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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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PS and t-SNARE binding properties of syt I mutations. (A) A sample cosedimentation gel for syt I mutants bound to the indicated concentrations of liposomes (containing 25% PS). [Ca2+] = 1 mM. (B) Percentage of protein bound to liposomes plotted versus liposome concentration. (C) Kd-cs values for syt mutations derived from plots in B. (D) A sample cofloatation gel for syt-t-SNARE binding. (E) Syt-t-SNARE binding in the presence of Ca2+ or EGTA. (F) Fold-increase in t-SNARE binding (binding in Ca2+/binding in EGTA) computed as the ratio from the values in E. Data from at least three independent experiments. *, p < 0.05; ***, p < 0.001. Error bars represent SEM.
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Figure 5: PS and t-SNARE binding properties of syt I mutations. (A) A sample cosedimentation gel for syt I mutants bound to the indicated concentrations of liposomes (containing 25% PS). [Ca2+] = 1 mM. (B) Percentage of protein bound to liposomes plotted versus liposome concentration. (C) Kd-cs values for syt mutations derived from plots in B. (D) A sample cofloatation gel for syt-t-SNARE binding. (E) Syt-t-SNARE binding in the presence of Ca2+ or EGTA. (F) Fold-increase in t-SNARE binding (binding in Ca2+/binding in EGTA) computed as the ratio from the values in E. Data from at least three independent experiments. *, p < 0.05; ***, p < 0.001. Error bars represent SEM.

Mentions: Many syts bind to PS-containing lipid bilayers and to SNARE proteins in a Ca2+-dependent manner (Brunger, 2005; Chapman, 2008). We therefore examined these interactions in the syt mutants and isoforms investigated above with the goal of relating changes in exocytotic function to changes in in vitro interactions. Our cosedimentation measurements with PS-containing liposomes showed that syt I(R399A) had impaired lipid binding, while coflotation measurements confirmed that its t-SNARE interaction was indistinguishable from wild type (Gaffaney et al., 2008) (Figure 5). Thus, the decreases in secretion rate and PSF lifetime seen with this mutant suggest that impairing syt-PS binding reduces the rate of fusion and destabilizes open fusion pores (Figures 2, A–C and 3, B and C). Syt I(T328A) had reduced PS binding activity, but t-SNARE binding increased, both in the presence and absence of Ca2+ (Figure 5). Thus, the T328A mutation has opposite effects on PS binding and t-SNARE binding. We note that the t-SNARE binding in EGTA evident in Figures 5E and 6E confirms earlier studies demonstrating a Ca2+-independent interaction between syntaxin and synaptotagmin (Bennett et al., 1992; Gaffaney et al., 2008). Here, we will evaluate the functional significance of both of these interactions in parallel.


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

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

PS and t-SNARE binding properties of syt I mutations. (A) A sample cosedimentation gel for syt I mutants bound to the indicated concentrations of liposomes (containing 25% PS). [Ca2+] = 1 mM. (B) Percentage of protein bound to liposomes plotted versus liposome concentration. (C) Kd-cs values for syt mutations derived from plots in B. (D) A sample cofloatation gel for syt-t-SNARE binding. (E) Syt-t-SNARE binding in the presence of Ca2+ or EGTA. (F) Fold-increase in t-SNARE binding (binding in Ca2+/binding in EGTA) computed as the ratio from the values in E. Data from at least three independent experiments. *, p < 0.05; ***, p < 0.001. Error bars represent SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2921110&req=5

Figure 5: PS and t-SNARE binding properties of syt I mutations. (A) A sample cosedimentation gel for syt I mutants bound to the indicated concentrations of liposomes (containing 25% PS). [Ca2+] = 1 mM. (B) Percentage of protein bound to liposomes plotted versus liposome concentration. (C) Kd-cs values for syt mutations derived from plots in B. (D) A sample cofloatation gel for syt-t-SNARE binding. (E) Syt-t-SNARE binding in the presence of Ca2+ or EGTA. (F) Fold-increase in t-SNARE binding (binding in Ca2+/binding in EGTA) computed as the ratio from the values in E. Data from at least three independent experiments. *, p < 0.05; ***, p < 0.001. Error bars represent SEM.
Mentions: Many syts bind to PS-containing lipid bilayers and to SNARE proteins in a Ca2+-dependent manner (Brunger, 2005; Chapman, 2008). We therefore examined these interactions in the syt mutants and isoforms investigated above with the goal of relating changes in exocytotic function to changes in in vitro interactions. Our cosedimentation measurements with PS-containing liposomes showed that syt I(R399A) had impaired lipid binding, while coflotation measurements confirmed that its t-SNARE interaction was indistinguishable from wild type (Gaffaney et al., 2008) (Figure 5). Thus, the decreases in secretion rate and PSF lifetime seen with this mutant suggest that impairing syt-PS binding reduces the rate of fusion and destabilizes open fusion pores (Figures 2, A–C and 3, B and C). Syt I(T328A) had reduced PS binding activity, but t-SNARE binding increased, both in the presence and absence of Ca2+ (Figure 5). Thus, the T328A mutation has opposite effects on PS binding and t-SNARE binding. We note that the t-SNARE binding in EGTA evident in Figures 5E and 6E confirms earlier studies demonstrating a Ca2+-independent interaction between syntaxin and synaptotagmin (Bennett et al., 1992; Gaffaney et al., 2008). Here, we will evaluate the functional significance of both of these interactions in parallel.

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