scholarly journals Syncollin inhibits regulated corticotropin secretion from AtT-20 cells through a reduction in the secretory vesicle population

2004 ◽  
Vol 380 (3) ◽  
pp. 897-905 ◽  
Author(s):  
Barbara WÄSLE ◽  
Lori B. HAYS ◽  
Christopher J. RHODES ◽  
J. Michael EDWARDSON
Keyword(s):  
Fluorescent Protein ◽  
Close Association ◽  
Secretory Vesicle ◽  
Neuroendocrine Cells ◽  
Zymogen Granule ◽  
Secretory Vesicles ◽  
Granule Membrane ◽  
Constitutive Secretion ◽  
Cell Processes ◽  
Green Fluorescent

Syncollin is a 13 kDa protein that is highly expressed in the exocrine pancreas. Syncollin normally exists as a doughnut-shaped homo-oligomer (quite probably a hexamer) in close association with the luminal surface of the zymogen granule membrane. In the present study, we examine the effect of expression of syncollin in AtT-20 neuroendocrine cells, which do not normally express this protein. Efficient expression was achieved by infection of the cells with adenoviral constructs encoding either untagged or GFP (green fluorescent protein)-tagged syncollin. Both forms of the protein were sorted into corticotropin (ACTH)-positive secretory vesicles present mainly at the tips of cell processes. Neither form affected basal corticotropin secretion or the constitutive secretion of exogenously expressed secreted alkaline phosphatase. In contrast, regulated secretion of corticotropin was inhibited (by 49%) by untagged but not by GFP-tagged syncollin. In parallel, untagged syncollin caused a 46% reduction in the number of secretory vesicles present at the tips of the cell processes. Syncollin–GFP was without effect. We could also show that native syncollin purified from rat pancreas was capable of permeabilizing erythrocytes. We suggest that syncollin may induce uncontrolled permeabilization of corticotropin-containing vesicles and subsequently destabilize them. Both forms of syncollin were tightly membrane-associated and appeared to exist as homooligomers. Hence, the lack of effect of syncollin–GFP on regulated exocytosis suggests that the GFP tag interferes in a subtler manner with the properties of the assembled protein.

scholarly journals Distinct Subcellular Localization of a Group of Synaptobrevin-Like SNAREs in Paramecium tetraurelia and Effects of Silencing SNARE-Specific Chaperone NSF

2009 ◽  
Vol 9 (2) ◽  
pp. 288-305 ◽  
Author(s):  
Christina Schilde ◽  
Barbara Schönemann ◽  
Ivonne M. Sehring ◽  
Helmut Plattner
Keyword(s):  
Cell Surface ◽  
Membrane Trafficking ◽  
Fluorescent Protein ◽  
Close Association ◽  
Secretory Vesicle ◽  
Food Vacuole ◽  
Paramecium Tetraurelia ◽  
Content Type ◽  
Early Endosomes ◽  
Green Fluorescent

ABSTRACT We have identified new synaptobrevin-like SNAREs and localized the corresponding gene products with green fluorescent protein (GFP)-fusion constructs and specific antibodies at the light and electron microscope (EM) levels. These SNAREs, named Paramecium tetraurelia synaptobrevins 8 to 12 (PtSyb8 to PtSyb12), showed mostly very restricted, specific localization, as they were found predominantly on structures involved in endo- or phagocytosis. In summary, we found PtSyb8 and PtSyb9 associated with the nascent food vacuole, PtSyb10 near the cell surface, at the cytostome, and in close association with ciliary basal bodies, and PtSyb11 on early endosomes and on one side of the cytostome, while PtSyb12 was found in the cytosol. PtSyb4 and PtSyb5 (identified previously) were localized on small vesicles, PtSyb5 probably being engaged in trichocyst (dense core secretory vesicle) processing. PtSyb4 and PtSyb5 are related to each other and are the furthest deviating of all SNAREs identified so far. Because they show no similarity with any other R-SNAREs outside ciliates, they may represent a ciliate-specific adaptation. PtSyb10 forms small domains near ciliary bases, and silencing slows down cell rotation during depolarization-induced ciliary reversal. NSF silencing supports a function of cell surface SNAREs by revealing vesicles along the cell membrane at sites normally devoid of vesicles. The distinct distributions of these SNAREs emphasize the considerable differentiation of membrane trafficking, particularly along the endo-/phagocytic pathway, in this protozoan.

scholarly journals Myosin Va Transports Dense Core Secretory Vesicles in Pancreatic MIN6 β-Cells

2005 ◽  
Vol 16 (6) ◽  
pp. 2670-2680 ◽  
Author(s):  
Aniko Varadi ◽  
Takashi Tsuboi ◽  
Guy A. Rutter
Keyword(s):  
Fluorescent Protein ◽  
Dominant Negative ◽  
Neuroendocrine Cells ◽  
Secretory Vesicles ◽  
Tail Domain ◽  
Cortical Actin ◽  
Β Cells ◽  
Protein Levels ◽  
Myosin Va ◽  
Green Fluorescent

The role of unconventional myosins in neuroendocrine cells is not fully understood, with involvement suggested in the movement of both secretory vesicles and mitochondria. Here, we demonstrate colocalization of myosin Va (MyoVa) with insulin in pancreatic β-cells and show that MyoVa copurifies with insulin in density gradients and with the vesicle marker phogrin-enhanced green fluorescent protein upon fluorescence-activated sorting of vesicles. By contrast, MyoVa immunoreactivity was poorly colocalized with mitochondrial or other markers. Demonstrating an important role for MyoVa in the recruitment of secretory vesicles to the cell surface, a reduction of MyoVa protein levels achieved by RNA interference caused a significant decrease in glucose- or depolarization-stimulated insulin secretion. Similarly, expression of the dominant-negative–acting globular tail domain of MyoVa decreased by ∼50% the number of vesicles docked at the plasma membrane and by 87% the number of depolarization-stimulated exocytotic events detected by total internal reflection fluorescence microscopy. We conclude that MyoVa-driven movements of vesicles along the cortical actin network are essential for the terminal stages of regulated exocytosis in β-cells.

Microtubule-dependent transport of secretory vesicles visualized in real time with a GFP-tagged secretory protein

1997 ◽  
Vol 110 (13) ◽  
pp. 1453-1463 ◽  
Author(s):  
I. Wacker ◽  
C. Kaether ◽  
A. Kromer ◽  
A. Migala ◽  
W. Almers ◽  
...  
Keyword(s):  
Real Time ◽  
Protein Transport ◽  
Fluorescent Protein ◽  
Vero Cells ◽  
Secretory Vesicle ◽  
Secretory Protein ◽  
Secretory Vesicles ◽  
Chromogranin B ◽  
Directed Movement ◽  
Green Fluorescent

Biosynthetic transport from the trans-Golgi network (TGN) to the plasma membrane (PM) is mediated by secretory vesicles. We analyzed secretory vesicle transport in real time using a GFP-tagged secretory protein, hCgB-GFP, consisting of human chromogranin B (hCgB) and green fluorescent protein (GFP). The fusion protein was expressed transiently in Vero cells or in a stable clone after induction with butyrate. After arrest of the biosynthetic protein transport at 20 degrees C, fluorescent hCgB-GFP colocalized with TGN38, a marker of the TGN. Subsequent release of the secretion block at 37 degrees C led to the formation of green fluorescent vesicles. Confocal analysis revealed that these vesicles were devoid of TGN38 and of Texas Red-coupled transferrin and cathepsin D, markers of the endosomal/lysosomal pathway. As determined by fluorometry and metabolic labelling hCgB-GFP was secreted from the TGN to the PM with a t(1/2) of 20–30 minutes. Video-microscope analysis of green fluorescent vesicles showed brief periods of rapid directed movement with maximal velocities of 1 microm/second. Vesicle movement occurred in all directions, centrifugal, centripetal and circumferential, and 50% of the vesicles analyzed reversed their direction of movement at least once within an observation period of 45 seconds. In the presence of nocodazole the movement of fluorescent vesicles ceased. Concomitantly, secretion of hCgB-GFP was slowed but not completely blocked. We suggest that microtubules (MT) facilitate the delivery of secretory vesicles to the PM by a stochastic transport, thereby increasing the probability for a vesicle/target membrane encounter.

Synaptotagmin-1: A Multi-Functional Protein that Mediates Vesicle Docking, Priming, and Fusion

2021 ◽  
Vol 22 ◽  
Author(s):  
Najeeb Ullah ◽  
Ezzouhra El Maaiden ◽  
Md. Sahab Uddin ◽  
Ghulam Md Ashraf
Keyword(s):  
Plasma Membrane ◽  
Secretory Granules ◽  
Secretory Vesicle ◽  
Neuroendocrine Cells ◽  
Snare Complex ◽  
Secretory Vesicles ◽  
Functional Protein ◽  
Vesicle Docking ◽  
Synaptotagmin 1

: The fusion of secretory vesicles with the plasma membrane depends on the assembly of v-SNAREs (VAMP2/synaptobrevin2) and t-SNAREs (SNAP25/syntaxin1) into the SNARE complex. Vesicles go through several upstream steps, referred to as docking and priming, to gain fusion competence. The vesicular protein synaptotagmin-1 (Syt-1) is the principal Ca2+ sensor for fusion in several central nervous system neurons and neuroendocrine cells and part of the docking complex for secretory granules. Syt-1 binds to the acceptor complex such as synaxin1, SNAP-25 on the plasma membrane to facilitate secretory vesicle docking, and upon Ca2+-influx promotes vesicle fusion. This review assesses the role of the Syt-1 protein involved in the secretory vesicle docking, priming, and fusion.

scholarly journals Intragranular targeting of syncollin, but not a syncollinGFP chimera, inhibits regulated insulin exocytosis in pancreatic β-cells

2005 ◽  
Vol 185 (1) ◽  
pp. 57-67 ◽  
Author(s):  
L B Hays ◽  
B Wicksteed ◽  
Y Wang ◽  
J F McCuaig ◽  
L H Philipson ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Fluorescent Protein ◽  
Zymogen Granule ◽  
Β Cells ◽  
Rat Islets ◽  
Intracellular Ca ◽  
Specific Localization ◽  
Glucagon Like Peptide 1 ◽  
Green Fluorescent ◽  
Insulin Exocytosis

Several proteins play a role in the mechanism of insulin exocytosis. However, these ‘exocytotic proteins’ have yet to account for the regulated aspect of insulin exocytosis, and other factors are involved. In pancreatic exocrine cells, the intralumenal zymogen granule protein, syncollin, is required for efficient regulated exocytosis, but it is not known whether intragranular peptides similarly influence regulated insulin exocytosis. Here, this issue has been addressed using expression of syncollin and a syncollin-green fluorescent protein (syncollinGFP) chimera in rat islet β-cells as experimental tools. Syncollin is not normally expressed in β-cells but adenoviral-mediated expression of both syncollin and syncollinGFP indicated that these were specifically targeted to the lumen of β-granules. Syncollin expression in isolated rat islets had no effect on basal insulin secretion but significantly inhibited regulated insulin secretion stimulated by glucose (16.7 mM), glucagon-like peptide-1 (GLP-1) (10 nM) and glyburide (5μM). Consistent with specific localization of syncollin to β-granules, constitutive secretion was unchanged by syncollin expression in rat islets. Syncollin-mediated inhibition of insulin secretion was not due to inadequate insulin production. Moreover, secretagogue-induced increases in cytosolic intracellular Ca2+, which is a prerequisite for triggering insulin exocytosis, were unaffected in syncollin-expressing islets. Therefore, syncollin was most likely acting downstream of secondary signals at the level of insulin exocytosis. Thus, syncollin expression in β-cells has highlighted the importance of intralumenal β-granule peptide factors playing a role in the control of insulin exocytosis. In contrast to syncollin, syncollinGFP had no effect on insulin secretion, underlining its usefulness as a ‘fluorescent tag’ to track β-granule transport and exocytosis in real time.

scholarly journals In vivo analysis of mouse gastrin gene regulation in enhanced GFP-BAC transgenic mice

2011 ◽  
Vol 300 (2) ◽  
pp. G334-G344 ◽  
Author(s):  
Shigeo Takaishi ◽  
Wataru Shibata ◽  
Hiroyuki Tomita ◽  
Guangchun Jin ◽  
Xiangdong Yang ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Fluorescent Protein ◽  
Artificial Chromosome ◽  
Transitional Zone ◽  
Anterior Lobe ◽  
Gastric Antrum ◽  
Neuroendocrine Cells ◽  
Adult Mice ◽  
Green Fluorescent

Gastrin is secreted from a subset of neuroendocrine cells residing in the gastric antrum known as G cells, but low levels are also expressed in fetal pancreas and intestine and in many solid malignancies. Although past studies have suggested that antral gastrin is transcriptionally regulated by inflammation, gastric pH, somatostatin, and neoplastic transformation, the transcriptional regulation of gastrin has not previously been demonstrated in vivo. Here, we describe the creation of an enhanced green fluorescent protein reporter (mGAS-EGFP) mouse using a bacterial artificial chromosome that contains the entire mouse gastrin gene. Three founder lines expressed GFP signals in the gastric antrum and the transitional zone to the corpus. In addition, GFP(+) cells could be detected in the fetal pancreatic islets and small intestinal villi, but not in these organs of the adult mice. The administration of acid-suppressive reagents such as proton pump inhibitor omeprazole and gastrin/CCK-2 receptor antagonist YF476 significantly increased GFP signal intensity and GFP(+) cell numbers in the antrum, whereas these parameters were decreased by overnight fasting, octreotide (long-lasting somatostatin ortholog) infusion, and Helicobacter felis infection. GFP(+) cells were also detected in the anterior lobe of the pituitary gland and importantly in the colonic tumor cells induced by administration with azoxymethane and dextran sulfate sodium salt. This transgenic mouse provides a useful tool to study the regulation of mouse gastrin gene in vivo, thus contributing to our understanding of the mechanisms involved in transcriptional control of the gastrin gene.

Cleavage of progastrin-derived peptides is modulated by intravesicular PH: Studies using PH-sensitive green fluorescent protein directed to secretory vesicles

2000 ◽  
Vol 118 (4) ◽  
pp. A712
Author(s):  
Colin Blackmore ◽  
Lisa Bishop ◽  
Andrea Varro ◽  
Rod Dimaline ◽  
David V. Gallacher ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ph Sensitive ◽  
Secretory Vesicles ◽  
Green Fluorescent

scholarly journals The class V myosin motor, myosin 5c, localizes to mature secretory vesicles and facilitates exocytosis in lacrimal acini

2008 ◽  
Vol 295 (1) ◽  
pp. C13-C28 ◽  
Author(s):  
Ronald R. Marchelletta ◽  
Damon T. Jacobs ◽  
Joel E. Schechter ◽  
Richard E. Cheney ◽  
Sarah F. Hamm-Alvarez
Keyword(s):  
Lacrimal Gland ◽  
Fluorescent Protein ◽  
Secretory Vesicle ◽  
Exocrine Secretion ◽  
Secretory Component ◽  
Immunogold Electron Microscopy ◽  
Secretory Vesicles ◽  
Muscarinic Agonist ◽  
Tail Domain ◽  
Myosin Motor

We investigated the role of the actin-based myosin motor, myosin 5c (Myo5c) in vesicle transport in exocrine secretion. Lacrimal gland acinar cells (LGAC) are the major source for the regulated secretion of proteins from the lacrimal gland into the tear film. Confocal fluorescence and immunogold electron microscopy revealed that Myo5c was associated with secretory vesicles in primary rabbit LGAC. Upon stimulation of secretion with the muscarinic agonist, carbachol, Myo5c was also detected in association with actin-coated fusion intermediates. Adenovirus-mediated expression of green fluorescent protein (GFP) fused to the tail domain of Myo5c (Ad-GFP-Myo5c-tail) showed that this protein was localized to secretory vesicles. Furthermore, its expression induced a significant ( P ≤ 0.05) decrease in carbachol-stimulated release of two secretory vesicle content markers, secretory component and syncollin-GFP. Adenovirus-mediated expression of GFP appended to the full-length Myo5c (Ad-GFP-Myo5c-full) was used in parallel with adenovirus-mediated expression of GFP-Myo5c-tail in LGAC to compare various parameters of secretory vesicles labeled with either GFP-labeled protein in resting and stimulated LGAC. These studies revealed that the carbachol-stimulated increase in secretory vesicle diameter associated with compound fusion of secretory vesicles that was also exhibited by vesicles labeled with GFP-Myo5c-full was impaired in vesicles labeled with GFP-Myo5c-tail. A significant decrease in GFP labeling of actin-coated fusion intermediates was also seen in carbachol-stimulated LGAC transduced with GFP-Myo5c-tail relative to LGAC transduced with GFP-Myo5c-full. These results suggest that Myo5c participates in apical exocytosis of secretory vesicles.

Live pancreatic acinar imaging of exocytosis using syncollin-pHluorin

2011 ◽  
Vol 300 (6) ◽  
pp. C1513-C1523 ◽  
Author(s):  
Nestor A. Fernandez ◽  
Tao Liang ◽  
Herbert Y. Gaisano
Keyword(s):  
Fusion Protein ◽  
Protein Expression ◽  
Fluorescent Protein ◽  
Acinar Cells ◽  
Three Dimensions ◽  
Imaging Method ◽  
Zymogen Granule ◽  
Ph Dependent ◽  
Green Fluorescent ◽  
Superior Approach

In this report, a novel live acinar exocytosis imaging technique is described. An adenovirus was engineered, encoding for an endogenous zymogen granule (ZG) protein (syncollin) fused to pHluorin, a pH-dependent green fluorescent protein (GFP). Short-term culture of mouse acini infected with this virus permits exogenous adenoviral protein expression while retaining acinar secretory competence and cell polarity. The syncollin-pHluorin fusion protein was shown to be correctly localized to ZGs, and the pH-dependent fluorescence of pHluorin was retained. Coupled with the use of a spinning disk confocal microscope, the syncollin-pHluorin fusion protein exploits the ZG luminal pH changes that occur during exocytosis to visualize exocytic events of live acinar cells in real-time with high spatial resolution in three dimensions. Apical and basolateral exocytic events were observed on stimulation of acinar cells with maximal and supramaximal cholecystokinin concentrations, respectively. Sequential exocytic events were also observed. Coupled with the use of transgenic mice and/or adenovirus-mediated protein expression, this syncollin-pHluorin imaging method offers a superior approach to studying pancreatic acinar exocytosis. This assay can also be applied to acinar disease models to elucidate the mechanisms implicated in pancreatitis.

Novel Secretory Vesicle Serpins, Endopin 1 and Endopin 2: Endogenous Protease Inhibitors with Distinct Target Protease Specificities

2002 ◽  
Vol 383 (7-8) ◽  
pp. 1067-1074 ◽  
Author(s):  
V. Y. H. Hook ◽  
S.-R. Hwang
Keyword(s):  
Protease Inhibitors ◽  
Serine Proteases ◽  
Chromaffin Cells ◽  
Cysteine Proteases ◽  
Secretory Vesicle ◽  
Proteolytic Processing ◽  
Neuroendocrine Cells ◽  
Secretory Vesicles ◽  
Subcellular Compartment ◽  
Endogenous Protease

Abstract Secretory vesicles of neuroendocrine cells possess multiple proteases for proteolytic processing of proteins into biologically active peptide components, such as peptide hormones and neurotransmitters. The importance of proteases within secretory vesicles predicts the presence of endogenous protease inhibitors in this subcellular compartment. Notably, serpins represent a diverse class of endogenous protease inhibitors that possess selective target protease specificities, defined by the reactive site loop domains (RSL). In the search for endogenous serpins in model secretory vesicles of neuroendocrine chromaffin cells, the presence of serpins related to α1-antichymotrypsin (ACT) was detected by Western blots with antiACT. Molecular cloning revealed the primary structures of two unique serpins, endopin 1 and endopin 2, that possess homology to ACT. Of particular interest was the observation that distinct RSL domains of these new serpins predicted that endopin 1 would inhibit trypsinlike serine proteases cleaving at basic residues, and endopin 2 would inhibit both elastase and papain that represent serine and cysteine proteases, respectively. Endopin 1 showed selective inhibition of trypsin, but did not inhibit chymotrypsin, elastase, or subtilisin. Endopin 2 demonstrated crossclass inhibition of the cysteine protease papain and the serine protease elastase. Endopin 2 did not inhibit chymotrypsin, trypsin, plasmin, thrombin, furin, or cathepsin B. Endopin 1 and endopin 2 each formed SDSstable complexes with target proteases, a characteristic property of serpins. In neuroendocrine chromaffin cells from adrenal medulla, endopin 1 and endopin 2 were both localized to secretory vesicles. Moreover, the inhibitory activity of endopin 2 was optimized under reducing conditions, which required reduced Cys-374; this property is consistent with the presence of endogenous reducing agents in secretory vesicles in vivo. These new findings demonstrate the presence of unique secretory vesicle serpins, endopin 1 and endopin 2, which possess distinct target protease selectivities. Endopin 1 inhibits trypsinlike proteases; endopin 2 possesses crossclass inhibition for inhibition of papainlike cysteine proteases and elastaselike serine proteases. It will be of interest in future studies to define the endogenous protease targets of these two novel secretory vesicle serpins.

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