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Identification of synaptotagmin effectors via acute inhibition of secretion from cracked PC12 cells.

Tucker WC, Edwardson JM, Bai J, Kim HJ, Martin TF, Chapman ER - J. Cell Biol. (2003)

Bottom Line: Several putative Ca2+-syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown.Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I-XI to interfere with endogenous syt-effector interactions during Ca2+-triggered exocytosis from cracked PC12 cells.As expected, if syts I and IX function as Ca2+ sensors, fragments from these isoforms blocked secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Wisconsin, Madison, WI 53706, USA.

ABSTRACT
The synaptotagmins (syts) are a family of membrane proteins proposed to regulate membrane traffic in neuronal and nonneuronal cells. In neurons, the Ca2+-sensing ability of syt I is critical for fusion of docked synaptic vesicles with the plasma membrane in response to stimulation. Several putative Ca2+-syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown. Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I-XI to interfere with endogenous syt-effector interactions during Ca2+-triggered exocytosis from cracked PC12 cells. Inhibition was closely correlated with syntaxin-SNAP-25 and phosphatidylinositol 4,5-bisphosphate (PIP2)-binding activity. Moreover, we measured the expression levels of endogenous syts in PC12 cells; the major isoforms are I and IX, with trace levels of VII. As expected, if syts I and IX function as Ca2+ sensors, fragments from these isoforms blocked secretion. These data suggest that syts trigger fusion via their Ca2+-regulated interactions with t-SNAREs and PIP2, target molecules known to play critical roles in exocytosis.

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Inhibitory C2A domains bind t-SNAREs. (A) t-SNARE–binding profiles of the C2A domains derived from syts I–XI. Immobilized GST–C2A fusions (20 μg in 150 μl HBS plus 0.5% Triton X-100) and 5 μM t-SNARE hetero-dimer were incubated in 2 mM EGTA (− Ca2+) or 1 mM CaCl2 (+ Ca2+). 20% of bound protein and 3% of total t-SNARE was subjected to SDS-PAGE and stained with Coomassie blue. (B) The C2A domains exhibit different degrees of Ca2+-dependent t-SNARE binding. The t-SNARE–binding assay shown in A was quantified by densitometry, and relative optical density (O.D.) was plotted in the presence (black bars) or absence (white bars) of Ca2+. (C) Inhibition of catecholamine release by C2A domains correlates with t-SNARE binding. The percentage of inhibition was plotted as a function of Ca2+-dependent t-SNARE binding.
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fig3: Inhibitory C2A domains bind t-SNAREs. (A) t-SNARE–binding profiles of the C2A domains derived from syts I–XI. Immobilized GST–C2A fusions (20 μg in 150 μl HBS plus 0.5% Triton X-100) and 5 μM t-SNARE hetero-dimer were incubated in 2 mM EGTA (− Ca2+) or 1 mM CaCl2 (+ Ca2+). 20% of bound protein and 3% of total t-SNARE was subjected to SDS-PAGE and stained with Coomassie blue. (B) The C2A domains exhibit different degrees of Ca2+-dependent t-SNARE binding. The t-SNARE–binding assay shown in A was quantified by densitometry, and relative optical density (O.D.) was plotted in the presence (black bars) or absence (white bars) of Ca2+. (C) Inhibition of catecholamine release by C2A domains correlates with t-SNARE binding. The percentage of inhibition was plotted as a function of Ca2+-dependent t-SNARE binding.

Mentions: As noted in the Introduction, syt also forms direct contacts with syntaxin and SNAP-25, two components of the fusion complex. This interaction provides a compelling connection between the Ca2+ sensor for exocytosis and the fusion apparatus. We explored the idea that C2A domains might inhibit release by blocking syt–SNARE interactions by assaying the ability of C2A from syts I–XI to bind purified SNAP-25–syntaxin heterodimers (Fig. 3). Isoforms III, V, VII, and X exhibited robust Ca2+-promoted binding activity, whereas binding was much weaker or devoid for the other isoforms (Fig. 3 B). The correlation between the ability of C2A domains to bind neuronal t-SNAREs in the presence of Ca2+ and to inhibit release was striking (Fig. 3 C; r2 = 0.93); the extent of inhibition by a C2A domain is directly proportional to its ability to engage t-SNAREs. These results suggest that inhibitory C2A domains inhibit catecholamine release from PC12 cells by disruption of endogenous syt–SNARE interactions and by perturbing syt–PIP2 interactions.


Identification of synaptotagmin effectors via acute inhibition of secretion from cracked PC12 cells.

Tucker WC, Edwardson JM, Bai J, Kim HJ, Martin TF, Chapman ER - J. Cell Biol. (2003)

Inhibitory C2A domains bind t-SNAREs. (A) t-SNARE–binding profiles of the C2A domains derived from syts I–XI. Immobilized GST–C2A fusions (20 μg in 150 μl HBS plus 0.5% Triton X-100) and 5 μM t-SNARE hetero-dimer were incubated in 2 mM EGTA (− Ca2+) or 1 mM CaCl2 (+ Ca2+). 20% of bound protein and 3% of total t-SNARE was subjected to SDS-PAGE and stained with Coomassie blue. (B) The C2A domains exhibit different degrees of Ca2+-dependent t-SNARE binding. The t-SNARE–binding assay shown in A was quantified by densitometry, and relative optical density (O.D.) was plotted in the presence (black bars) or absence (white bars) of Ca2+. (C) Inhibition of catecholamine release by C2A domains correlates with t-SNARE binding. The percentage of inhibition was plotted as a function of Ca2+-dependent t-SNARE binding.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172790&req=5

fig3: Inhibitory C2A domains bind t-SNAREs. (A) t-SNARE–binding profiles of the C2A domains derived from syts I–XI. Immobilized GST–C2A fusions (20 μg in 150 μl HBS plus 0.5% Triton X-100) and 5 μM t-SNARE hetero-dimer were incubated in 2 mM EGTA (− Ca2+) or 1 mM CaCl2 (+ Ca2+). 20% of bound protein and 3% of total t-SNARE was subjected to SDS-PAGE and stained with Coomassie blue. (B) The C2A domains exhibit different degrees of Ca2+-dependent t-SNARE binding. The t-SNARE–binding assay shown in A was quantified by densitometry, and relative optical density (O.D.) was plotted in the presence (black bars) or absence (white bars) of Ca2+. (C) Inhibition of catecholamine release by C2A domains correlates with t-SNARE binding. The percentage of inhibition was plotted as a function of Ca2+-dependent t-SNARE binding.
Mentions: As noted in the Introduction, syt also forms direct contacts with syntaxin and SNAP-25, two components of the fusion complex. This interaction provides a compelling connection between the Ca2+ sensor for exocytosis and the fusion apparatus. We explored the idea that C2A domains might inhibit release by blocking syt–SNARE interactions by assaying the ability of C2A from syts I–XI to bind purified SNAP-25–syntaxin heterodimers (Fig. 3). Isoforms III, V, VII, and X exhibited robust Ca2+-promoted binding activity, whereas binding was much weaker or devoid for the other isoforms (Fig. 3 B). The correlation between the ability of C2A domains to bind neuronal t-SNAREs in the presence of Ca2+ and to inhibit release was striking (Fig. 3 C; r2 = 0.93); the extent of inhibition by a C2A domain is directly proportional to its ability to engage t-SNAREs. These results suggest that inhibitory C2A domains inhibit catecholamine release from PC12 cells by disruption of endogenous syt–SNARE interactions and by perturbing syt–PIP2 interactions.

Bottom Line: Several putative Ca2+-syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown.Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I-XI to interfere with endogenous syt-effector interactions during Ca2+-triggered exocytosis from cracked PC12 cells.As expected, if syts I and IX function as Ca2+ sensors, fragments from these isoforms blocked secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Wisconsin, Madison, WI 53706, USA.

ABSTRACT
The synaptotagmins (syts) are a family of membrane proteins proposed to regulate membrane traffic in neuronal and nonneuronal cells. In neurons, the Ca2+-sensing ability of syt I is critical for fusion of docked synaptic vesicles with the plasma membrane in response to stimulation. Several putative Ca2+-syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown. Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I-XI to interfere with endogenous syt-effector interactions during Ca2+-triggered exocytosis from cracked PC12 cells. Inhibition was closely correlated with syntaxin-SNAP-25 and phosphatidylinositol 4,5-bisphosphate (PIP2)-binding activity. Moreover, we measured the expression levels of endogenous syts in PC12 cells; the major isoforms are I and IX, with trace levels of VII. As expected, if syts I and IX function as Ca2+ sensors, fragments from these isoforms blocked secretion. These data suggest that syts trigger fusion via their Ca2+-regulated interactions with t-SNAREs and PIP2, target molecules known to play critical roles in exocytosis.

Show MeSH
Related in: MedlinePlus