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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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Screening the C2A domains of syt I–IX for inhibition of exocytosis. (A) A model of syt function during exocytosis; Ca2+–syt triggers release via interactions with effector molecules. Addition of exogenous C2 domains derived from different syt isoforms competes with endogenous syt for effector interactions, thereby inhibiting fusion. (B) C2A domains derived from syts I–XI exhibit different abilities to disrupt catecholamine release from PC12 cells. The indicated C2A domain (10 μM) was added to the reaction chamber 1 min before release was triggered with 100 μM CaCl2. Control samples lacking recombinant protein (buffer alone) were analyzed in parallel and used to calculate the percentage of inhibition. We note that some experiments were performed at the lowest [Ca2+] at which we could reliably measure secretion; in these experiments, the C2A domains of syt III and VII exhibited the same inhibitory activity that was observed at 100 μM Ca2+ (unpublished data). (C) Inhibition of catecholamine release by C2A-III and C2A-VII occurs at all times during the release process. C2A-I, -III, and -VII (10 μM) were added at the indicated time (relative to Ca2+ addition). For each timing experiment, release profiles were superimposed along with a control trace (buffer only).
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fig1: Screening the C2A domains of syt I–IX for inhibition of exocytosis. (A) A model of syt function during exocytosis; Ca2+–syt triggers release via interactions with effector molecules. Addition of exogenous C2 domains derived from different syt isoforms competes with endogenous syt for effector interactions, thereby inhibiting fusion. (B) C2A domains derived from syts I–XI exhibit different abilities to disrupt catecholamine release from PC12 cells. The indicated C2A domain (10 μM) was added to the reaction chamber 1 min before release was triggered with 100 μM CaCl2. Control samples lacking recombinant protein (buffer alone) were analyzed in parallel and used to calculate the percentage of inhibition. We note that some experiments were performed at the lowest [Ca2+] at which we could reliably measure secretion; in these experiments, the C2A domains of syt III and VII exhibited the same inhibitory activity that was observed at 100 μM Ca2+ (unpublished data). (C) Inhibition of catecholamine release by C2A-III and C2A-VII occurs at all times during the release process. C2A-I, -III, and -VII (10 μM) were added at the indicated time (relative to Ca2+ addition). For each timing experiment, release profiles were superimposed along with a control trace (buffer only).

Mentions: We reasoned that C2 domains derived from multiple syt isoforms would inhibit secretion from cracked PC12 cells based on their ability to disrupt endogenous syt–effector interactions that are critical for large dense core vesicle (LDCV) fusion with the plasma membrane (Fig. 1 A). Since many syt isoforms are likely to function in the same manner, inhibitory C2 domains would be expected to interact with a common set of effectors, thus providing insight into the mechanism of syt function during Ca2+-triggered fusion. To address this, we generated C2A domains from syts I–XI and measured their ability to inhibit catecholamine release from cracked PC12 cells using a recently developed real-time voltammetry assay (Earles et al., 2001). C2A domains were chosen because of their relative ease of bacterial expression compared with the C2B or tandem C2A-C2B domains of the syt family. Furthermore, isolated C2 domains engage fewer effectors than the tandem C2 domains, simplifying screens for common effectors. We then correlated the ability of the C2 domains to inhibit release with their ability to engage a set of well-characterized syt-binding partners.


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)

Screening the C2A domains of syt I–IX for inhibition of exocytosis. (A) A model of syt function during exocytosis; Ca2+–syt triggers release via interactions with effector molecules. Addition of exogenous C2 domains derived from different syt isoforms competes with endogenous syt for effector interactions, thereby inhibiting fusion. (B) C2A domains derived from syts I–XI exhibit different abilities to disrupt catecholamine release from PC12 cells. The indicated C2A domain (10 μM) was added to the reaction chamber 1 min before release was triggered with 100 μM CaCl2. Control samples lacking recombinant protein (buffer alone) were analyzed in parallel and used to calculate the percentage of inhibition. We note that some experiments were performed at the lowest [Ca2+] at which we could reliably measure secretion; in these experiments, the C2A domains of syt III and VII exhibited the same inhibitory activity that was observed at 100 μM Ca2+ (unpublished data). (C) Inhibition of catecholamine release by C2A-III and C2A-VII occurs at all times during the release process. C2A-I, -III, and -VII (10 μM) were added at the indicated time (relative to Ca2+ addition). For each timing experiment, release profiles were superimposed along with a control trace (buffer only).
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Screening the C2A domains of syt I–IX for inhibition of exocytosis. (A) A model of syt function during exocytosis; Ca2+–syt triggers release via interactions with effector molecules. Addition of exogenous C2 domains derived from different syt isoforms competes with endogenous syt for effector interactions, thereby inhibiting fusion. (B) C2A domains derived from syts I–XI exhibit different abilities to disrupt catecholamine release from PC12 cells. The indicated C2A domain (10 μM) was added to the reaction chamber 1 min before release was triggered with 100 μM CaCl2. Control samples lacking recombinant protein (buffer alone) were analyzed in parallel and used to calculate the percentage of inhibition. We note that some experiments were performed at the lowest [Ca2+] at which we could reliably measure secretion; in these experiments, the C2A domains of syt III and VII exhibited the same inhibitory activity that was observed at 100 μM Ca2+ (unpublished data). (C) Inhibition of catecholamine release by C2A-III and C2A-VII occurs at all times during the release process. C2A-I, -III, and -VII (10 μM) were added at the indicated time (relative to Ca2+ addition). For each timing experiment, release profiles were superimposed along with a control trace (buffer only).
Mentions: We reasoned that C2 domains derived from multiple syt isoforms would inhibit secretion from cracked PC12 cells based on their ability to disrupt endogenous syt–effector interactions that are critical for large dense core vesicle (LDCV) fusion with the plasma membrane (Fig. 1 A). Since many syt isoforms are likely to function in the same manner, inhibitory C2 domains would be expected to interact with a common set of effectors, thus providing insight into the mechanism of syt function during Ca2+-triggered fusion. To address this, we generated C2A domains from syts I–XI and measured their ability to inhibit catecholamine release from cracked PC12 cells using a recently developed real-time voltammetry assay (Earles et al., 2001). C2A domains were chosen because of their relative ease of bacterial expression compared with the C2B or tandem C2A-C2B domains of the syt family. Furthermore, isolated C2 domains engage fewer effectors than the tandem C2 domains, simplifying screens for common effectors. We then correlated the ability of the C2 domains to inhibit release with their ability to engage a set of well-characterized syt-binding partners.

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