Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibitory fragments of syt I and IX bind t-SNAREs. (A) GST-C2A, -C2B, or -C2A-C2B (3 μM) from syts I and IX was immobilized on beads and assayed for t-SNARE heterodimer- (5 μM) binding activity, as described in Materials and methods, in the presence of EGTA (2 mM) or Ca2+ (1 mM). Proteins were visualized by staining with Coomassie blue. (B) Quantitation of t-SNARE–binding activity was determined by densitometry of bands shown in A. EGTA, white bars; Ca2+, black bars. (C) Inhibition of secretion correlates with t-SNARE–binding activity. The percentage of inhibition (Fig. 5) is plotted as a function of Ca2+-dependent t-SNARE–binding activity (B). (D) Coimmunoprecipitation of syt I and IX with t-SNAREs. Soluble syt fragments (2 μM) were incubated with soluble t-SNARE heterodimer (2 μM) in 2 mM EGTA or 1 mM Ca2+ for 1 h. Syntaxin was then immunoprecipitated by adding mAb HPC-1 and protein G–Sepharose. Immunoprecipitates were subjected to SDS-PAGE; gels were stained with Coomassie blue. (E) Quantitation of t-SNARE–binding assayed by coimmunoprecipitation. Quantitation was performed by densitometry as described in B. (F) The percentage of inhibition (Fig. 5) was plotted as a function of Ca2+-dependent t-SNARE binding as determined by coimmunoprecipitation in E.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172790&req=5

fig7: Inhibitory fragments of syt I and IX bind t-SNAREs. (A) GST-C2A, -C2B, or -C2A-C2B (3 μM) from syts I and IX was immobilized on beads and assayed for t-SNARE heterodimer- (5 μM) binding activity, as described in Materials and methods, in the presence of EGTA (2 mM) or Ca2+ (1 mM). Proteins were visualized by staining with Coomassie blue. (B) Quantitation of t-SNARE–binding activity was determined by densitometry of bands shown in A. EGTA, white bars; Ca2+, black bars. (C) Inhibition of secretion correlates with t-SNARE–binding activity. The percentage of inhibition (Fig. 5) is plotted as a function of Ca2+-dependent t-SNARE–binding activity (B). (D) Coimmunoprecipitation of syt I and IX with t-SNAREs. Soluble syt fragments (2 μM) were incubated with soluble t-SNARE heterodimer (2 μM) in 2 mM EGTA or 1 mM Ca2+ for 1 h. Syntaxin was then immunoprecipitated by adding mAb HPC-1 and protein G–Sepharose. Immunoprecipitates were subjected to SDS-PAGE; gels were stained with Coomassie blue. (E) Quantitation of t-SNARE–binding assayed by coimmunoprecipitation. Quantitation was performed by densitometry as described in B. (F) The percentage of inhibition (Fig. 5) was plotted as a function of Ca2+-dependent t-SNARE binding as determined by coimmunoprecipitation in E.

Mentions: We next determined whether the ability of syts I and IX fragments to block release was also correlated with their ability to bind t-SNAREs. In a GST pull-down assay, the C2B and the C2A-C2B domains of syts I and IX exhibited strong binding to syntaxin–SNAP-25 t-SNARE heterodimers (Fig. 7, A and B). Consistent with previous reports, GST-C2A exhibited little t-SNARE–binding activity (Chapman et al., 1996; Davis et al., 1999). There was a strong correlation between t-SNARE–binding activity and inhibition of secretion by the syts I and IX fragments (Fig. 7 C; r2 = 0.78). These data further support a model in which syt–SNARE and syt–PIP2 interactions are essential steps in Ca2+-triggered exocytosis. Furthermore, these studies establish the idea that the biochemical activity of a given C2 domain cannot be predicted by its position in a parent polypeptide. In some cases, an activity that is localized to the first C2 domain (C2A) in one isoform might only be manifest in the second C2 domain (C2B) of another isoform.


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 fragments of syt I and IX bind t-SNAREs. (A) GST-C2A, -C2B, or -C2A-C2B (3 μM) from syts I and IX was immobilized on beads and assayed for t-SNARE heterodimer- (5 μM) binding activity, as described in Materials and methods, in the presence of EGTA (2 mM) or Ca2+ (1 mM). Proteins were visualized by staining with Coomassie blue. (B) Quantitation of t-SNARE–binding activity was determined by densitometry of bands shown in A. EGTA, white bars; Ca2+, black bars. (C) Inhibition of secretion correlates with t-SNARE–binding activity. The percentage of inhibition (Fig. 5) is plotted as a function of Ca2+-dependent t-SNARE–binding activity (B). (D) Coimmunoprecipitation of syt I and IX with t-SNAREs. Soluble syt fragments (2 μM) were incubated with soluble t-SNARE heterodimer (2 μM) in 2 mM EGTA or 1 mM Ca2+ for 1 h. Syntaxin was then immunoprecipitated by adding mAb HPC-1 and protein G–Sepharose. Immunoprecipitates were subjected to SDS-PAGE; gels were stained with Coomassie blue. (E) Quantitation of t-SNARE–binding assayed by coimmunoprecipitation. Quantitation was performed by densitometry as described in B. (F) The percentage of inhibition (Fig. 5) was plotted as a function of Ca2+-dependent t-SNARE binding as determined by coimmunoprecipitation in E.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172790&req=5

fig7: Inhibitory fragments of syt I and IX bind t-SNAREs. (A) GST-C2A, -C2B, or -C2A-C2B (3 μM) from syts I and IX was immobilized on beads and assayed for t-SNARE heterodimer- (5 μM) binding activity, as described in Materials and methods, in the presence of EGTA (2 mM) or Ca2+ (1 mM). Proteins were visualized by staining with Coomassie blue. (B) Quantitation of t-SNARE–binding activity was determined by densitometry of bands shown in A. EGTA, white bars; Ca2+, black bars. (C) Inhibition of secretion correlates with t-SNARE–binding activity. The percentage of inhibition (Fig. 5) is plotted as a function of Ca2+-dependent t-SNARE–binding activity (B). (D) Coimmunoprecipitation of syt I and IX with t-SNAREs. Soluble syt fragments (2 μM) were incubated with soluble t-SNARE heterodimer (2 μM) in 2 mM EGTA or 1 mM Ca2+ for 1 h. Syntaxin was then immunoprecipitated by adding mAb HPC-1 and protein G–Sepharose. Immunoprecipitates were subjected to SDS-PAGE; gels were stained with Coomassie blue. (E) Quantitation of t-SNARE–binding assayed by coimmunoprecipitation. Quantitation was performed by densitometry as described in B. (F) The percentage of inhibition (Fig. 5) was plotted as a function of Ca2+-dependent t-SNARE binding as determined by coimmunoprecipitation in E.
Mentions: We next determined whether the ability of syts I and IX fragments to block release was also correlated with their ability to bind t-SNAREs. In a GST pull-down assay, the C2B and the C2A-C2B domains of syts I and IX exhibited strong binding to syntaxin–SNAP-25 t-SNARE heterodimers (Fig. 7, A and B). Consistent with previous reports, GST-C2A exhibited little t-SNARE–binding activity (Chapman et al., 1996; Davis et al., 1999). There was a strong correlation between t-SNARE–binding activity and inhibition of secretion by the syts I and IX fragments (Fig. 7 C; r2 = 0.78). These data further support a model in which syt–SNARE and syt–PIP2 interactions are essential steps in Ca2+-triggered exocytosis. Furthermore, these studies establish the idea that the biochemical activity of a given C2 domain cannot be predicted by its position in a parent polypeptide. In some cases, an activity that is localized to the first C2 domain (C2A) in one isoform might only be manifest in the second C2 domain (C2B) of another isoform.

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