Two distinct secretory vesicle–priming steps in adrenal chromaffin cells

Institut für Physiologie, Universität des Saarlandes, 66421 Homburg, Germany.
The Journal of Cell Biology (Impact Factor: 9.83). 09/2010; 190(6):1067-77. DOI: 10.1083/jcb.201001164
Source: PubMed


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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Available from: Jeongseop Rhee, Jan 16, 2014
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    • "Synapsin I has also been reported in cultured astrocytes [38], liver, NRK epithelial cells [39] [40], and osteoblasts [41]. Rab3a and Munc13, whose respective functions in synaptic vesicle targeting and priming in neurons are well known [42] [43] also regulate secretion in bovine chromaffin cells [44] [45] [46] and insulin release [47] [48]. The interaction and functions of several key secretory regulators have been investigated in acrosomal exocytosis in human sperm [49] [50] [51]. "
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    ABSTRACT: Proteins known to function during cell-cell communication and exocytosis in neurons and other secretory cells have recently been reported in human sperm. Synapsins are a group of proteins that have been very well characterized in neurons, but little is known about synapsin function in other cell types. Based upon previous findings and the known function of synapsin, we tested the hypothesis that synapsin I was present in human sperm. Washed, capacitated, and acrosome induced sperm preparations were used to evaluate the functional distribution of synapsin I using immunocytochemistry. Protein extracts from mouse brain, mouse testis/epididymis, and human semen were used for protein blotting techniques. Immunolocalization revealed synapsin I was enriched in the sperm equatorial segment. Protein extracts from mouse brain, mouse testis/epididymis, and human semen were positive for synapsin I using several different antibodies, and dot blot results were confirmed by Western blot analyses. Finally, treatment of capacitated and acrosome reaction induced samples with anti-synapsin antibodies significantly reduced sperm motility. Localization of synapsin I in human sperm is a novel finding. The association of synapsin I with the sperm equatorial segment and effects on motility are suggestive of a role associated with capacitation and/or acrosome reaction, processes that render sperm capable of fertilizing an oocyte.
    Full-text · Article · Nov 2015 · FEBS Open Bio
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    • "Mammals express two CAPS genes, CAPS-1 and CAPS-2, which are complementarily expressed in brain (Speidel et al., 2003) and are essential for synaptic transmission (Jockusch et al., 2007). In adrenal chromaffin cells, CAPS-1 deletion affects catecholamine uptake in chromaffin granules (Speidel et al., 2005; Brunk et al., 2009) and deletion of CAPS-1 and CAPS-2 abolishes their fusion without affecting docking (Liu et al., 2010). CAPS-2 is important for cerebellar development and neuron survival (Sadakata et al., 2004, 2007a), and deletion of CAPS-1 in cerebellar neurons perturbs DCV trafficking (Sadakata et al., 2010, 2013). "
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    ABSTRACT: Neuropeptides released from dense-core vesicles (DCVs) modulate neuronal activity, but the molecules driving DCV secretion in mammalian neurons are largely unknown. We studied the role of calcium-activator protein for secretion (CAPS) proteins in neuronal DCV secretion at single vesicle resolution. Endogenous CAPS-1 co-localized with synaptic markers but was not enriched at every synapse. Deletion of CAPS-1 and CAPS-2 did not affect DCV biogenesis, loading, transport or docking, but DCV secretion was reduced by 70% in CAPS-1/CAPS-2 double null mutant (DKO) neurons and remaining fusion events required prolonged stimulation. CAPS deletion specifically reduced secretion of stationary DCVs. CAPS-1-EYFP expression in DKO neurons restored DCV secretion, but CAPS-1-EYFP and DCVs rarely traveled together. Synaptic localization of CAPS-1-EYFP in DKO neurons was calcium dependent and DCV fusion probability correlated with synaptic CAPS-1-EYFP expression. These data indicate that CAPS-1 promotes fusion competence of immobile (tethered) DCVs in presynaptic terminals and that CAPS-1 localization to DCVs is probably not essential for this role. DOI:
    Full-text · Article · Feb 2015 · eLife Sciences
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    • "The absence of both CAPS isoforms leads to a selective loss of the RRP, with the SRP remaining unaffected (Liu et al., 2008, 2010), an almost complete loss of the sustained release component, which represents ongoing priming and fusion of LDCVs in the presence of high intracellular Ca 2+ concentrations , and a deficit in catecholamine loading of LDCVs (Speidel et al., 2005). All of these phenotypic changes are rescued by viral re-expression of CAPS1 (Liu et al., 2008) or rat CAPS2b (Liu et al., 2010). As expected, both mouse CAPS2a and mouse CAPS2b led to a full rescue of secretory defects in CAPS1/CAPS2 DKO chromaffin cells (Figures 2A and 2B). "
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    ABSTRACT: Priming of secretory vesicles is a prerequisite for their Ca(2+)-dependent fusion with the plasma membrane. The key vesicle priming proteins, Munc13s and CAPSs, are thought to mediate vesicle priming by regulating the conformation of the t-SNARE syntaxin, thereby facilitating SNARE complex assembly. Munc13s execute their priming function through their MUN domain. Given that the MUN domain of Ca(2+)-dependent activator protein for secretion (CAPS) also binds syntaxin, it was assumed that CAPSs prime vesicles through the same mechanism as Munc13s. We studied naturally occurring splice variants of CAPS2 in CAPS1/CAPS2-deficient cells and found that CAPS2 primes vesicles independently of its MUN domain. Instead, the pleckstrin homology domain of CAPS2 seemingly is essential for its priming function. Our findings indicate a priming mode for secretory vesicles. This process apparently requires membrane phospholipids, does not involve the binding or direct conformational regulation of syntaxin by MUN domains of CAPSs, and is therefore not redundant with Munc13 action. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Nov 2014 · Cell Reports
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