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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Available from: Robert H Chow, Oct 04, 2015
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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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    • "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.
    PLoS ONE 10/2012; 7(10):e47921. DOI:10.1371/journal.pone.0047921 · 3.23 Impact Factor
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    • "It was frequently observed that the percentage of hGH secreted from the small fraction of transfected cells was more than the percentage of insulin secreted from the bulk of the cells. This may suggest that newly assembled secretory granules are preferentially secreted rather than older secretory granules, as reported in other cell types [31]. These data indicate that the amount of secreted hGH induced by 16 mM glucose reflected the ability of secretion in transfected β-cells in islets. "
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    ABSTRACT: In glucose-induced insulin secretion from pancreatic β-cells, a population of insulin granules fuses with the plasma membrane without the typical docking process (newcomer granule fusions), however, its mechanism is unclear. In this study, we investigated the PI3K signaling pathways involved in the upregulation of newcomer granule fusions. Acute treatment with the class IA-selective PI3K inhibitors, PIK-75 and PI-103, enhanced the glucose-induced insulin secretion. Total internal reflection fluorescent microscopy revealed that the PI3K inhibitors increased the fusion events from newcomer granules. We developed a new system for transfection into pancreatic islets and demonstrated the usefulness of this system in order for evaluating the effect of transfected genes on the glucose-induced secretion in primary cultured pancreatic islets. Using this transfection system together with a series of constitutive active mutants, we showed that the PI3K-3-phosphoinositide dependent kinase-1 (PDK1)-Akt pathway mediated the potentiation of insulin secretion. The Akt inhibitor also enhanced the glucose-induced insulin secretion in parallel with the upregulation of newcomer granule fusions, probably via increased motility of intracellular insulin granules. These data suggest that the PI3K-PDK1-Akt pathway plays a significant role in newcomer granule fusions, probably through an alteration of the dynamics of the intracellular insulin granules.
    PLoS ONE 10/2012; 7(10):e47381. DOI:10.1371/journal.pone.0047381 · 3.23 Impact Factor
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