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

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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Available from: Jeongseop Rhee, Jan 16, 2014
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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:
    eLife Sciences 02/2015; 4(4):e05438. DOI:10.7554/eLife.05438 · 9.32 Impact Factor
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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.
    Cell Reports 11/2014; 9(3):902-9. DOI:10.1016/j.celrep.2014.09.050 · 8.36 Impact Factor
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    • "MUN domains are also found in Unc31/CAPS, proteins with functions related to but distinct from those of Unc13/Munc13s (James et al., 2009; Liu et al., 2010), and in diverse proteins from plants and fungi (Koch et al., 2000; Basu et al., 2005). Notably, recent sequence analyses revealed remote homology between MUN domains and subunits of tethering complexes from diverse membrane compartments, such as the exocyst, GARP, Cog and Dsl1p complexes (Pei et al., 2009), and structural data is establishing similarities among these tethering factors (Sivaram et al., 2006; Tripathi et al., 2009). "
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    ABSTRACT: Unc13/Munc13s play a crucial function in neurotransmitter release through their MUN domain, which mediates the transition from the Syntaxin-1/Munc18-1 complex to the SNARE complex. The MUN domain was suggested to be related to tethering factors, but no MUN-domain structure is available to experimentally validate this notion and address key unresolved questions about the interactions and minimal structural unit required for Unc13/Munc13 function. Here we identify an autonomously folded module within the MUN domain (MUN-CD) and show that its crystal structure is remarkably similar to several tethering factors. We also show that the activity in promoting the Syntaxin-1/Munc18-1 to SNARE complex transition is strongly impaired in MUN-CD. These results show that MUN domains and tethering factors indeed belong to the same family and may have a common role in membrane trafficking. We propose a model whereby the MUN-CD module is central for Munc13 function but full activity requires adjacent sequences.
    Structure 10/2011; 19(10):1443-55. DOI:10.1016/j.str.2011.07.012 · 5.62 Impact Factor
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