scholarly journals Electron microscopic immunocytochemical evidence for the involvement of the convertases PC1 and PC2 in the processing of proinsulin in pancreatic beta-cells.

1995 ◽  
Vol 43 (1) ◽  
pp. 11-19 ◽  
Author(s):  
D Malide ◽  
N G Seidah ◽  
M Chrétien ◽  
M Bendayan
Keyword(s):  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Protein A ◽  
Gold Complex ◽  
Immunocytochemical Study ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Endoproteolytic Cleavage

Endoproteolytic cleavage of pairs of basic amino acids is the key mechanism in the specific processing of precursor hormone molecules. Two endoproteases, PC1 (or PC3) and PC2, have recently been implicated in the conversion of proinsulin. Using antibodies against these proteases and proinsulin, followed by protein A-gold complex, we performed an immunocytochemical study for precise identification of the subcellular compartments involved in the processing of insulin. Both PC1 and PC2 immunoreactivities followed a pattern of gradually increasing density along the secretory pathway, being higher in the immature granules. Proinsulin labeling was detected in the Golgi apparatus and in the coated immature secretory granules located mainly in the Golgi area. Using double labeling, we demonstrated the presence of PC1 and/or PC2 in the majority of proinsulin-rich granules. In addition, we provided evidence that PC1 and PC2 are co-localized within the same granules. Co-expression of PC1 and PC2 with proinsulin in islet beta-cells indicates that these proteases are actively involved, probably in a sequential manner, in the conversion of proinsulin into insulin.

scholarly journals The Secretory Granule and Pro-opiomelanocortin Processing in Xenopus Melanotrope Cells During Background Adaptation

1997 ◽  
Vol 45 (12) ◽  
pp. 1673-1682 ◽  
Author(s):  
Constance A.F.M. Berghs ◽  
Shigeyasu Tanaka ◽  
Frank J.C. Van Strien ◽  
Shingo Kurabuchi ◽  
Eric W. Roubos
Keyword(s):  
Synthetic Peptide ◽  
Secretory Granules ◽  
Immunocytochemical Study ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Dense Granules ◽  
Black Background ◽  
Background Adaptation ◽  
Granule Maturation ◽  
Electron Lucent

In this immunocytochemical study, we used light and electron microscopic observations in combination with morphometry to analyze the processing of pro-opiomelanocortin (POMC) in melanotrope cells of the intermediate pituitary of Xenopus laevis adapted to either a white or a black background. An antiserum was raised against a synthetic peptide including the cleavage site between ACTH and β-lipotropic hormone in Xenopus. Western blotting revealed that this antiserum recognizes only a 38-kD protein, the POMC prohormone, from extracts of Xenopus neurointermediate pituitary. Light immunocytochemistry showed differential immunostaining for anti-POMC compared to anti-α-MSH. Anti-POMC was predominantly found in the perinuclear region, whereas anti-α-MSH yielded staining throughout the cytoplasm. Immunogold double labeling revealed that electron-dense secretory granules (DGs) show high immunoreactivity for anti-POMC and low immunoreactivity for anti-α-MSH. Electron-lucent granules (LGs) are immunoreactive to anti-α-MSH only. Moderately electron-dense granules (MGs) revealed intermediate reactivity compared to DGs and LGs. Background light intensity has significant effects on the morphology and the immunoreactivity of the secretory granules. Black-adapted animals have 4.5 times as many DGs and MGs as white-adapted animals. In addition, the MGs in black animals show 42% more anti-α-MSH immunogold than the MGs in white animals. Together, these findings indicate that the three granule types represent subsequent stages in granule maturation. Adaptation to a black background stimulates the formation of young immature granules, while at the same time the processing rate during granule maturation increases.

scholarly journals Distinct molecular mechanisms for protein sorting within immature secretory granules of pancreatic beta-cells.

1994 ◽  
Vol 126 (1) ◽  
pp. 77-86 ◽  
Author(s):  
R Kuliawat ◽  
P Arvan
Keyword(s):  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Secretory Granules ◽  
Protein Sorting ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Lysosomal Hydrolases ◽  
Storage Granules

In the beta-cells of pancreatic islets, insulin is stored as the predominant protein within storage granules that undergo regulated exocytosis in response to glucose. By pulse-chase analysis of radiolabeled protein condensation in beta-cells, the formation of insoluble aggregates of regulated secretory protein lags behind the conversion of proinsulin to insulin. Condensation occurs within immature granules (IGs), accounting for passive protein sorting as demonstrated by constitutive-like secretion of newly synthesized C-peptide in stoichiometric excess of insulin (Kuliawat, R., and P. Arvan. J. Cell Biol. 1992. 118:521-529). Experimental manipulation of condensation conditions in vivo reveals a direct relationship between sorting of regulated secretory protein and polymer assembly within IGs. By contrast, entry from the trans-Golgi network into IGs does not appear especially selective for regulated secretory proteins. Specifically, in normal islets, lysosomal enzyme precursors enter the stimulus-dependent secretory pathway with comparable efficiency to that of proinsulin. However, within 2 h after synthesis (the same period during which proinsulin processing occurs), newly synthesized hydrolases are fairly efficiently relocated out of the stimulus-dependent pathway. In tunicamycin-treated islets, while entry of new lysosomal enzymes into the regulated secretory pathway continues unperturbed, exit of nonglycosylated hydrolases from this pathway does not occur. Consequently, the ultimate targeting of nonglycosylated hydrolases in beta-cells is to storage granules rather than lysosomes. These results implicate a post-Golgi mechanism for the active removal of lysosomal hydrolases away from condensed granule contents during the storage process for regulated secretory proteins.

scholarly journals Immunolabeling of adenohypophysial cells with protein A-colloidal gold--antibody complex for electron microscopy: use of the freeze-substitution technique in tissue preparation.

1984 ◽  
Vol 32 (7) ◽  
pp. 705-711 ◽  
Author(s):  
S Hisano ◽  
T Adachi ◽  
S Daikoku
Keyword(s):  
Colloidal Gold ◽  
Secretory Granules ◽  
Protein A ◽  
Freeze Substitution ◽  
Pituitary Cells ◽  
Tissue Preparation ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Cytoplasmic Matrix ◽  
Gold Particles

The value of the freeze-substitution (FS) method for preparing tissues for electron microscopic immunohistochemistry was studied by comparing anterior pituitary cells prepared by this method and by a conventional method. Ultrathin sections of tissues embedded in Epon were subjected to immunostaining. The antigens adrenocorticotropin (ACTH) and prolactin (PRL) in a single ultrathin section were demonstrated by a simple double-labeling technique using a protein A-colloidal gold-antibody (pAG-Ab) complex. The preservation of cellular ultrastructure was superior in preparations obtained by FS. Gold-labeling was seen over secretory granules, and in ACTH cells also over the cytoplasmic matrix. The labeling was more intense in preparations obtained by FS, judging from the numbers of gold particles. In the double-labeling procedure, in which the pA-small colloidal gold-anti-PRL complex and pA-large colloidal gold-anti-ACTH complex were applied sequentially to sections, no cross-labeling with small and large gold particles was observed. It is concluded that if the antisera are sufficiently specific, the use of FS and the pAG-Ab complex is very effective in peptide immunohistochemistry. However, in the double-labeling procedure it is essential that the Fc-binding sites of pAG are saturated by the use of excess amounts of antibodies.

scholarly journals MyRIP interaction with MyoVa on secretory granules is controlled by the cAMP-PKA pathway

2012 ◽  
Vol 23 (22) ◽  
pp. 4444-4455 ◽  
Author(s):  
Flora Brozzi ◽  
Sophie Lajus ◽  
Frederique Diraison ◽  
Shavanthi Rajatileka ◽  
Katy Hayward ◽  
...  
Keyword(s):  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Secretory Granules ◽  
Hormone Secretion ◽  
Motor Protein ◽  
Functional Protein ◽  
Interacting Protein ◽  
Myosin Va ◽  
Glucose Stimulated Insulin Secretion

Myosin- and Rab-interacting protein (MyRIP), which belongs to the protein kinase A (PKA)–anchoring family, is implicated in hormone secretion. However, its mechanism of action is not fully elucidated. Here we investigate the role of MyRIP in myosin Va (MyoVa)-dependent secretory granule (SG) transport and secretion in pancreatic beta cells. These cells solely express the brain isoform of MyoVa (BR-MyoVa), which is a key motor protein in SG transport. In vitro pull-down, coimmunoprecipitation, and colocalization studies revealed that MyRIP does not interact with BR-MyoVa in glucose-stimulated pancreatic beta cells, suggesting that, contrary to previous notions, MyRIP does not link this motor protein to SGs. Glucose-stimulated insulin secretion is augmented by incretin hormones, which increase cAMP levels and leads to MyRIP phosphorylation, its interaction with BR-MyoVa, and phosphorylation of the BR-MyoVa receptor rabphilin-3A (Rph-3A). Rph-3A phosphorylation on Ser-234 was inhibited by small interfering RNA knockdown of MyRIP, which also reduced cAMP-mediated hormone secretion. Demonstrating the importance of this phosphorylation, nonphosphorylatable and phosphomimic Rph-3A mutants significantly altered hormone release when PKA was activated. These data suggest that MyRIP only forms a functional protein complex with BR-MyoVa on SGs when cAMP is elevated and under this condition facilitates phosphorylation of SG-associated proteins, which in turn can enhance secretion.

Colocalization of chaperone Cpn60, proinsulin and convertase PC1 within immature secretory granules of insulin-secreting cells suggests a role for Cpn60 in insulin processing

2000 ◽  
Vol 113 (11) ◽  
pp. 2075-2083 ◽  
Author(s):  
A.E. Arias ◽  
C.S. Velez-Granell ◽  
G. Mayer ◽  
M. Bendayan
Keyword(s):  
Islet Cell ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Double Labeling ◽  
In Vitro Binding ◽  
Cell Extracts ◽  
Vitro Binding ◽  
Preferential Binding ◽  
Insulin Secreting Cells

Many of the mechanisms that control insulin processing and packaging by interaction with different elements along the secretory pathway remain poorly understood. We have investigated the possibility that Cpn60, a member of the heat shock protein family, may be present in rat insulin-secreting cells, participating in the proinsulin-insulin maturation process. Immunofluorescence and high resolution immunocytochemical studies revealed the presence of the Cpn60 protein all along the insulin secretory pathway, being particularly abundant over the proinsulin-containing immature secretory granules. Double-labeling experiments showed associations between Cpn60 and proinsulin, as well as between Cpn60 and PC1 convertase, with a preferential binding to proinsulin. These findings paralleled those of coimmunoprecipitation studies showing the Cpn60 chaperone and the mature form of the PC1 convertase in proinsulin immunoprecipitates, as well as the PC1 in Cpn60 immunoprecipitates from total islet cell extracts. In vitro binding of Cpn60 to proinsulin, insulin and glucagon was also documented. Cpn60, significantly abundant in proinsulin-containing secretory granules where conversion of proinsulin to insulin takes place, and the colocalization of the chaperone with proinsulin and PC1 convertase suggest that the Cpn60 protein may play a role directing precise molecular interactions during insulin processing and/or packaging.

scholarly journals An antibody against secretogranin I (chromogranin B) is packaged into secretory granules.

1989 ◽  
Vol 109 (1) ◽  
pp. 17-34 ◽  
Author(s):  
P Rosa ◽  
U Weiss ◽  
R Pepperkok ◽  
W Ansorge ◽  
C Niehrs ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Secretory Granules ◽  
Intracellular Localization ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Chromogranin B ◽  
Double Labeling ◽  
Protein Protein Interaction ◽  
And Function ◽  
Secretogranin I

We have investigated the sorting and packaging of secretory proteins into secretory granules by an immunological approach. An mAb against secretogranin I (chromogranin B), a secretory protein costored with various peptide hormones and neuropeptides in secretory granules of many endocrine cells and neurons, was expressed by microinjection of its mRNA into the secretogranin I-producing cell line PC12. An mAb against the G protein of vesicular stomatitis virus--i.e., against an antigen not present in PC12 cells--was expressed as a control. The intracellular localization and the secretion of the antibodies was studied by double-labeling immunofluorescence using the conventional and the confocal microscope, as well as by pulse-chase experiments. The secretogranin I antibody, like the control antibody, was transported along the secretory pathway to the Golgi complex. However, in contrast to the control antibody, which was secreted via the constitutive pathway, the secretogranin I antibody formed an immunocomplex with secretogranin I, was packaged into secretory granules, and was released by regulated exocytosis. Our results show that a constitutive secretory protein, unaltered by genetic engineering, can be diverted to the regulated pathway of secretion by its protein-protein interaction with a regulated secretory protein. The data also provide the basis for immunologically studying the role of luminally exposed protein domains in the biogenesis and function of regulated secretory vesicles.

scholarly journals Localization of lysosomal and peroxisomal enzymes in the specific granules of rat intestinal eosinophil leukocytes revealed by immunoelectron microscopic techniques.

1984 ◽  
Vol 32 (3) ◽  
pp. 267-274 ◽  
Author(s):  
S Yokota ◽  
H Tsuji ◽  
K Kato
Keyword(s):  
Quantitative Analysis ◽  
Intestinal Mucosa ◽  
Cathepsin D ◽  
Lysosomal Enzymes ◽  
Protein A ◽  
Gold Complex ◽  
Double Labeling ◽  
Specific Granules ◽  
Acyl Coa Oxidase ◽  
Microscopic Techniques

Immunoelectron microscopic localization of lysosomal and peroxisomal enzymes in the eosinophil leukocytes of rat intestinal mucosa was studied by use of rabbit antibodies to the enzymes coupled to protein A-gold complex. Gold particle labeling for the lysosomal enzymes, beta-glucuronidase and cathepsin D, was present on specific granules, with a heavy concentration on their paracrystalline cores. The peroxisomal enzymes, acyl-CoA oxidase and catalase, were also found on these granules. The double labeling procedures using two different combination of anti-acyl-CoA oxidase and anti-beta-glucuronidase or anti-catalase and anti-cathepsin D revealed that these enzymes were simultaneously present in specific granules of the intestinal eosinophils. Quantitative analysis of the labeling on subcellular compartments confirmed that all enzymes examined are significantly localized within specific granules and that there is no significant labeling on other compartments such as the nucleus and cytoplasm. In the control sections incubated with an immunoglobulin G fraction from nonimmunized rabbits, no specific labeling was seen on the granules or other organelles. These findings indicate that enzymes which previously have been identified in some organs as lysosomal and in others as peroxisomal can be found together in eosinophil granules.

scholarly journals Detection of the Neu5 Ac (alpha 2,3) Gal (beta 1,4) GlcNAc sequence with the leukoagglutinin from Maackia amurensis: light and electron microscopic demonstration of differential tissue expression of terminal sialic acid in alpha 2,3- and alpha 2,6-linkage.

1989 ◽  
Vol 37 (11) ◽  
pp. 1577-1588 ◽  
Author(s):  
T Sata ◽  
P M Lackie ◽  
D J Taatjes ◽  
W Peumans ◽  
J Roth
Keyword(s):  
Sialic Acid ◽  
High Specificity ◽  
Histochemical Staining ◽  
Gold Complex ◽  
Differential Distribution ◽  
Electron Microscopic ◽  
Gold Complexes ◽  
Double Labeling ◽  
Maackia Amurensis ◽  
Maackia Amurensis Lectin

The Maackia amurensis leukoagglutinin has been shown to react specifically with the Neu5Ac (alpha 2,3) Gal sequence of asparagine-linked complex type oligosaccharides. We report here the preparation of Maackia amurensis lectin-gold complexes and their application for light and electron microscopic detection of the Neu5 Ac (alpha 2,3) Gal sequence in various tissues. The use of the lectin directly gold labeled was superior to a two-step cytochemical affinity technique using a fetuin-gold complex. The Maackia amurensis lectin-gold staining was inhibited by pre-incubation of the lectin-gold complexes with 50 mM alpha 2,3 sialyllactose, whereas alpha 2,6 sialyllactose up to concentrations of 1 M had no effect, thus demonstrating the high specificity of the histochemical staining. In addition to N-glycanase-sensitive asparagine-linked oligosaccharides, beta-elimination-sensitive serine/threonine-linked oligosaccharides could be detected. Data are presented which show that cellular staining patterns obtained with Maackia amurensis lectin-gold complexes may differ from those with elderberry bark lectin-gold, which detects the Neu5 Ac (alpha 2,6) Gal/GalN Ac sequence. Electron microscopic double labeling for direct study of the differential distribution of the Neu5 Ac (alpha 2,3) Gal and Neu5 Ac (alpha 2,6) Gal sequences is reported. Therefore, the availability of two sialic acid binding lectins with different linkage specificity for histochemistry provides the first opportunity to study tissue and cell type expression of these terminal sequences of glycoproteins.

Localization of cellubrevin-related peptide, endobrevin, in the early endosome in pancreatic beta cells and its physiological function in exo-endocytosis of secretory granules

2001 ◽  
Vol 114 (1) ◽  
pp. 219-227 ◽  
Author(s):  
S. Nagamatsu ◽  
Y. Nakamichi ◽  
T. Watanabe ◽  
S. Matsushima ◽  
S. Yamaguchi ◽  
...  
Keyword(s):  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Cellular Localization ◽  
Secretory Granules ◽  
Physiological Role ◽  
Early Endosome ◽  
Binding Studies ◽  
Min6 Cells ◽  
Related Peptide ◽  
Early Endosomes

Cellubrevins are integral membrane proteins expressed in a wide variety of tissues and usually localized in recycling vesicles. Here, we investigated the cellular localization of a cellubrevin-related peptide, endobrevin, in pancreatic (beta) cells and its implication in the exo-endocytosis of insulin and (gamma)-amino butyric acid (GABA). Immunocytochemistry showed that endobrevin is associated with tubulo-vesicular structures, which are colocalized with early endosomes labeled by early endosome antigen (EEA)-1 in insulinoma MIN6 cells. To determine the cellular localization of endobrevin, we appended the green fluorescent protein (GFP) to endobrevin and the fusion protein was introduced into MIN6 cells. The subcellular localization of GFP-endobrevin was visualized by confocal laser microscopy. Colocalization study based on the expressed GFP-endobrevin and endocytosed Texas-Red(Tx-R) labeled transferrin receptor and immunocytochemistry with anti-EEA1 antibody revealed that endobrevin was preferentially localized in the early endosome. Then, we examined the functional role of endobrevin in the exocytosis of insulin and GABA from pancreatic (beta) cells. Endobrevin overexpression increased the amount of GABA released from MIN6 cells; in contrast, it decreased the glucose-stimulated insulin release from rat islets, MIN6 and INS1-D cells to approximately 50% of the control levels. Both in vitro and in vivo binding studies showed that endobrevin binds to syntaxin 1. Finally, using the fluorescent probe FM4-64, it was revealed that endobrevin overexpression accelerates vesicle recycling. We conclude that (1) endobrevin is localized in the early endosome in pancreatic (beta) cells and (2) endobrevin plays a physiological role in the exo-endocytosis of insulin and GABA from pancreatic (beta) cells, probably via an interaction between endocytic vesicles and the endosome.

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