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Regulation of exocytosis and fusion pores by synaptotagmin-effector interactions.

Zhang Z, Hui E, Chapman ER, Jackson MB - Mol. Biol. Cell (2010)

Bottom Line: Syt binds to phosphatidylserine (PS)-containing lipid bilayers as well as to soluble N-ethylmaleimide sensitive factor receptors (SNAREs) and promotes SNARE assembly.All these interactions are regulated by Ca(2+), but their specific roles in distinct kinetic steps of exocytosis are not well understood.By contrast, syt-SNARE interactions and syt-induced SNARE assembly were uncorrelated with rates of exocytosis.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Neurological Disorders and Stroke, Bethesda, MD 20892, USA.

ABSTRACT
Synaptotagmin (syt) serves as a Ca(2+) sensor in the release of neurotransmitters and hormones. This function depends on the ability of syt to interact with other molecules. Syt binds to phosphatidylserine (PS)-containing lipid bilayers as well as to soluble N-ethylmaleimide sensitive factor receptors (SNAREs) and promotes SNARE assembly. All these interactions are regulated by Ca(2+), but their specific roles in distinct kinetic steps of exocytosis are not well understood. To explore these questions we used amperometry recording from PC12 cells to investigate the kinetics of exocytosis. Syt isoforms and syt I mutants were overexpressed to perturb syt-PS and syt-SNARE interactions to varying degrees and evaluate the effects on fusion event frequency and the rates of fusion pore transitions. Syt I produced more rapid dilation of fusion pores than syt VII or syt IX, consistent with its role in synchronous synaptic release. Stronger syt-PS interactions were accompanied by a higher frequency of fusion events and more stable fusion pores. By contrast, syt-SNARE interactions and syt-induced SNARE assembly were uncorrelated with rates of exocytosis. This associates the syt-PS interaction with two distinct kinetic steps in Ca(2+) triggered exocytosis and supports a role for the syt-PS interaction in stabilizing open fusion pores.

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Related in: MedlinePlus

Test for correlation between in vitro interactions and exocytosis in syt I mutants. Plots of secretion rate versus PS-liposome binding Kd-cs (A), fold-increase in t-SNARE binding (Ca2+ binding/EGTA binding) (B), and syt-induced t-SNARE assembly (C). Plots of PSF lifetime versus Kd-cs (D), fold-increase in t-SNARE binding (binding in Ca2+/binding in EGTA) (E), and syt-induced t-SNARE assembly (F). A statistically significant correlation was found only for PSF duration versus Kd-cs (D).
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Figure 8: Test for correlation between in vitro interactions and exocytosis in syt I mutants. Plots of secretion rate versus PS-liposome binding Kd-cs (A), fold-increase in t-SNARE binding (Ca2+ binding/EGTA binding) (B), and syt-induced t-SNARE assembly (C). Plots of PSF lifetime versus Kd-cs (D), fold-increase in t-SNARE binding (binding in Ca2+/binding in EGTA) (E), and syt-induced t-SNARE assembly (F). A statistically significant correlation was found only for PSF duration versus Kd-cs (D).

Mentions: To evaluate the binding results in terms of functional roles, the secretion rate and fusion pore lifetime were plotted versus various measurements of syt-effector interactions. For mutants, these plots showed that the secretion rate decreased slightly with increases in the dissociation constant for syt-PS binding (Kd-cs), but this change was not statistically significant (Figure 8, A and D; p = 0.20). In plots of fusion pore lifetime versus Kd-cs we observed a significant decline with a slope of −0.89 (Figure 8D; p = 0.03). Plots of secretion rate and fusion pore lifetime versus syt-t-SNARE interactions in the presence of EGTA or Ca2+, as well as the fold increase in SNARE binding (binding in Ca2+/binding in EGTA), exhibited no correlations (Figure 8, B and E, and Supplemental Figure 2), supporting the idea that syt-PS interactions play a more important role than either Ca2+-dependent or Ca2+-independent syt-t-SNARE interactions. The results shown in Figure 8 suggest that alterations in the strength of the syt-PS interaction dominate over alterations in the strength of the syt-t-SNARE interaction in the regulation of secretion and fusion pore stability.


Regulation of exocytosis and fusion pores by synaptotagmin-effector interactions.

Zhang Z, Hui E, Chapman ER, Jackson MB - Mol. Biol. Cell (2010)

Test for correlation between in vitro interactions and exocytosis in syt I mutants. Plots of secretion rate versus PS-liposome binding Kd-cs (A), fold-increase in t-SNARE binding (Ca2+ binding/EGTA binding) (B), and syt-induced t-SNARE assembly (C). Plots of PSF lifetime versus Kd-cs (D), fold-increase in t-SNARE binding (binding in Ca2+/binding in EGTA) (E), and syt-induced t-SNARE assembly (F). A statistically significant correlation was found only for PSF duration versus Kd-cs (D).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2921110&req=5

Figure 8: Test for correlation between in vitro interactions and exocytosis in syt I mutants. Plots of secretion rate versus PS-liposome binding Kd-cs (A), fold-increase in t-SNARE binding (Ca2+ binding/EGTA binding) (B), and syt-induced t-SNARE assembly (C). Plots of PSF lifetime versus Kd-cs (D), fold-increase in t-SNARE binding (binding in Ca2+/binding in EGTA) (E), and syt-induced t-SNARE assembly (F). A statistically significant correlation was found only for PSF duration versus Kd-cs (D).
Mentions: To evaluate the binding results in terms of functional roles, the secretion rate and fusion pore lifetime were plotted versus various measurements of syt-effector interactions. For mutants, these plots showed that the secretion rate decreased slightly with increases in the dissociation constant for syt-PS binding (Kd-cs), but this change was not statistically significant (Figure 8, A and D; p = 0.20). In plots of fusion pore lifetime versus Kd-cs we observed a significant decline with a slope of −0.89 (Figure 8D; p = 0.03). Plots of secretion rate and fusion pore lifetime versus syt-t-SNARE interactions in the presence of EGTA or Ca2+, as well as the fold increase in SNARE binding (binding in Ca2+/binding in EGTA), exhibited no correlations (Figure 8, B and E, and Supplemental Figure 2), supporting the idea that syt-PS interactions play a more important role than either Ca2+-dependent or Ca2+-independent syt-t-SNARE interactions. The results shown in Figure 8 suggest that alterations in the strength of the syt-PS interaction dominate over alterations in the strength of the syt-t-SNARE interaction in the regulation of secretion and fusion pore stability.

Bottom Line: Syt binds to phosphatidylserine (PS)-containing lipid bilayers as well as to soluble N-ethylmaleimide sensitive factor receptors (SNAREs) and promotes SNARE assembly.All these interactions are regulated by Ca(2+), but their specific roles in distinct kinetic steps of exocytosis are not well understood.By contrast, syt-SNARE interactions and syt-induced SNARE assembly were uncorrelated with rates of exocytosis.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Neurological Disorders and Stroke, Bethesda, MD 20892, USA.

ABSTRACT
Synaptotagmin (syt) serves as a Ca(2+) sensor in the release of neurotransmitters and hormones. This function depends on the ability of syt to interact with other molecules. Syt binds to phosphatidylserine (PS)-containing lipid bilayers as well as to soluble N-ethylmaleimide sensitive factor receptors (SNAREs) and promotes SNARE assembly. All these interactions are regulated by Ca(2+), but their specific roles in distinct kinetic steps of exocytosis are not well understood. To explore these questions we used amperometry recording from PC12 cells to investigate the kinetics of exocytosis. Syt isoforms and syt I mutants were overexpressed to perturb syt-PS and syt-SNARE interactions to varying degrees and evaluate the effects on fusion event frequency and the rates of fusion pore transitions. Syt I produced more rapid dilation of fusion pores than syt VII or syt IX, consistent with its role in synchronous synaptic release. Stronger syt-PS interactions were accompanied by a higher frequency of fusion events and more stable fusion pores. By contrast, syt-SNARE interactions and syt-induced SNARE assembly were uncorrelated with rates of exocytosis. This associates the syt-PS interaction with two distinct kinetic steps in Ca(2+) triggered exocytosis and supports a role for the syt-PS interaction in stabilizing open fusion pores.

Show MeSH
Related in: MedlinePlus