The R2 from linear regression (red) were as indicated. Total 105–242 PSF events.

Mentions: We found that in all groups, these mean amplitude distributions for putative KR events (the signals with peak amplitudes of 2–3.5 pA corresponding to mean amplitudes of 0.3–2 pA) can be fitted by a single Gaussian distribution (Fig. 4A), suggesting that these square-like events arise from a single distinct population as reported previously [6]. Moreover, the mean amplitude of putative KR events (Fig. 4A) were comparable to those of PSF (Fig. 2D), suggesting that the KR events were from the non-dilating fusion pore. In contrast, the mean amplitude distributions for the events ≥3.5 pA were relatively skewed (the signals with peak amplitudes ≥3.5 pA corresponding to mean amplitudes >1 pA) (Figure S3), similar to the previous reports for spike height or area [6], [40], [41], [42], [43].


Phosphomimetic mutation of cysteine string protein-α increases the rate of regulated exocytosis by modulating fusion pore dynamics in PC12 cells.

Chiang N, Hsiao YT, Yang HJ, Lin YC, Lu JC, Wang CT - PLoS ONE (2014)

KR events in cells overexpressing CSP and its phosphomutants.A, Mean amplitude histograms of KR events fitted by Gaussian distributions (red) for all groups. The mean values and R2 from Gaussian distributions were as indicated. B, Scatter plots of mean amplitudes vs. durations for KR events. The R2 from linear regression (red) were as indicated. For A and B, n = 797–1112 events. C, Scatter plots of PSF mean amplitudes vs. durations. The R2 from linear regression (red) were as indicated. Total 105–242 PSF events.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4067274&req=5

pone-0099180-g004: KR events in cells overexpressing CSP and its phosphomutants.A, Mean amplitude histograms of KR events fitted by Gaussian distributions (red) for all groups. The mean values and R2 from Gaussian distributions were as indicated. B, Scatter plots of mean amplitudes vs. durations for KR events. The R2 from linear regression (red) were as indicated. For A and B, n = 797–1112 events. C, Scatter plots of PSF mean amplitudes vs. durations. The R2 from linear regression (red) were as indicated. Total 105–242 PSF events.
Mentions: We found that in all groups, these mean amplitude distributions for putative KR events (the signals with peak amplitudes of 2–3.5 pA corresponding to mean amplitudes of 0.3–2 pA) can be fitted by a single Gaussian distribution (Fig. 4A), suggesting that these square-like events arise from a single distinct population as reported previously [6]. Moreover, the mean amplitude of putative KR events (Fig. 4A) were comparable to those of PSF (Fig. 2D), suggesting that the KR events were from the non-dilating fusion pore. In contrast, the mean amplitude distributions for the events ≥3.5 pA were relatively skewed (the signals with peak amplitudes ≥3.5 pA corresponding to mean amplitudes >1 pA) (Figure S3), similar to the previous reports for spike height or area [6], [40], [41], [42], [43].

Bottom Line: These seemingly conflicting results lead to a hypothesis that CSPα may undergo a modification that switches its function in regulating neurotransmitter and hormone secretion.We found that the secretion rate was significantly increased in cells overexpressing S10D or S10E 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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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 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/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.

Show MeSH
Related in: MedlinePlus