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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

Protein backbone structure of the cytoplasmic domain of syt I C2AB in ribbon format, with the membrane sketched below. The solution structures of C2A and C2B were modified from (Shao et al., 1998) and (Fernandez et al., 2001). Red spheres depict bound Ca2+. The residues mutated for this study, R399 and T328, are indicated and their side chains are rendered in blue.
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Figure 1: Protein backbone structure of the cytoplasmic domain of syt I C2AB in ribbon format, with the membrane sketched below. The solution structures of C2A and C2B were modified from (Shao et al., 1998) and (Fernandez et al., 2001). Red spheres depict bound Ca2+. The residues mutated for this study, R399 and T328, are indicated and their side chains are rendered in blue.

Mentions: In the present study we have attempted to address the question of the roles of syt-effector interactions by evaluating both syt I mutants and syt isoforms. We utilized in vitro assays to test Ca2+-stimulated PS binding, Ca2+-stimulated (target-SNARE [t-SNARE]) binding, and Ca2+-stimulated t-SNARE assembly of two syt I mutants (R399A and T328A, Figure 1A), selected to produce very different alterations in these interactions (Gaffaney et al., 2008). We also evaluated syt isoforms with widely divergent interactions. Using amperometry to measure norepinephrine release and to resolve distinct kinetic steps of exocytosis (Chow and von Rüden, 1995; Travis and Wightman, 1998), we found that PC12 cells overexpressing these syt variants showed alterations in the overall rate of exocytosis, as well as fusion pore stability. In agreement with previous work on cells with different PS levels (Zhang et al., 2009), our results suggest that Ca2+-triggered PS binding has multiple functions in exocytosis, including a stabilization of open fusion pores by retarding the rate of fusion pore dilation. By contrast, variations in Ca2+-triggered SNARE binding and assembly do not appear to be correlated with the steps of exocytosis tested here.


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

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

Protein backbone structure of the cytoplasmic domain of syt I C2AB in ribbon format, with the membrane sketched below. The solution structures of C2A and C2B were modified from (Shao et al., 1998) and (Fernandez et al., 2001). Red spheres depict bound Ca2+. The residues mutated for this study, R399 and T328, are indicated and their side chains are rendered in blue.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Protein backbone structure of the cytoplasmic domain of syt I C2AB in ribbon format, with the membrane sketched below. The solution structures of C2A and C2B were modified from (Shao et al., 1998) and (Fernandez et al., 2001). Red spheres depict bound Ca2+. The residues mutated for this study, R399 and T328, are indicated and their side chains are rendered in blue.
Mentions: In the present study we have attempted to address the question of the roles of syt-effector interactions by evaluating both syt I mutants and syt isoforms. We utilized in vitro assays to test Ca2+-stimulated PS binding, Ca2+-stimulated (target-SNARE [t-SNARE]) binding, and Ca2+-stimulated t-SNARE assembly of two syt I mutants (R399A and T328A, Figure 1A), selected to produce very different alterations in these interactions (Gaffaney et al., 2008). We also evaluated syt isoforms with widely divergent interactions. Using amperometry to measure norepinephrine release and to resolve distinct kinetic steps of exocytosis (Chow and von Rüden, 1995; Travis and Wightman, 1998), we found that PC12 cells overexpressing these syt variants showed alterations in the overall rate of exocytosis, as well as fusion pore stability. In agreement with previous work on cells with different PS levels (Zhang et al., 2009), our results suggest that Ca2+-triggered PS binding has multiple functions in exocytosis, including a stabilization of open fusion pores by retarding the rate of fusion pore dilation. By contrast, variations in Ca2+-triggered SNARE binding and assembly do not appear to be correlated with the steps of exocytosis tested here.

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