Duncan, R.R. et al. Functional and spatial segregation of secretory vesicle pools according to vesicle age. Nature 422, 176-180

The University of Edinburgh, Edinburgh, Scotland, United Kingdom
Nature (Impact Factor: 41.46). 04/2003; 422(6928):176-80. DOI: 10.1038/nature01389
Source: PubMed


Synaptic terminals and neuroendocrine cells are packed with secretory vesicles, only a few of which are docked at the plasma membrane and readily releasable. The remainder are thought to constitute a large cytoplasmic reserve pool awaiting recruitment into the readily releasable pool (RRP) for exocytosis. How vesicles are prioritized in recruitment is still unknown: the choice could be random, or else the oldest or the newest ones might be favoured. Here we show, using a fluorescent cargo protein that changes colour with time, that vesicles in bovine adrenal chromaffin cells segregate into distinct populations, based on age. Newly assembled vesicles are immobile (morphologically docked) at the plasma membrane shortly after biogenesis, whereas older vesicles are mobile and located deeper in the cell. Different secretagogues selectively release vesicles from the RRP or, surprisingly, selectively from the deeper cytoplasmic pool. Thus, far from being equal, vesicles are segregated functionally and spatially according to age.

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    • "Prior in vitro studies detected robust release of ANF-GFP from single DCVs in chromaffin cells, undifferentiated PC12 cells, and differentiated PC12 cell growth cones and more limited ANF-GFP release from cultured hippocampal neurons (Han et al., 1999; Barg et al., 2002; Duncan et al., 2003; Ng et al., 2003; Xia et al., 2009). However, the stimuli used in these studies are not amenable to imaging transgenically expressed ANF-GFP at the native Drosophila NMJ: bath-applied K + directly evokes muscle contraction that "
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    ABSTRACT: Neurons release neuropeptides, enzymes and neurotrophins by exocytosis of dense-core vesicles (DCVs). Peptide release from individual DCVs has been imaged in vitro with endocrine cells and at the neuron soma, growth cones, neurites, axons, and dendrites, but not at nerve terminals where peptidergic neurotransmission occurs. Single presynaptic DCVs have, however, been tracked in native terminals with SPAIM (simultaneous photobleaching and imaging) to show that DCVs undergo anterograde and retrograde capture as they circulate through en passant boutons. Here dynamin (encoded by the shibire gene) is shown to enhance activity-evoked peptide release at the Drosophila neuromuscular junction. Then SPAIM demonstrates that activity depletes only a portion of a single presynaptic DCV's content. Activity initiates exocytosis within seconds, but subsequent release occurs slowly. Synaptic neuropeptide release is further sustained by DCVs undergoing multiple rounds of exocytosis. Synaptic neuropeptide release is surprisingly similar regardless of anterograde or retrograde DCV transport into boutons, bouton location and time of arrival in the terminal. Thus, vesicle circulation and bidirectional capture supplies synapses with functionally competent DCVs. These results show that activity-evoked synaptic neuropeptide release is independent of a DCV's past traffic and occurs by slow dynamin-dependent partial emptying of DCVs suggestive of kiss and run exocytosis. © 2015 by The American Society for Cell Biology.
    Molecular biology of the cell 04/2015; 26(13). DOI:10.1091/mbc.E15-01-0002 · 4.47 Impact Factor
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    • "Cargo sorting into LDCVs is a very complicated and highly regulated process. It has been postulated that there different LDCV populations exist, which might have different constituents (Duncan et al., 2003; Grabner et al., 2005; Watanabe et al., 1991). The segregation of CgA and SgII in different populations implicates that there exist different sorting machineries for these granins (Watanabe et al., 1991). "
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    ABSTRACT: The large dense-core vesicle (LDCV), a type of lysosome-related organelle, is involved in the secretion of hormones and neuropeptides in specialized secretory cells. The granin family is a driving force in LDCV biogenesis, but the machinery for granin sorting to this biogenesis pathway is largely unknown. The mu mutant mouse, which carries a spontaneous null mutation on the Muted gene (also known as Bloc1s5) that encodes a subunit of lysosome-related organelles complex-1 (BLOC-1), is a mouse model of Hermansky-Pudlak syndrome. We here found that LDCVs were enlarged in mu adrenal chromaffin cells. Chromogranin A (CgA) was increased in mu adrenals and muted-knockdown cells. The increased CgA in mu mice was likely due to the failure of its sorting-out, which impairs LDCV maturation and docking. In mu chromaffin cells, the size of readily releasable pool and the vesicle release frequency were reduced. Our studies suggest that the muted protein is involved in the sorting-out of CgA during the biogenesis of LDCVs.
    Journal of Cell Science 02/2015; 128(7). DOI:10.1242/jcs.161414 · 5.43 Impact Factor
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    • "Our previous (successful) use of the SFV system to induce rescue within 6–8 h was focused on proteins directly involved in exocytosis, including the SNAREs and syt-1. We suspected that rescue with vti1a might require longer times, because of its role in vesicle generation, which has a longer turnover time (Duncan et al, 2003). Therefore, we used the lentiviral (LV) expression system, which allows stable and long-term expression. "
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    ABSTRACT: The SNARE protein vti1a is proposed to drive fusion of intracellular organelles, but recent data also implicated vti1a in exocytosis. Here we show that vti1a is absent from mature secretory vesicles in adrenal chromaffin cells, but localizes to a compartment near the trans-Golgi network, partially overlapping with syntaxin-6. Exocytosis is impaired in vti1a null cells, partly due to fewer Ca(2+)-channels at the plasma membrane, partly due to fewer vesicles of reduced size and synaptobrevin-2 content. In contrast, release kinetics and Ca(2+)-sensitivity remain unchanged, indicating that the final fusion reaction leading to transmitter release is unperturbed. Additional deletion of the closest related SNARE, vti1b, does not exacerbate the vti1a phenotype, and vti1b null cells show no secretion defects, indicating that vti1b does not participate in exocytosis. Long-term re-expression of vti1a (days) was necessary for restoration of secretory capacity, whereas strong short-term expression (hours) was ineffective, consistent with vti1a involvement in an upstream step related to vesicle generation, rather than in fusion. We conclude that vti1a functions in vesicle generation and Ca(2+)-channel trafficking, but is dispensable for transmitter release.
    The EMBO Journal 06/2014; 33(15). DOI:10.15252/embj.201387549 · 10.43 Impact Factor
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