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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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Syts I and IX are the major syt isoforms in PC12 cells. (A) Peptides corresponding to the indicated regions of syts III, IV, VII, and IX were used to produce polyclonal antibodies as described in Materials and methods. (B) Specificity of the anti-syt (α-syt) antibodies. 100 ng of the indicated GST–syt fragments lacking the C2B domain (αI, III, IV, and VII) or composed of only the cytoplasmic domain (αIX) were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with the indicated antibody. Truncated syt fragments were used due to their relative ease of expression and isolation compared with full-length syts. Recombinant protein preparations often contained multiple proteolytic fragments as a result of bacterial expression; quantification of recombinant standards was based on the abundance of the band corresponding to the molecular weight of the intact protein. Each of the antibodies is specific for the syt it is raised against. For αI, mAb 41.1 was used (Chapman and Jahn, 1994). (C) Syt expression levels in PC12 cells. The indicated amounts of recombinant standards and postnuclear PC12 cell membranes (total protein) were subjected to SDS-PAGE and immunoblot analysis using the indicated antibodies. (D) Syt VII is a low abundance Ca2+ sensor in PC12 cells. Immunoblots for syts III, IV, and VII were performed as described in C with reduced amounts of standards and increased amounts of cell membranes as indicated. (E) Blots shown in C and D were quantified by densitometry and plotted as a percentage of total protein from membranes. Since multiple bands were detected for each isoform expressed, quantitation was based on total immunoreactivity from cell extracts by summing the density of all bands. (F) Anti–syt-III and -VII antibodies detect syt III and VII expressed in fibroblasts. HEK cells were transfected with expression constructs (pCI-neo; Promega) for syt isoforms I, III, or VII (+), or empty vector (−), and probed with their respective antibody.
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fig4: Syts I and IX are the major syt isoforms in PC12 cells. (A) Peptides corresponding to the indicated regions of syts III, IV, VII, and IX were used to produce polyclonal antibodies as described in Materials and methods. (B) Specificity of the anti-syt (α-syt) antibodies. 100 ng of the indicated GST–syt fragments lacking the C2B domain (αI, III, IV, and VII) or composed of only the cytoplasmic domain (αIX) were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with the indicated antibody. Truncated syt fragments were used due to their relative ease of expression and isolation compared with full-length syts. Recombinant protein preparations often contained multiple proteolytic fragments as a result of bacterial expression; quantification of recombinant standards was based on the abundance of the band corresponding to the molecular weight of the intact protein. Each of the antibodies is specific for the syt it is raised against. For αI, mAb 41.1 was used (Chapman and Jahn, 1994). (C) Syt expression levels in PC12 cells. The indicated amounts of recombinant standards and postnuclear PC12 cell membranes (total protein) were subjected to SDS-PAGE and immunoblot analysis using the indicated antibodies. (D) Syt VII is a low abundance Ca2+ sensor in PC12 cells. Immunoblots for syts III, IV, and VII were performed as described in C with reduced amounts of standards and increased amounts of cell membranes as indicated. (E) Blots shown in C and D were quantified by densitometry and plotted as a percentage of total protein from membranes. Since multiple bands were detected for each isoform expressed, quantitation was based on total immunoreactivity from cell extracts by summing the density of all bands. (F) Anti–syt-III and -VII antibodies detect syt III and VII expressed in fibroblasts. HEK cells were transfected with expression constructs (pCI-neo; Promega) for syt isoforms I, III, or VII (+), or empty vector (−), and probed with their respective antibody.

Mentions: Peptides corresponding to nonconserved segments of syt III, VII, and IX were used to raise isoform-specific antibodies (Fig. 4 A). In the case of syt VII, the peptide corresponded to a region present in all predicted splice variants (Sugita et al., 2001). Antibodies to syt IV, which was previously reported to be expressed at low levels in PC12 cells (Zhang et al., 2002), were also generated. After affinity purification, antibodies were screened for specificity. mAb 41.1 was used to detect syt I (Chapman and Jahn, 1994). As shown in Fig. 4 B, all five antibodies recognized only the appropriate syt isoform. The antibodies were then used to carry out quantitative immunoblot analysis of PC12 cell membranes using recombinant protein standards (Fig. 4 C). Syts I and IX were readily detected and accounted for ∼0.26 and 0.15%, respectively, of the total membrane protein (Fig. 4, C and E). The ratio of syt I to syt IX (∼1.6) is similar to the value (∼1.3) reported previously (Zhang et al., 2002). Syts III, IV, and VII were not detected at the level of sensitivity adequate for isoforms I and IX (Fig. 4 C). In an effort to detect these isoforms, we decreased the levels of the standards loaded onto the blots, and we increased the level of PC12 cell membranes (Fig. 4 D). Under these conditions, the syt VII antibody yielded bands at ∼45, 40, and 26 kD. Together, these bands represent ∼0.007% of the total protein (Fig. 4 E) and is 37 times less abundant that syt I. The multiple syt VII bands are likely to reflect splice variants (Sugita et al., 2001) or degradation products. A similar banding pattern was seen in HEK cells transfected with full-length syt VII, although the intensity of the 26-kD band was significantly reduced relative to the higher molecular weight bands (Fig. 4 F). In brain extracts, higher molecular weight bands (∼76 kD) and a virtually identical banding pattern to that reported by Sugita et al. (2001) were observed (unpublished data), indicating that these antibodies recognize the same antigens. A faint band at ∼26 kD became apparent in the high sensitivity syt III blots, but bands expected for the full-length protein were not observed (Fig. 4 D). It is not clear whether the 26-kD band is the result of nonspecific binding, cross reactivity, or a degradation product of the full-length protein. Transfection of syt III in HEK cells gave rise to a single band at ∼80 kD (Fig. 4 F), consistent with a previous report (Fukuda et al., 1999). Hence, this antibody is able to recognize syt III expressed in mammalian cells. On some blots, a faint syt IV band was present, but the level of expression was too low to quantify.


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)

Syts I and IX are the major syt isoforms in PC12 cells. (A) Peptides corresponding to the indicated regions of syts III, IV, VII, and IX were used to produce polyclonal antibodies as described in Materials and methods. (B) Specificity of the anti-syt (α-syt) antibodies. 100 ng of the indicated GST–syt fragments lacking the C2B domain (αI, III, IV, and VII) or composed of only the cytoplasmic domain (αIX) were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with the indicated antibody. Truncated syt fragments were used due to their relative ease of expression and isolation compared with full-length syts. Recombinant protein preparations often contained multiple proteolytic fragments as a result of bacterial expression; quantification of recombinant standards was based on the abundance of the band corresponding to the molecular weight of the intact protein. Each of the antibodies is specific for the syt it is raised against. For αI, mAb 41.1 was used (Chapman and Jahn, 1994). (C) Syt expression levels in PC12 cells. The indicated amounts of recombinant standards and postnuclear PC12 cell membranes (total protein) were subjected to SDS-PAGE and immunoblot analysis using the indicated antibodies. (D) Syt VII is a low abundance Ca2+ sensor in PC12 cells. Immunoblots for syts III, IV, and VII were performed as described in C with reduced amounts of standards and increased amounts of cell membranes as indicated. (E) Blots shown in C and D were quantified by densitometry and plotted as a percentage of total protein from membranes. Since multiple bands were detected for each isoform expressed, quantitation was based on total immunoreactivity from cell extracts by summing the density of all bands. (F) Anti–syt-III and -VII antibodies detect syt III and VII expressed in fibroblasts. HEK cells were transfected with expression constructs (pCI-neo; Promega) for syt isoforms I, III, or VII (+), or empty vector (−), and probed with their respective antibody.
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fig4: Syts I and IX are the major syt isoforms in PC12 cells. (A) Peptides corresponding to the indicated regions of syts III, IV, VII, and IX were used to produce polyclonal antibodies as described in Materials and methods. (B) Specificity of the anti-syt (α-syt) antibodies. 100 ng of the indicated GST–syt fragments lacking the C2B domain (αI, III, IV, and VII) or composed of only the cytoplasmic domain (αIX) were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with the indicated antibody. Truncated syt fragments were used due to their relative ease of expression and isolation compared with full-length syts. Recombinant protein preparations often contained multiple proteolytic fragments as a result of bacterial expression; quantification of recombinant standards was based on the abundance of the band corresponding to the molecular weight of the intact protein. Each of the antibodies is specific for the syt it is raised against. For αI, mAb 41.1 was used (Chapman and Jahn, 1994). (C) Syt expression levels in PC12 cells. The indicated amounts of recombinant standards and postnuclear PC12 cell membranes (total protein) were subjected to SDS-PAGE and immunoblot analysis using the indicated antibodies. (D) Syt VII is a low abundance Ca2+ sensor in PC12 cells. Immunoblots for syts III, IV, and VII were performed as described in C with reduced amounts of standards and increased amounts of cell membranes as indicated. (E) Blots shown in C and D were quantified by densitometry and plotted as a percentage of total protein from membranes. Since multiple bands were detected for each isoform expressed, quantitation was based on total immunoreactivity from cell extracts by summing the density of all bands. (F) Anti–syt-III and -VII antibodies detect syt III and VII expressed in fibroblasts. HEK cells were transfected with expression constructs (pCI-neo; Promega) for syt isoforms I, III, or VII (+), or empty vector (−), and probed with their respective antibody.
Mentions: Peptides corresponding to nonconserved segments of syt III, VII, and IX were used to raise isoform-specific antibodies (Fig. 4 A). In the case of syt VII, the peptide corresponded to a region present in all predicted splice variants (Sugita et al., 2001). Antibodies to syt IV, which was previously reported to be expressed at low levels in PC12 cells (Zhang et al., 2002), were also generated. After affinity purification, antibodies were screened for specificity. mAb 41.1 was used to detect syt I (Chapman and Jahn, 1994). As shown in Fig. 4 B, all five antibodies recognized only the appropriate syt isoform. The antibodies were then used to carry out quantitative immunoblot analysis of PC12 cell membranes using recombinant protein standards (Fig. 4 C). Syts I and IX were readily detected and accounted for ∼0.26 and 0.15%, respectively, of the total membrane protein (Fig. 4, C and E). The ratio of syt I to syt IX (∼1.6) is similar to the value (∼1.3) reported previously (Zhang et al., 2002). Syts III, IV, and VII were not detected at the level of sensitivity adequate for isoforms I and IX (Fig. 4 C). In an effort to detect these isoforms, we decreased the levels of the standards loaded onto the blots, and we increased the level of PC12 cell membranes (Fig. 4 D). Under these conditions, the syt VII antibody yielded bands at ∼45, 40, and 26 kD. Together, these bands represent ∼0.007% of the total protein (Fig. 4 E) and is 37 times less abundant that syt I. The multiple syt VII bands are likely to reflect splice variants (Sugita et al., 2001) or degradation products. A similar banding pattern was seen in HEK cells transfected with full-length syt VII, although the intensity of the 26-kD band was significantly reduced relative to the higher molecular weight bands (Fig. 4 F). In brain extracts, higher molecular weight bands (∼76 kD) and a virtually identical banding pattern to that reported by Sugita et al. (2001) were observed (unpublished data), indicating that these antibodies recognize the same antigens. A faint band at ∼26 kD became apparent in the high sensitivity syt III blots, but bands expected for the full-length protein were not observed (Fig. 4 D). It is not clear whether the 26-kD band is the result of nonspecific binding, cross reactivity, or a degradation product of the full-length protein. Transfection of syt III in HEK cells gave rise to a single band at ∼80 kD (Fig. 4 F), consistent with a previous report (Fukuda et al., 1999). Hence, this antibody is able to recognize syt III expressed in mammalian cells. On some blots, a faint syt IV band was present, but the level of expression was too low to quantify.

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