PKCε controls the fusion of secretory vesicles in mast cells in a phosphatidic acid-dependent mode

2021 ◽  
Author(s):  
Emilio M. Serrano-López ◽  
David López-Martínez ◽  
Juan C. Gómez-Fernández ◽  
Antonio Luis Egea-Jimenez ◽  
Senena Corbalán-García
Keyword(s):  
Mast Cells ◽  
Phosphatidic Acid ◽  
Secretory Vesicles

Stimulated release of fluorescently labeled IgE fragments that efficiently accumulate in secretory granules after endocytosis in RBL-2H3 mast cells

1998 ◽  
Vol 111 (16) ◽  
pp. 2385-2396 ◽  
Author(s):  
K. Xu ◽  
R.M. Williams ◽  
D. Holowka ◽  
B. Baird
Keyword(s):  
Mast Cells ◽  
Fluorescence Microscopy ◽  
High Efficiency ◽  
Immunoglobulin E ◽  
Secretory Granules ◽  
Dose Dependence ◽  
Secretory Vesicles ◽  
Release Process ◽  
Intracellular Compartments ◽  
Stimulation Of

Sensitization of RBL-2H3 mast cells with monomeric fluorescein-5-isothiocyanate (FITC)-labeled immunoglobulin E (IgE) results in slow but highly efficient accumulation of labeled IgE fragments in a pool of acidic peripheral vesicles that are visible by fluorescence microscopy after raising endosomal pH with ammonium chloride. Stimulation of cells containing these FITC-IgE fragments by aggregation of high affinity receptors for IgE (FcepsilonRI) or by Ca2+ ionophore and phorbol 12-myristate 13-acetate results in release of FITC fluorescence from the cells, which can be monitored continuously with a spectrofluorometer. The fluorescence release process corresponds to cellular degranulation: it is prevented under conditions that prevent stimulated beta-hexosaminidase release, and these two processes exhibit the same antigen dose-dependence and kinetics. Pulse-chase labeling reveals that aggregation of FITC-IgE bound to FcepsilonRI at the cell surface causes internalization and delivery to the regulated secretory vesicles with a high efficiency similar to monomeric IgE-FcepsilonRI, but more rapidly. Binding of Cy3-modified IgE to FcepsilonRI results in labeling of the same secretory vesicles as in FITC-IgE-sensitized cells, and these Cy3-labeled vesicles can be observed by fluorescence microscopy without neutralization of intracellular compartments. Simultaneous three-photon microscopy of serotonin fluorescence and two-photon microscopy of Cy3 fluorescence reveals that these Cy3-labeled vesicles coincide with serotonin-labeled secretory granules. After stimulation of the cells via aggregation of IgE-FcepsilonRI or addition of Ca2+ ionophore and phorbol 12-myristate 13-acetate, depletion of the Cy3 label from the intracellular vesicles is observed with confocal microscopy. These results provide strong evidence for the lysosomal nature of secretory granules in these cells. In addition, they provide the basis for a direct, real-time method for monitoring single cell degranulation.

Possible Involvement of Calcium, Calmodulin and Intramembrane Particle Arrays in Regulation of Secretion

1980 ◽  
Vol 38 ◽  
pp. 590-593
Author(s):  
Birgit H. Satir
Keyword(s):  
Salivary Gland ◽  
Mast Cells ◽  
Membrane Fusion ◽  
Single Cell ◽  
Common Denominator ◽  
Secretory Vesicles ◽  
Intramembrane Particle ◽  
Contraction Coupling ◽  
Stimulus Secretion Coupling ◽  
Regulation Of Secretion

Ca2+ is found to be a common denominator involved in the control of membrane fusion and release in many biological secretory systems ranging from the classical mammalian secretory systems such as the pancreas, salivary gland, neurosecretion, single cell “glands” such as mast cells, to protozoa such as Tetrahymena and Paramecium with their release of mucocysts and trichocysts, respectively. In all of these systems, Ca2+ has been shown to be an important ion in the process of stimulus-secretion-coupling, a term coined by Douglas (1968), in analogy to excitation-contraction coupling in muscle. Individual degrees of specificity are shown by each of these systems as to exactly which membrane the secretory vesicles migrate to and dock prior to release.

scholarly journals Enlargement of Secretory Vesicles by Protein Tyrosine Phosphatase PTP-MEG2 in Rat Basophilic Leukemia Mast Cells and Jurkat T Cells

2002 ◽  
Vol 168 (9) ◽  
pp. 4612-4619 ◽  
Author(s):  
Xiaodong Wang ◽  
Huong Huynh ◽  
Anette Gjörloff-Wingren ◽  
Edvard Monosov ◽  
Mats Stridsberg ◽  
...  
Keyword(s):  
T Cells ◽  
Mast Cells ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Secretory Vesicles ◽  
Jurkat T Cells ◽  
Protein Tyrosine ◽  
Basophilic Leukemia

scholarly journals Neomycin is a potent secretagogue of mast cells that directly activates a GTP-binding protein involved in exocytosis.

1990 ◽  
Vol 111 (6) ◽  
pp. 2885-2891 ◽  
Author(s):  
M Aridor ◽  
R Sagi-Eisenberg
Keyword(s):  
Mast Cells ◽  
Phosphatidic Acid ◽  
Rat Brain ◽  
G Protein ◽  
Gtp Binding Protein ◽  
Histamine Secretion ◽  
Gtp Binding ◽  
Rat Brain Membranes ◽  
Gamma S ◽  
Dose Dependent

When loaded alongside GTP-gamma-S into ATP-permeabilized cells, neomycin, at concentrations below 1 mM, inhibits GTP-gamma-S-induced histamine secretion and phosphatidic acid formation (Cockcroft, S., and B. D. Gomperts, 1985. Nature (Lond.). 314: 534-536; Aridor, M., L. M. Traub, and R. Sagi-Eisenberg. 1990. J. Cell Biol. 111:909-917). However, at higher concentrations internally applied neomycin induces histamine secretion in a process that is: (a) dose dependent; (b) dependent on the internal application of GTP; (c) independent of phosphoinositide breakdown; and (d) inhibited by pertussis toxin (PtX) treatment. These results indicate that neomycin can stimulate histamine secretion in a mechanism that bypasses phospholipase C (PLC) activation and yet involves a PtX-sensitive GTP-binding protein (G protein). Unlike its dual effects, when internally applied, neomycin induces histamine secretion from intact mast cells in a dose-dependent manner. Half-maximal and maximal effects are obtained at 0.5 and 1 mM neomycin, respectively. This process is rapid (approximately 30 s), is independent of external Ca2+, and is associated with phosphatidic acid formation, implying that neomycin can activate histamine secretion by a mechanism similar to that utilized by other basic secretagogues of mast cells. Neomycin stimulates fourfold the GTPase activity of cholate-solubilized rat brain membranes in a PtX-inhibitable manner. In addition neomycin, as well as the basic secretagogues of mast cells, compound 48/80, and mastoparan, significantly reduce (by approximately 80%) the ADP ribosylation of PtX substrates present in rat brain membranes. Taken together these data suggest that neomycin can stimulate secretion from mast cells by directly activating G proteins that play a role in stimulus-secretion coupling. When internally applied, neomycin presumably stimulates secretion by activating a G protein that is located downstream to PLC. This G protein serves as a substrate for PtX.

scholarly journals Continual Production of Phosphatidic Acid by Phospholipase D Is Essential for Antigen-stimulated Membrane Ruffling in Cultured Mast Cells

2002 ◽  
Vol 13 (10) ◽  
pp. 3730-3746 ◽  
Author(s):  
Niamh O'Luanaigh ◽  
Raul Pardo ◽  
Amanda Fensome ◽  
Victoria Allen-Baume ◽  
David Jones ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Mast Cells ◽  
Phosphatidic Acid ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Permeabilized Cells ◽  
Membrane Ruffling ◽  
Green Fluorescent ◽  
Cytoskeletal Rearrangements ◽  
Adp Ribosylation Factor

Phospholipase Ds (PLDs) are regulated enzymes that generate phosphatidic acid (PA), a putative second messenger implicated in the regulation of vesicular trafficking and cytoskeletal reorganization. Mast cells, when stimulated with antigen, show a dramatic alteration in their cytoskeleton and also release their secretory granules by exocytosis. Butan-1-ol, which diverts the production of PA generated by PLD to the corresponding phosphatidylalcohol, was found to inhibit membrane ruffling when added together with antigen or when added after antigen. Inhibition by butan-1-ol was completely reversible because removal of butan-1-ol restored membrane ruffling. Measurements of PLD activation by antigen indicate a requirement for continual PA production during membrane ruffling, which was maintained for at least 30 min. PLD1 and PLD2 are both expressed in mast cells and green fluorescent protein-tagged proteins were used to identify PLD2 localizing to membrane ruffles of antigen-stimulated mast cells together with endogenous ADP ribosylation factor 6 (ARF6). In contrast, green fluorescent protein-PLD1 localized to intracellular vesicles and remained in this location after stimulation with antigen. Membrane ruffling was independent of exocytosis of secretory granules because phorbol 12-myristate 13-acetate increased membrane ruffling in the absence of exocytosis. Antigen or phorbol 12-myristate 13-acetate stimulation increased both PLD1 and PLD2 activity when expressed individually in RBL-2H3 cells. Although basal activity of PLD2-overexpressing cells is very high, membrane ruffling was still dependent on antigen stimulation. In permeabilized cells, antigen-stimulated phosphatidylinositol(4,5)bisphosphate synthesis was dependent on both ARF6 and PA generated from PLD. We conclude that both activation of ARF6 by antigen and a continual PLD2 activity are essential for local phosphatidylinositol(4,5)bisphosphate generation that regulates dynamic actin cytoskeletal rearrangements.

Ultrastructural Analysis of the Salivary Glands of Gromphadorhina Portentosa (Schaum)

1977 ◽  
Vol 35 ◽  
pp. 638-639
Author(s):  
P.J. Dailey
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Salivary Glands ◽  
Secretory Vesicles ◽  
The Past ◽  
Transmission Electron ◽  
Means Of Transmission ◽  
Gromphadorhina Portentosa ◽  
Scanning Electron ◽  
Topographical Relief

The structure of insect salivary glands has been extensively investigated during the past decade; however, none have attempted scanning electron microscopy (SEM) in ultrastructural examinations of these secretory organs. This study correlates fine structure by means of SEM cryofractography with that of thin-sectioned epoxy embedded material observed by means of transmission electron microscopy (TEM).Salivary glands of Gromphadorhina portentosa were excised and immediately submerged in cold (4°C) paraformaldehyde-glutaraldehyde fixative1 for 2 hr, washed and post-fixed in 1 per cent 0s04 in phosphosphate buffer (4°C for 2 hr). After ethanolic dehydration half of the samples were embedded in Epon 812 for TEM and half cryofractured and subsequently critical point dried for SEM. Dried specimens were mounted on aluminum stubs and coated with approximately 150 Å of gold in a cold sputtering apparatus.Figure 1 shows a cryofractured plane through a salivary acinus revealing topographical relief of secretory vesicles.

Cytochemical analysis of mast cell granules after poly-dl-lysine action

1978 ◽  
Vol 36 (2) ◽  
pp. 278-279
Author(s):  
R. Courtoy ◽  
L.J. Simar ◽  
J. Christophe
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Chemical Compounds ◽  
Cellular Membrane ◽  
Thin Sections ◽  
Organic Bases ◽  
Ferric Thiocyanate ◽  
Cytochemical Analysis ◽  
Mast Cell Granule ◽  
Amine Liberation

Several chemical compounds induce amine liberation from mast cells but do not necessarily provoque the granule expulsion. For example, poly-dl-lysine induces modifications of the cellular membrane permeability which promotes ion exchange at the level of mast cell granules. Few of them are expulsed but the majority remains in the cytoplasm and appears less dense to the electrons. A cytochemical analysis has been performed to determine the composition of these granules after the polylysine action.We have previously reported that it was possible to demonstrate polyanions on epon thin sections using a cetylpyridinium ferric thiocyanate method. Organic bases are selectively stained with cobalt thiocyanate and the sulfhydryle groups are characterized with a silver methenamine reaction. These techniques permit to reveal the mast cell granule constituents, i.e. heparin, biogenic amines and basic proteins.

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