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

PIP2-binding activity and inhibition of exocytosis are correlated and localized to the C2B domains of syts I and IX. (A) PS-binding activity of the indicated syt I/IX constructs was assayed as described in the legend to Fig. 2 C. (B) PS-binding activity does not correlate with inhibition of secretion. The percentage of inhibition of catecholamine release (Fig. 5) was plotted as a function of Ca2+-dependent PS/PC liposome-binding activity and fitted by linear regression. For syts I and IX, PS binding is not necessary for inhibition since the C2B domains bind PS weakly yet inhibit release. (C) PIP2-binding activity of the indicated syt I/IX constructs was assayed as described in the legend to Fig. 2 A. (D) PIP2 binding is correlated with inhibition of secretion. The percentage of inhibition (Fig. 5) was plotted as a function of Ca2+-dependent PIP2 binding (C) and fitted by linear regression.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172790&req=5

fig6: PIP2-binding activity and inhibition of exocytosis are correlated and localized to the C2B domains of syts I and IX. (A) PS-binding activity of the indicated syt I/IX constructs was assayed as described in the legend to Fig. 2 C. (B) PS-binding activity does not correlate with inhibition of secretion. The percentage of inhibition of catecholamine release (Fig. 5) was plotted as a function of Ca2+-dependent PS/PC liposome-binding activity and fitted by linear regression. For syts I and IX, PS binding is not necessary for inhibition since the C2B domains bind PS weakly yet inhibit release. (C) PIP2-binding activity of the indicated syt I/IX constructs was assayed as described in the legend to Fig. 2 A. (D) PIP2 binding is correlated with inhibition of secretion. The percentage of inhibition (Fig. 5) was plotted as a function of Ca2+-dependent PIP2 binding (C) and fitted by linear regression.

Mentions: Ca2+ triggers the penetration of the Ca2+-binding loops of syt into the surface of lipid bilayers that contain anionic phospholipids (e.g., phosphatidylserine [PS]), and this interaction has been proposed to function as a coupling step in excitation-secretion coupling (Bai et al., 2002). Whereas PS-binding activity has been characterized for the isolated C2A domains of syts I–VIII (Li et al., 1995; Sugita et al., 2002), little is known concerning the interaction of syts with other lipids, particularly PIP2, which has been shown to bind syt I (Schiavo et al., 1996). PIP2 is a plasma membrane lipid that is essential for exocytosis of LDCVs (Eberhard et al., 1990; Hay and Martin, 1993; Hay et al., 1995), potentially via its interactions with syt. Therefore, we screened the C2A domains used in the inhibition studies for PS- and PIP2-binding activity (Fig. 2, A and C). With the exception of isoforms IV, VIII, and XI, the C2A domains of the syt family bound PS-containing liposomes in response to Ca2+ (Fig. 2 C). The PIP2-binding profile, however, was dramatically different (Fig. 2 A). PIP2-containing liposomes bound C2A-III and C2A-VII and to a lesser extent C2A-V and C2A-X. Hence, PIP2–C2A interactions are restricted to fewer syt isoforms than is PS-binding activity. However, as detailed below, in some syt isoforms PIP2 binding is mediated by the C2B domain (see Fig. 6; Schiavo et al., 1996).


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)

PIP2-binding activity and inhibition of exocytosis are correlated and localized to the C2B domains of syts I and IX. (A) PS-binding activity of the indicated syt I/IX constructs was assayed as described in the legend to Fig. 2 C. (B) PS-binding activity does not correlate with inhibition of secretion. The percentage of inhibition of catecholamine release (Fig. 5) was plotted as a function of Ca2+-dependent PS/PC liposome-binding activity and fitted by linear regression. For syts I and IX, PS binding is not necessary for inhibition since the C2B domains bind PS weakly yet inhibit release. (C) PIP2-binding activity of the indicated syt I/IX constructs was assayed as described in the legend to Fig. 2 A. (D) PIP2 binding is correlated with inhibition of secretion. The percentage of inhibition (Fig. 5) was plotted as a function of Ca2+-dependent PIP2 binding (C) and fitted by linear regression.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: PIP2-binding activity and inhibition of exocytosis are correlated and localized to the C2B domains of syts I and IX. (A) PS-binding activity of the indicated syt I/IX constructs was assayed as described in the legend to Fig. 2 C. (B) PS-binding activity does not correlate with inhibition of secretion. The percentage of inhibition of catecholamine release (Fig. 5) was plotted as a function of Ca2+-dependent PS/PC liposome-binding activity and fitted by linear regression. For syts I and IX, PS binding is not necessary for inhibition since the C2B domains bind PS weakly yet inhibit release. (C) PIP2-binding activity of the indicated syt I/IX constructs was assayed as described in the legend to Fig. 2 A. (D) PIP2 binding is correlated with inhibition of secretion. The percentage of inhibition (Fig. 5) was plotted as a function of Ca2+-dependent PIP2 binding (C) and fitted by linear regression.
Mentions: Ca2+ triggers the penetration of the Ca2+-binding loops of syt into the surface of lipid bilayers that contain anionic phospholipids (e.g., phosphatidylserine [PS]), and this interaction has been proposed to function as a coupling step in excitation-secretion coupling (Bai et al., 2002). Whereas PS-binding activity has been characterized for the isolated C2A domains of syts I–VIII (Li et al., 1995; Sugita et al., 2002), little is known concerning the interaction of syts with other lipids, particularly PIP2, which has been shown to bind syt I (Schiavo et al., 1996). PIP2 is a plasma membrane lipid that is essential for exocytosis of LDCVs (Eberhard et al., 1990; Hay and Martin, 1993; Hay et al., 1995), potentially via its interactions with syt. Therefore, we screened the C2A domains used in the inhibition studies for PS- and PIP2-binding activity (Fig. 2, A and C). With the exception of isoforms IV, VIII, and XI, the C2A domains of the syt family bound PS-containing liposomes in response to Ca2+ (Fig. 2 C). The PIP2-binding profile, however, was dramatically different (Fig. 2 A). PIP2-containing liposomes bound C2A-III and C2A-VII and to a lesser extent C2A-V and C2A-X. Hence, PIP2–C2A interactions are restricted to fewer syt isoforms than is PS-binding activity. However, as detailed below, in some syt isoforms PIP2 binding is mediated by the C2B domain (see Fig. 6; Schiavo et al., 1996).

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