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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

Characteristics of the PSF in cells overexpressing CSP and its phosphomutants.A, The amperometric recording for a full-fusion event from a single vesicle. The prespike foot (PSF, shown as shaded area) reports the initial opening of a fusion pore and the spike begins when the fusion pore enters into the dilation phase. The PSF lifetime was measured from onset (the current rising to 1×RMS noise above the baseline current) to end point (the intersection between the baseline and the red line going through the rise phase from 35 to 60% of the peak). The features of PSF and spikes analyzed in this study are illustrated (see Materials and Methods for detail). B, PSF lifetime distributions were constructed for the indicated groups. Distributions were fitted by the single-exponential decay function N(t) = N(0)×exp (−t/τ) to yield the mean PSF duration τ (the goodness of the fits: R2∼0.90). C, τ obtained in (B) for each group. Numbers in bars indicate the number of PSF used for each calculation. D, PSF mean amplitudes calculated by the cellular means method. E, PSF areas calculated by the cellular means method. For D and E, numbers in bars indicate the number of cells used for each calculation.
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pone-0099180-g002: Characteristics of the PSF in cells overexpressing CSP and its phosphomutants.A, The amperometric recording for a full-fusion event from a single vesicle. The prespike foot (PSF, shown as shaded area) reports the initial opening of a fusion pore and the spike begins when the fusion pore enters into the dilation phase. The PSF lifetime was measured from onset (the current rising to 1×RMS noise above the baseline current) to end point (the intersection between the baseline and the red line going through the rise phase from 35 to 60% of the peak). The features of PSF and spikes analyzed in this study are illustrated (see Materials and Methods for detail). B, PSF lifetime distributions were constructed for the indicated groups. Distributions were fitted by the single-exponential decay function N(t) = N(0)×exp (−t/τ) to yield the mean PSF duration τ (the goodness of the fits: R2∼0.90). C, τ obtained in (B) for each group. Numbers in bars indicate the number of PSF used for each calculation. D, PSF mean amplitudes calculated by the cellular means method. E, PSF areas calculated by the cellular means method. For D and E, numbers in bars indicate the number of cells used for each calculation.

Mentions: In amperometric recordings, the small rising signal that precedes a steep spike is referred to as a PSF, and it represents the transient opening of the initial fusion pore prior to dilation (Fig. 2A, shaded area). To examine if CSPα phosphomutation affects the opening of this initial fusion pore, we analyzed the characteristics of the PSF associated with spikes. For each transfection group, we constructed the histograms of PSF lifetimes in a semi-logarithmic plot (termed PSF lifetime distributions) and fitted the data by a single-exponential decay function (Fig. 2B) [3], [6]. The fitted lines gave the mean PSF duration, τ (Fig. 2B and C). The WT group's τ was similar to that for Ctrl and S10A groups. The S10D and S10E groups had comparable τ's but both of these were higher than those for other groups: the S10D group's τ was significantly higher than that for S10A (p<0.05) and WT (p<0.05) groups; the S10E's τ was significantly higher than that for S10A (p<0.001), WT (p<0.01), and Ctrl (p<0.05) groups. These results suggest that CSPα phosphomimetic mutation prolongs the opening of the fusion pore that leads to dilation. Notably, the mean amplitude of the PSF showed no significant difference across groups (Fig. 2D), suggesting that CSPα phosphomutation does not affect the flux through the initial fusion pore. In comparison, the PSF area was significantly increased by phosphomimetic mutants: the S10D group's PSF area was significantly higher than that for WT (p<0.05) groups; the S10E's PSF area was significantly higher than that for S10A (p<0.05), WT (p<0.05), and Ctrl (p<0.05) groups (Fig. 2E). Thus, CSPα phosphomimetic mutation may alter the kinetics of the initial fusion pore that forms prior to dilation.


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)

Characteristics of the PSF in cells overexpressing CSP and its phosphomutants.A, The amperometric recording for a full-fusion event from a single vesicle. The prespike foot (PSF, shown as shaded area) reports the initial opening of a fusion pore and the spike begins when the fusion pore enters into the dilation phase. The PSF lifetime was measured from onset (the current rising to 1×RMS noise above the baseline current) to end point (the intersection between the baseline and the red line going through the rise phase from 35 to 60% of the peak). The features of PSF and spikes analyzed in this study are illustrated (see Materials and Methods for detail). B, PSF lifetime distributions were constructed for the indicated groups. Distributions were fitted by the single-exponential decay function N(t) = N(0)×exp (−t/τ) to yield the mean PSF duration τ (the goodness of the fits: R2∼0.90). C, τ obtained in (B) for each group. Numbers in bars indicate the number of PSF used for each calculation. D, PSF mean amplitudes calculated by the cellular means method. E, PSF areas calculated by the cellular means method. For D and E, numbers in bars indicate the number of cells used for each calculation.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099180-g002: Characteristics of the PSF in cells overexpressing CSP and its phosphomutants.A, The amperometric recording for a full-fusion event from a single vesicle. The prespike foot (PSF, shown as shaded area) reports the initial opening of a fusion pore and the spike begins when the fusion pore enters into the dilation phase. The PSF lifetime was measured from onset (the current rising to 1×RMS noise above the baseline current) to end point (the intersection between the baseline and the red line going through the rise phase from 35 to 60% of the peak). The features of PSF and spikes analyzed in this study are illustrated (see Materials and Methods for detail). B, PSF lifetime distributions were constructed for the indicated groups. Distributions were fitted by the single-exponential decay function N(t) = N(0)×exp (−t/τ) to yield the mean PSF duration τ (the goodness of the fits: R2∼0.90). C, τ obtained in (B) for each group. Numbers in bars indicate the number of PSF used for each calculation. D, PSF mean amplitudes calculated by the cellular means method. E, PSF areas calculated by the cellular means method. For D and E, numbers in bars indicate the number of cells used for each calculation.
Mentions: In amperometric recordings, the small rising signal that precedes a steep spike is referred to as a PSF, and it represents the transient opening of the initial fusion pore prior to dilation (Fig. 2A, shaded area). To examine if CSPα phosphomutation affects the opening of this initial fusion pore, we analyzed the characteristics of the PSF associated with spikes. For each transfection group, we constructed the histograms of PSF lifetimes in a semi-logarithmic plot (termed PSF lifetime distributions) and fitted the data by a single-exponential decay function (Fig. 2B) [3], [6]. The fitted lines gave the mean PSF duration, τ (Fig. 2B and C). The WT group's τ was similar to that for Ctrl and S10A groups. The S10D and S10E groups had comparable τ's but both of these were higher than those for other groups: the S10D group's τ was significantly higher than that for S10A (p<0.05) and WT (p<0.05) groups; the S10E's τ was significantly higher than that for S10A (p<0.001), WT (p<0.01), and Ctrl (p<0.05) groups. These results suggest that CSPα phosphomimetic mutation prolongs the opening of the fusion pore that leads to dilation. Notably, the mean amplitude of the PSF showed no significant difference across groups (Fig. 2D), suggesting that CSPα phosphomutation does not affect the flux through the initial fusion pore. In comparison, the PSF area was significantly increased by phosphomimetic mutants: the S10D group's PSF area was significantly higher than that for WT (p<0.05) groups; the S10E's PSF area was significantly higher than that for S10A (p<0.05), WT (p<0.05), and Ctrl (p<0.05) groups (Fig. 2E). Thus, CSPα phosphomimetic mutation may alter the kinetics of the initial fusion pore that forms prior to dilation.

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