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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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Syt I mutations and isoforms affect fusion pore stability. (A) Sample trace with an expanded view of a single vesicle release event showing the prespike foot (PSF). (B) Fusion pore lifetime distributions for wild-type syt I and two mutants, and (C) their mean fusion pore lifetimes. (D) Fusion pore lifetime distributions for control, syt VII, and syt IX (for syt I see B), and (E) their mean fusion pore lifetimes. 315-1021 prespike feet from 42 to 125 cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars represent SEM.
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Figure 3: Syt I mutations and isoforms affect fusion pore stability. (A) Sample trace with an expanded view of a single vesicle release event showing the prespike foot (PSF). (B) Fusion pore lifetime distributions for wild-type syt I and two mutants, and (C) their mean fusion pore lifetimes. (D) Fusion pore lifetime distributions for control, syt VII, and syt IX (for syt I see B), and (E) their mean fusion pore lifetimes. 315-1021 prespike feet from 42 to 125 cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars represent SEM.

Mentions: Secretion rate and amperometry spikes were analyzed as described previously (Zhang and Jackson, 2008). Spikes with peak amplitudes ≥2 pA were counted in the determination of secretion rate. Cells with extremely low (<3) or high (>100) spike numbers during 20 s for a single application of KCl solution were excluded. Fusion pore lifetimes were taken as the duration of prespike feet (PSF) measured for spikes with amplitudes ≥20 pA (see Figure 3A). Kiss-and-run events arising from fusion pores that also give rise to PSF were recognized as rectangular events with peak amplitudes between 2 and 3.5 pA (Wang et al., 2006) (see Figure 4A). The Student's t test was used to evaluate statistical significance. One-way ANOVA and Dunnett's test were also applied to evaluate statistical significance, and the results were very similar to the t test. Numbers of cells and events analyzed are presented in the figure legends. For evaluation of statistical significance in event frequency measurements we used the number of cells per recordings as the number of measurements; for fusion pore kinetics we have previously shown that there is no dependence on cells per recordings, so the number of PSF was used (Wang et al., 2006).


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

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

Syt I mutations and isoforms affect fusion pore stability. (A) Sample trace with an expanded view of a single vesicle release event showing the prespike foot (PSF). (B) Fusion pore lifetime distributions for wild-type syt I and two mutants, and (C) their mean fusion pore lifetimes. (D) Fusion pore lifetime distributions for control, syt VII, and syt IX (for syt I see B), and (E) their mean fusion pore lifetimes. 315-1021 prespike feet from 42 to 125 cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars represent SEM.
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Related In: Results  -  Collection

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

Figure 3: Syt I mutations and isoforms affect fusion pore stability. (A) Sample trace with an expanded view of a single vesicle release event showing the prespike foot (PSF). (B) Fusion pore lifetime distributions for wild-type syt I and two mutants, and (C) their mean fusion pore lifetimes. (D) Fusion pore lifetime distributions for control, syt VII, and syt IX (for syt I see B), and (E) their mean fusion pore lifetimes. 315-1021 prespike feet from 42 to 125 cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars represent SEM.
Mentions: Secretion rate and amperometry spikes were analyzed as described previously (Zhang and Jackson, 2008). Spikes with peak amplitudes ≥2 pA were counted in the determination of secretion rate. Cells with extremely low (<3) or high (>100) spike numbers during 20 s for a single application of KCl solution were excluded. Fusion pore lifetimes were taken as the duration of prespike feet (PSF) measured for spikes with amplitudes ≥20 pA (see Figure 3A). Kiss-and-run events arising from fusion pores that also give rise to PSF were recognized as rectangular events with peak amplitudes between 2 and 3.5 pA (Wang et al., 2006) (see Figure 4A). The Student's t test was used to evaluate statistical significance. One-way ANOVA and Dunnett's test were also applied to evaluate statistical significance, and the results were very similar to the t test. Numbers of cells and events analyzed are presented in the figure legends. For evaluation of statistical significance in event frequency measurements we used the number of cells per recordings as the number of measurements; for fusion pore kinetics we have previously shown that there is no dependence on cells per recordings, so the number of PSF was used (Wang et al., 2006).

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