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.
    Preview · Article · Apr 2015 · Molecular biology of the cell
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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.
    Full-text · Article · Feb 2015 · Journal of Cell Science
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    • "R110-labeled old insulin-HT signals showed a punctate pattern throughout the cytoplasm that largely did not overlap with new insulin-HT TMR signals (Fig. 3A). This result is consistent with previous observations using atrial natriuretic factor-tagged fluorescent timer protein, in which secretory vesicles segregated into distinct populations according to age in bovine adrenal chromaffin cells [17]. In contrast to chromaffin cells, in which young rather than older secretory vesicles preferentially docked to the plasma membrane, newly synthesized insulin-HT TMR signals were rarely observed in sections either from the top and bottom z-axis stages in MIN6 cells, whereas R110 signals from older insulin were present throughout the stages (Figs. "
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    ABSTRACT: Newly synthesized hormones have been suggested to be preferentially secreted by various neuroendocrine cells. This observation indicates that there is a distinct population of secretory granules containing new and old hormones. Recent development of fluorescent timer proteins used in bovine adrenal chromaffin cells revealed that secretory vesicles segregate into distinct age-dependent populations. Here, we verify the preferential release of newly synthesized insulin in the pancreatic β-cell line, MIN6, using a combination of multi-labeling reporter systems with both fluorescent and biochemical procedures. This system allows hormones or granules of any age to be labeled, in contrast to the timer proteins, which require fluorescence shift time. Pulse-chase labeling with different color probes distinguishes insulin secretory granules by age, with younger granules having a predominantly intracellular localization rather than at the cell periphery.
    Full-text · Article · Oct 2012 · PLoS ONE
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