scholarly journals Synaptotagmin-7 places dense-core vesicles at the cell membrane to promote Munc13-2- and Ca2+-dependent priming

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Bassam Tawfik ◽  
Joana S Martins ◽  
Sébastien Houy ◽  
Cordelia Imig ◽  
Paulo S Pinheiro ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Chromaffin Cells ◽  
Priming Effect ◽  
Vesicle Fusion ◽  
Dense Core ◽  
Competitive Interactions ◽  
Secretory Vesicles ◽  
Dense Core Vesicles ◽  
Calcium Dependence ◽  
Synaptotagmin 1

Synaptotagmins confer calcium-dependence to the exocytosis of secretory vesicles, but how coexpressed synaptotagmins interact remains unclear. We find that synaptotagmin-1 and synaptotagmin-7 when present alone act as standalone fast and slow Ca2+-sensors for vesicle fusion in mouse chromaffin cells. When present together, synaptotagmin-1 and synaptotagmin-7 are found in largely non-overlapping clusters on dense-core vesicles. Synaptotagmin-7 stimulates Ca2+-dependent vesicle priming and inhibits depriming, and it promotes ubMunc13-2- and phorbolester-dependent priming, especially at low resting calcium concentrations. The priming effect of synaptotagmin-7 increases the number of vesicles fusing via synaptotagmin-1, while negatively affecting their fusion speed, indicating both synergistic and competitive interactions between synaptotagmins. Synaptotagmin-7 places vesicles in close membrane apposition (<6 nm); without it, vesicles accumulate out of reach of the fusion complex (20–40 nm). We suggest that a synaptotagmin-7-dependent movement toward the membrane is involved in Munc13-2/phorbolester/Ca2+-dependent priming as a prelude to fast and slow exocytosis triggering.

scholarly journals Synaptotagmin-7 places dense-core vesicles at the cell membrane to promote Munc13-2- and Ca2+-dependent priming

2020 ◽  
Author(s):  
Bassam Tawfik ◽  
Joana S. Martins ◽  
Sébastien Houy ◽  
Cordelia Imig ◽  
Paulo S. Pinheiro ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Chromaffin Cells ◽  
Priming Effect ◽  
Vesicle Fusion ◽  
Synergistic Action ◽  
Dense Core ◽  
Dense Core Vesicles ◽  
Readily Releasable Pool ◽  
Synaptotagmin 1 ◽  
Functional Consequences

AbstractThe functional consequences of the co-expression of synaptotagmin-1 and synaptotagmin-7 are unclear. We show that when present separately, synaptotagmin-1 and synaptotagmin-7 act as standalone fast and slow Ca2+-sensors for vesicle fusion in mouse chromaffin cells. When present together, synaptotagmin-7 stimulates Ca2+-dependent vesicle priming and inhibits depriming. The priming effect of Synaptotagmin-7 extends to the Readily Releasable Pool, whose fusion is executed by synaptotagmin-1, indicating synergistic action of the two Ca2+-sensors, although they are only partially colocalized. Synaptotagmin-7 promotes ubMunc13-2-dependent priming and the absence of synaptotagmin-7 renders phorbolesters less effective in stimulating priming, although synaptotagmin-7 independent priming is also observed. Morphologically, synaptotagmin-7 places vesicles in close membrane apposition (< 6 nm); in its absence vesicles accumulate out of reach of the fusion complex (20-40 nm). We suggest that a synaptotagmin-7-dependent movement toward the membrane is involved in Munc13-2/phorbolester/Ca2+-dependent priming and sets the stage for fast and slow exocytosis triggering.

scholarly journals Trachynilysin mediates SNARE-dependent release of catecholamines from chromaffin cells via external and stored Ca2+

2000 ◽  
Vol 113 (7) ◽  
pp. 1119-1125 ◽  
Author(s):  
F.A. Meunier ◽  
C. Mattei ◽  
P. Chameau ◽  
G. Lawrence ◽  
C. Colasante ◽  
...  
Keyword(s):  
Chromaffin Cells ◽  
Motor Nerve ◽  
Neuroendocrine Cells ◽  
Dense Core ◽  
Frog Neuromuscular Junction ◽  
Dense Core Vesicles ◽  
Protein Receptor ◽  
Large Dense Core Vesicles ◽  
Quantal Transmitter Release ◽  
Bovine Chromaffin Cells

Trachynilysin, a 159 kDa dimeric protein purified from stonefish (Synanceia trachynis) venom, dramatically increases spontaneous quantal transmitter release at the frog neuromuscular junction, depleting small clear synaptic vesicles, whilst not affecting large dense core vesicles. The basis of this insensitivity of large dense core vesicles exocytosis was examined using a fluorimetric assay to determine whether the toxin could elicit catecholamine release from bovine chromaffin cells. Unlike the case of the motor nerve endings, nanomolar concentrations of trachynilysin evoked sustained Soluble N-ethylmaleimide-sensitive fusion protein Attachment Protein REceptor-dependent exocytosis of large dense core vesicles, but only in the presence of extracellular Ca2+. However, this response to trachynilysin does not rely on Ca2+ influx through voltage-activated Ca2+ channels because the secretion was only slightly affected by blockers of L, N and P/Q types. Instead, trachynilysin elicited a localized increase in intracellular fluorescence monitored with fluo-3/AM, that precisely co-localized with the increase of fluorescence resulting from caffeine-induced release of Ca2+ from intracellular stores. Moreover, depletion of the latter stores inhibited trachynilysin-induced exocytosis. Thus, the observed requirement of external Ca2+ for stimulation of large dense core vesicles exocytosis from chromaffin cells implicates plasma membrane channels that signal efflux of Ca2+ from intracellular stores. This study also suggests that the bases of exocytosis of large dense core vesicles from motor nerve terminals and neuroendocrine cells are distinct.

scholarly journals MicroRNA exocytosis by large dense-core vesicle fusion

2016 ◽  
Author(s):  
Alican Gümürdü ◽  
Ramazan Yildiz ◽  
Erden Eren ◽  
Gökhan Karakülah ◽  
Turgay Ünver ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Vesicle Fusion ◽  
Dense Core ◽  
Snare Complex ◽  
Dense Core Vesicle ◽  
Next Generation ◽  
Dense Core Vesicles ◽  
Large Dense Core Vesicles ◽  
Extracellular Mirna ◽  
Generation Sequencing

AbstractNeurotransmitters and peptide hormones are secreted into outside the cell by a vesicle fusion process. Although non-coding RNA (ncRNA) that include microRNA (miRNA) regulates gene expression inside the cell where they are transcribed, extracellular miRNA has been recently discovered outside the cells, proposing that miRNA might be released to participate in cell-to-cell communication. Despite its importance of extracellular miRNA, the molecular mechanisms by which miRNA can be stored in vesicles and released by vesicle fusion remain enigmatic. Using next-generation sequencing, vesicle purification techniques, and synthetic neurotransmission, we observe that large dense-core vesicles (LDCVs) contain a variety of miRNAs including miR-375. Furthermore, miRNA exocytosis is mediated by the SNARE complex and accelerated by Ca2+. Our results suggest that miRNA can be a novel neuromodulator that can be stored in vesicles and released by vesicle fusion together with classical neurotransmitters.One Sentence SummaryUsing next-generation sequencing (NGS) for microRNA (miRNA) and synthetic neurotransmission, we observed that large dense-core vesicles (LDCVs) contain a variety of miRNA together with classical neurotransmitters, and that miRNA can be released by vesicle fusion mediated by SNARE.

Vesicular Exocytosis under Hypotonic Conditions Shows Two Distinct Populations of Dense Core Vesicles in Bovine Chromaffin Cells

ChemPhysChem ◽  
2007 ◽  
Vol 8 (4) ◽  
pp. 578-585 ◽  
Author(s):  
Christian Amatore ◽  
Stéphane Arbault ◽  
Imelda Bonifas ◽  
Frédéric Lemaître ◽  
Yann Verchier
Keyword(s):  
Chromaffin Cells ◽  
Dense Core ◽  
Dense Core Vesicles ◽  
Bovine Chromaffin Cells ◽  
Vesicular Exocytosis

scholarly journals Synaptotagmin-7 endows a population of chromaffin cell vesicles with enhanced calcium sensing and delayed content release properties

2019 ◽  
Author(s):  
M Bendahmane ◽  
AJB Kreutzberger ◽  
A Chapman-Morales ◽  
J Philippe ◽  
N Schenk ◽  
...  
Keyword(s):  
Chromaffin Cells ◽  
Physiological Role ◽  
Calcium Sensitivity ◽  
Vesicle Fusion ◽  
Adrenal Chromaffin Cells ◽  
Supported Bilayers ◽  
Calcium Sensors ◽  
Calcium Sensing ◽  
Synaptotagmin 1 ◽  
Adrenal Chromaffin

Synaptotagmin-7 (Syt-7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin-1 (Syt-1). Despite its undoubted importance, questions remain as to the functional and physiological role of Syt-7 in secretion. We examined this issue using two distinct preparations - mouse chromaffin cells lacking endogenous Syt-7 (KO cells) and a reconstituted system employing cell-derived vesicles expressing either Syt-7 or Syt-1. First, we report using immunofluorescence that Syt-7 exhibits a punctate intracellular distribution consistent with its sorting to organelles, including dense core vesicles. We also find that the likelihood of vesicle fusion in KO cells is markedly lower than in WT cells. When fusion does occur, cargoes are discharged more rapidly when only Syt-1 is available to facilitate release. A distinctive characteristic of KO cells is that secretion runs down after prolonged cholinergic stimulation. In contrast, exocytosis persists in WT cells even with extended exposure to acetylcholine, suggesting a key role for Syt-7 in sustaining the secretory response. To determine the extent to which the aforementioned results are attributable purely to Syt-7, vesicles expressing only Syt-7 or Syt-1 were triggered to fuse on planar supported bilayers bearing plasma membrane SNARE proteins. Here, as in cells, Syt-7 confers substantially greater calcium sensitivity to vesicle fusion than Syt-1 and slows the rate at which cargos are released. Overall, this study demonstrates that by virtue of its high affinity for calcium, Syt-7 plays a central role in regulating secretory output from adrenal chromaffin cells.

The GTPase Rab3a is associated with large dense core vesicles in bovine chromaffin cells and rat PC12 cells

1995 ◽  
Vol 108 (4) ◽  
pp. 1639-1649 ◽  
Author(s):  
F. Darchen ◽  
J. Senyshyn ◽  
W.H. Brondyk ◽  
D.J. Taatjes ◽  
R.W. Holz ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Pc12 Cells ◽  
Immunoelectron Microscopy ◽  
Chromaffin Cells ◽  
Secretory Granules ◽  
Dense Core ◽  
Dense Core Vesicles ◽  
Large Dense Core Vesicles ◽  
Bovine Chromaffin Cells ◽  
Specific Association

Small GTPases of the rab family control intracellular vesicle traffic in eukaryotic cells. Although the molecular mechanisms underlying the activity of the Rab proteins have not been elucidated yet, it is known that the function of these proteins is dependent on their precise subcellular localization. It has been suggested that Rab3a, which is mainly expressed in neural and endocrine cells, might regulate exocytosis. Recently, direct experimental evidence supporting this hypothesis has been obtained. Consistent with such a role for Rab3a in regulated exocytosis was the previously reported specific association of Rab3a with synaptic vesicles and with secretory granules in adrenal chromaffin cells. Since the latter result, based on subcellular fractionation, has been controversial, we have re-investigated the subcellular localization of this GTP-binding protein by using a combination of morphological techniques. Bovine chromaffin cells were labelled with an affinity-purified polyclonal anti-Rab3a antibody and analyzed by confocal microcopy. Rab3a was found to colocalize partially with dopamine beta-hydroxylase, a chromaffin granule marker. In agreement with this observation, immunoelectron microscopy revealed a specific staining of chromaffin granules. In addition to large dense core vesicles, some small vesicles were labelled. To eliminate the possibility that the staining was due to a Rab3a-related protein, we investigated by immunoelectron microscopy the localization of an epitope-tagged Rab3a expressed in rat PC12 cells. Secretory granules were specifically labelled, whereas clear microvesicles were not. These results provide further evidence supporting a specific association of the GTPase Rab3a with large dense core secretory vesicles.

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