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Two distinct secretory vesicle-priming steps in adrenal chromaffin cells.

Liu Y, Schirra C, Edelmann L, Matti U, Rhee J, Hof D, Bruns D, Brose N, Rieger H, Stevens DR, Rettig J - J. Cell Biol. (2010)

Bottom Line: Priming of large dense-core vesicles (LDCVs) is a Ca(2+)-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin.Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps.Furthermore, the deficit in the readily releasable LDCV pool resulting from CAPS deletion is reversed by a constitutively open form of syntaxin but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Physiologie, Universität des Saarlandes, 66421 Homburg, Germany.

ABSTRACT
Priming of large dense-core vesicles (LDCVs) is a Ca(2+)-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin. Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps. CAPS is required for priming of the readily releasable LDCV pool and sustained secretion in the continued presence of high Ca(2+) concentrations. Either CAPS1 or CAPS2 can rescue secretion in cells lacking both CAPS isoforms. Furthermore, the deficit in the readily releasable LDCV pool resulting from CAPS deletion is reversed by a constitutively open form of syntaxin but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation. Our data indicate that CAPS functions downstream of Munc13s but also interacts functionally with Munc13s in the LDCV-priming process.

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Open syntaxin expression reduces the numbers of docked vesicles in chromaffin cells. (A and B) Representative electron micrographs of chromaffin cells from wild-type (WT) and CAPS DKO mice are shown. N, nucleus; M, mitochondria. (C and D) Representative electron micrographs of chromaffin cells from wild-type and CAPS DKO mice 6 h after pSFV1-syntaxinL165A/E166A-IRES-GFP infection are shown. (A–D) Bars, 2 µm. (E) Relative frequency distribution of the granule distances from the plasma membrane in chromaffin cells from CAPS DKO (n = 16) and wild-type mice (n = 21) and CAPS DKO with open syntaxin expression (n = 7) and wild-type with open syntaxin expression (n = 10). Although there was no difference in docked vesicles in CAPS DKO cells compared with wild-type cells, expression of open syntaxin led to a strong reduction in LDCVs adjacent to the membrane in both populations. Bin width was 60 nm. Error bars indicate mean ± SEM. (F) High magnification inset taken from the boxed region in A. Dashed line shows the distance of 60 nm from the plasma membrane that was taken to define docked vesicles. Bar, 200 nm.
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fig5: Open syntaxin expression reduces the numbers of docked vesicles in chromaffin cells. (A and B) Representative electron micrographs of chromaffin cells from wild-type (WT) and CAPS DKO mice are shown. N, nucleus; M, mitochondria. (C and D) Representative electron micrographs of chromaffin cells from wild-type and CAPS DKO mice 6 h after pSFV1-syntaxinL165A/E166A-IRES-GFP infection are shown. (A–D) Bars, 2 µm. (E) Relative frequency distribution of the granule distances from the plasma membrane in chromaffin cells from CAPS DKO (n = 16) and wild-type mice (n = 21) and CAPS DKO with open syntaxin expression (n = 7) and wild-type with open syntaxin expression (n = 10). Although there was no difference in docked vesicles in CAPS DKO cells compared with wild-type cells, expression of open syntaxin led to a strong reduction in LDCVs adjacent to the membrane in both populations. Bin width was 60 nm. Error bars indicate mean ± SEM. (F) High magnification inset taken from the boxed region in A. Dashed line shows the distance of 60 nm from the plasma membrane that was taken to define docked vesicles. Bar, 200 nm.

Mentions: We next analyzed the distribution of LDCVs in chromaffin cells to determine whether open syntaxin causes a docking defect such as the one reported for mutant mice expressing only open syntaxin (Gerber et al., 2008). We compared the distributions of LDCVs in untreated chromaffin cells from wild-type (n = 21) and CAPS DKO cells (n = 16) to those in wild-type cells after expression of open syntaxin (n = 10) and in CAPS DKO after expression of open syntaxin (n = 7). We determined the shortest distance from the plasma membrane of all identifiable LDCVs in chromaffin cells derived from wild-type or CAPS DKO embryonic day (E) 18/postnatal day (P) 0 mice either with or without expression of open syntaxin. Representative micrographs are shown in Fig. 5 (A–D). The distributions of measured distances (Fig. 5 E) showed a clear reduction in the fraction of LDCVs in close apposition to the membrane in open syntaxin–overexpressing CAPS DKO (62% reduction) and wild-type cells (77% reduction), as compared with untreated cells of CAPS DKO and wild-type mice. We conclude from these data that the reduction in sustained release by expression of open syntaxin is the result of a reduction in the transport of LDCVs to the plasma membrane, i.e., docking.


Two distinct secretory vesicle-priming steps in adrenal chromaffin cells.

Liu Y, Schirra C, Edelmann L, Matti U, Rhee J, Hof D, Bruns D, Brose N, Rieger H, Stevens DR, Rettig J - J. Cell Biol. (2010)

Open syntaxin expression reduces the numbers of docked vesicles in chromaffin cells. (A and B) Representative electron micrographs of chromaffin cells from wild-type (WT) and CAPS DKO mice are shown. N, nucleus; M, mitochondria. (C and D) Representative electron micrographs of chromaffin cells from wild-type and CAPS DKO mice 6 h after pSFV1-syntaxinL165A/E166A-IRES-GFP infection are shown. (A–D) Bars, 2 µm. (E) Relative frequency distribution of the granule distances from the plasma membrane in chromaffin cells from CAPS DKO (n = 16) and wild-type mice (n = 21) and CAPS DKO with open syntaxin expression (n = 7) and wild-type with open syntaxin expression (n = 10). Although there was no difference in docked vesicles in CAPS DKO cells compared with wild-type cells, expression of open syntaxin led to a strong reduction in LDCVs adjacent to the membrane in both populations. Bin width was 60 nm. Error bars indicate mean ± SEM. (F) High magnification inset taken from the boxed region in A. Dashed line shows the distance of 60 nm from the plasma membrane that was taken to define docked vesicles. Bar, 200 nm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3101601&req=5

fig5: Open syntaxin expression reduces the numbers of docked vesicles in chromaffin cells. (A and B) Representative electron micrographs of chromaffin cells from wild-type (WT) and CAPS DKO mice are shown. N, nucleus; M, mitochondria. (C and D) Representative electron micrographs of chromaffin cells from wild-type and CAPS DKO mice 6 h after pSFV1-syntaxinL165A/E166A-IRES-GFP infection are shown. (A–D) Bars, 2 µm. (E) Relative frequency distribution of the granule distances from the plasma membrane in chromaffin cells from CAPS DKO (n = 16) and wild-type mice (n = 21) and CAPS DKO with open syntaxin expression (n = 7) and wild-type with open syntaxin expression (n = 10). Although there was no difference in docked vesicles in CAPS DKO cells compared with wild-type cells, expression of open syntaxin led to a strong reduction in LDCVs adjacent to the membrane in both populations. Bin width was 60 nm. Error bars indicate mean ± SEM. (F) High magnification inset taken from the boxed region in A. Dashed line shows the distance of 60 nm from the plasma membrane that was taken to define docked vesicles. Bar, 200 nm.
Mentions: We next analyzed the distribution of LDCVs in chromaffin cells to determine whether open syntaxin causes a docking defect such as the one reported for mutant mice expressing only open syntaxin (Gerber et al., 2008). We compared the distributions of LDCVs in untreated chromaffin cells from wild-type (n = 21) and CAPS DKO cells (n = 16) to those in wild-type cells after expression of open syntaxin (n = 10) and in CAPS DKO after expression of open syntaxin (n = 7). We determined the shortest distance from the plasma membrane of all identifiable LDCVs in chromaffin cells derived from wild-type or CAPS DKO embryonic day (E) 18/postnatal day (P) 0 mice either with or without expression of open syntaxin. Representative micrographs are shown in Fig. 5 (A–D). The distributions of measured distances (Fig. 5 E) showed a clear reduction in the fraction of LDCVs in close apposition to the membrane in open syntaxin–overexpressing CAPS DKO (62% reduction) and wild-type cells (77% reduction), as compared with untreated cells of CAPS DKO and wild-type mice. We conclude from these data that the reduction in sustained release by expression of open syntaxin is the result of a reduction in the transport of LDCVs to the plasma membrane, i.e., docking.

Bottom Line: Priming of large dense-core vesicles (LDCVs) is a Ca(2+)-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin.Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps.Furthermore, the deficit in the readily releasable LDCV pool resulting from CAPS deletion is reversed by a constitutively open form of syntaxin but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Physiologie, Universität des Saarlandes, 66421 Homburg, Germany.

ABSTRACT
Priming of large dense-core vesicles (LDCVs) is a Ca(2+)-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin. Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps. CAPS is required for priming of the readily releasable LDCV pool and sustained secretion in the continued presence of high Ca(2+) concentrations. Either CAPS1 or CAPS2 can rescue secretion in cells lacking both CAPS isoforms. Furthermore, the deficit in the readily releasable LDCV pool resulting from CAPS deletion is reversed by a constitutively open form of syntaxin but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation. Our data indicate that CAPS functions downstream of Munc13s but also interacts functionally with Munc13s in the LDCV-priming process.

Show MeSH
Related in: MedlinePlus