Ultrastructural localization of cytoskeletal proteins in pancreatic secretory cells

1985 ◽  
Vol 63 (6) ◽  
pp. 680-690 ◽  
Author(s):  
Moïse Bendayan
Keyword(s):  
Secretory Granules ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Cytoskeletal Proteins ◽  
Secretory Cells ◽  
Secretory Proteins ◽  
Protein Maturation ◽  
Pancreatic Cells ◽  
Pancreatic Exocrine ◽  
Cell Web

Actin, myosin, and keratin immunoreactive sites have been localized with high resolution in pancreatic exocrine cells, by applying the protein A – gold technique on tissues processed at low temperature conditions. The labeling by gold particles was found at the level of the cell web and closely associated with the limiting membranes of the immature and mature secretory granules, as well as those of the "trans" cisternae of the Golgi apparatus. These results, together with those obtained in the study on the localization of secretory proteins in exocrine pancreatic cells, demonstrate that cytoskeletal proteins are present at sites where maturation and (or) concentration of the secretory proteins occur. Thus, besides the role that cytoskeletal proteins must play in the transport of the secretory granules from the Golgi to the plasma membrane, they may also be involved in the process of protein maturation and (or) concentration.

scholarly journals Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique.

1983 ◽  
Vol 31 (1) ◽  
pp. 101-109 ◽  
Author(s):  
M Bendayan ◽  
M Zollinger
Keyword(s):  
Secretory Granules ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Liver Mitochondria ◽  
Secretory Proteins ◽  
Additional Advantage ◽  
Sodium Metaperiodate ◽  
Thin Sections ◽  
Antigenic Sites ◽  
Structural Preservation

The protein A-gold immunocytochemical technique has been modified to allow labeling of cellular antigenic sites on osmium-fixed or postfixed tissues. Several strong oxidizing agents have been found able to restore protein antigenicity on osmicated tissue thin sections. According to the fine structural preservation and intensities of labeling, pretreatment with sodium metaperiodate gave optimal results. Pancreatic secretory proteins (and/or proproteins) as well as insulin (and/or proinsulin) were localized over perfectly preserved rough endoplasmic reticulum (rER), Golgi apparatus, and secretory granules of the corresponding pancreatic cells; carbamyl phosphate synthetase and catalase were revealed over liver mitochondria and peroxisomes, respectively. In addition to the higher resolution in the labeling obtained using osmium-fixed tissues, the present modification confers an additional advantage to the protein A-gold technique by allowing labeling on tissues processed for routine electron microscopy.

scholarly journals Localization of protein disulfide isomerase on plasma membranes of rat exocrine pancreatic cells.

1988 ◽  
Vol 36 (8) ◽  
pp. 1069-1074 ◽  
Author(s):  
S Akagi ◽  
A Yamamoto ◽  
T Yoshimori ◽  
R Masaki ◽  
R Ogawa ◽  
...  
Keyword(s):  
Protein Disulfide Isomerase ◽  
Plasma Membranes ◽  
Secretory Granules ◽  
Amorphous Material ◽  
Protein A ◽  
Disulfide Isomerase ◽  
Gold Particles ◽  
Pancreatic Cells ◽  
Acinar Lumen ◽  
Protein Disulfide

We investigated immunocytochemically the ultrastructural localization of protein disulfide isomerase (PDI) in rat pancreatic exocrine cells by use of the post-embedding protein A-gold technique. We found that not only the endoplasmic reticulum (ER) and nuclear envelope but also the trans-Golgi cisternae, secretory granules, and plasma membranes were heavily labeled with gold particles. Labeling density of the gold particles in the rough ER and plasma membranes of the exocrine pancreatic cells was twofold and twentyfold greater, respectively, than that of hepatocytes. In the acinar lumen, amorphous material presumably corresponding to the secreted zymogens was also labeled with gold particles. These results suggest that in rat exocrine pancreatic cells a significant amount of PDI is transported to the plasma membrane and secreted to the acinar lumen.

scholarly journals Immunohistochemical localization of pancreatic exocrine enzymes in normal and neoplastic pancreatic acinar epithelium of rat.

1981 ◽  
Vol 29 (2) ◽  
pp. 309-313 ◽  
Author(s):  
L J Hansen ◽  
M Mangkornkanok/Mark ◽  
J K Reddy
Keyword(s):  
Secretory Granules ◽  
Acinar Cells ◽  
Immunohistochemical Localization ◽  
Model Systems ◽  
Secretory Process ◽  
Secretory Proteins ◽  
Variable Extent ◽  
Immunofluorescent Technique ◽  
Pancreatic Exocrine ◽  
Indirect Immunofluorescent

The transplantable pancreatic acinar carcinomas of rat established recently provide useful model systems to examine the composition of secretory proteins as well as the secretory process in transformed pancreatic exocrine epithelium. The neoplastic acinar cells exhibit considerable variation in the extent of cytodifferentiation. In the present study the enzymatic profile of this heterogeneous tumor cell population has been investigated by the indirect immunofluorescent technique using antibodies against six pancreatic enzymes. By immunofluorescence, all neoplastic cells stained positively for the six enzymes tested: amylase, lipase, carboxypeptidase A, chymotrypsinogen, trypsinogen, and ribonuclease. Some variability in the intensity of immunofluorescence was noted, suggesting possible quantitative differences in the content of a given enzyme among tumor cells. These observations suggest that neoplastic acinar cells with or without secretory granules contain secretory proteins, but to a variable extent.

scholarly journals Ultrastructural localization of immunoreactive neurophysins using monoclonal antibodies and protein A-gold.

1985 ◽  
Vol 33 (10) ◽  
pp. 1015-1025 ◽  
Author(s):  
M Castel ◽  
J Morris ◽  
Y Ben-Barak ◽  
R Timberg ◽  
N Sivan ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Secretory Granules ◽  
Picric Acid ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Neurosecretory Granules ◽  
Sodium Metaperiodate ◽  
Background Staining ◽  
Axonal Varicosity

Using three different monoclonal antibodies against rat neurophysins (5), with protein A-gold as immunocytochemical marker (27), the murid hypothalamoneurohy-pophysial system was studied at the ultrastructural level. Postembedding staining was done on epoxy-embedded sections of supraoptic nuclei and posterior pituitaries. Specific immunolabeling of vasopressinergic and oxytocinergic neurosecretory granules was observed in tissues fixed with glutaraldehyde or glutaraldehyde mixtures (containing paraformaldehyde and picric acid), with or without osmium tetroxide postfixation and with or without sodium metaperiodate oxidation. Some autophagic vacuoles containing lysed neurosecretory granules were also neurophysin immunoreactive. Nonspecific background staining was extremely low. An attempt was made to appraise labeling intensities semiquantitatively by counting gold particles in relation to number of secretory granules per axonal varicosity. Immunoreactivity was measurably influenced by the mode of fixation, sodium metaperiodate oxidation, and titer and affinity of the antibody. The protein A-gold technique using monoclonal antibodies against neurophysins provides a superior means of ultrastructural analysis of the hypothalamoneurohypophysial system, both visually and morphometrically.

scholarly journals Ultrastructural localization of rat Clara cell 10 KD secretory protein by the immunogold technique using polyclonal and monoclonal antibodies.

1987 ◽  
Vol 35 (7) ◽  
pp. 789-794 ◽  
Author(s):  
C D Bedetti ◽  
J Singh ◽  
G Singh ◽  
S L Katyal ◽  
M L Wong-Chong
Keyword(s):  
Secretory Granules ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Secretory Protein ◽  
Polyclonal Antiserum ◽  
Clara Cell ◽  
Secretory Product ◽  
Clara Cells ◽  
Type Ii Pneumocytes ◽  
Pulmonary Cells

Using a monoclonal antibody and affinity-purified polyclonal antiserum against a 10 KD protein isolated from rat pulmonary lavage, we have localized the protein within Clara cells by a post-embedment protein A-gold technique. The gold particles were localized over the secretory granules of rat Clara cells. Ultrastructural immunolocalization was abolished when the primary antibodies were previously absorbed with purified 10 KD protein. Other pulmonary cells, including type II pneumocytes and ciliated cells, were negative with this technique. These results demonstrate the presence of the 10 KD protein in the secretory granules of the Clara cell and support the concept that this protein constitutes a specific and unique secretory product of Clara cells.

scholarly journals Ultrastructural localization of glucoside residues on tissue sections by applying the enzyme-gold approach.

1987 ◽  
Vol 35 (10) ◽  
pp. 1149-1155 ◽  
Author(s):  
M Bendayan ◽  
N Benhamou
Keyword(s):  
Secretory Granules ◽  
Ultrastructural Localization ◽  
Wall Material ◽  
Gold Complex ◽  
Plant Tissues ◽  
Fungal Cell ◽  
Tissue Sections ◽  
Pancreatic Cells ◽  
Beta Glucosidase ◽  
Labeling Pattern

The enzyme-gold approach was applied for ultrastructural localization of glucoside residues in animal and plant tissues. A beta-glucosidase-gold complex was prepared and used on thin tissue sections to reveal the corresponding substrate molecules by electron microscopy. Conditions for preparation of the complex, as well as for its application, were determined. Once applied on thin tissue sections, the glucosidase-gold complex yielded labeling over the rough endoplasmic reticulum, mainly on the ribosomal side of the membranes, and over the dense chromatin in the nucleus. Mitochondria, Golgi apparatus, and secretory granules in liver and pancreatic cells were free of gold particles. In plant cells, the labeling pattern was similar. In addition, the stroma regions of chloroplasts were densely labeled. In the extracellular space, labeling was found over the basal laminae of cells in animal tissues and over the fibrillar wall material bordering the intercellular space in plant tissues. Fungal cell cytoplasm was also labeled, as well as the membrane delineating mycoplasma-like organisms. Control conditions confirmed these labelings, demonstrating the possibility of revealing glucoside residues on tissue sections with high resolution and specificity.

scholarly journals Intermediate endocrine-acinar pancreatic cells in duct ligation conditions

1997 ◽  
Vol 273 (5) ◽  
pp. C1641-C1649 ◽  
Author(s):  
Eugenio Bertelli ◽  
Moïse Bendayan
Keyword(s):  
Laser Scanning ◽  
Secretory Granules ◽  
Laser Scanning Confocal Microscopy ◽  
Secretory Proteins ◽  
Zymogen Granules ◽  
Double Labeling ◽  
Scanning Confocal Microscopy ◽  
Pancreatic Cells ◽  
Duct Ligation

When tissues were subjected to 24 h of duct ligation, intermediate pancreatic cells simultaneously displaying endocrine and exocrine phenotypes appeared. Immunocytochemistry by laser scanning confocal microscopy revealed the appearance of a large number of these cells coexpressing insulin and amylase. These cells were located within the islets of Langerhans as well as in the acinar parenchyma. They were also detected in a culture system of isolated pancreatic cells. With the use of immunoelectron microscopy, two types of secretory granules were identified in these cells. One was insulin immunoreactive, whereas the other, resembling zymogen granules, contained amylase. Occasionally, some small granules displayed a double labeling for both secretory proteins. Numerous crinophagic bodies and autophagosomes containing insulin and/or amylase were also present. In situ hybridization, applied with the specific probes, confirmed the presence of both insulin and amylase mRNAs in these cells. Because duct ligation is known to induce insulin cell proliferation, the present results confirm that endocrine-acinar cells do appear in such condition and may represent intermediate steps in a transdifferentiating process.

scholarly journals The intracellular pathway of vitellogenin secretion in the frog hepatocyte as revealed by protein A-gold immunocytochemistry.

1984 ◽  
Vol 32 (7) ◽  
pp. 697-704 ◽  
Author(s):  
G H Herbener ◽  
M Bendayan ◽  
R C Feldhoff
Keyword(s):  
Golgi Apparatus ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Rana Catesbeiana ◽  
Secretory Granules ◽  
Plasma Concentrations ◽  
Protein A ◽  
Secretory Cells ◽  
Vitellogenin Synthesis ◽  
Cellular Compartments

The protein A-gold immunocytochemical technique was applied to the localization of vitellogenin in the hepatocyte of the bullfrog, Rana catesbeiana, eight days after treatment with estradiol-17 beta. Specific labeling was present in cellular compartments involved in protein secretion and was shown to progress in sequence through RER, Golgi apparatus, immature secretory granules, and mature secretory granules. Labeling intensities were quantitated and the values ranged from 34.6 to 172 gold particles/micron 2. In contrast, low background labeling was observed over mitochondria, nuclei, lipid droplets, and bile canaliculi. These observations support the hypothesis that vitellogenin synthesis and secretion in the frog hepatocyte lies exclusively along the RER-Golgi-granule secretory pathway. In addition to the cellular compartments involved in protein secretion, labeling was found over the majority of the lysosomes. The intensity of lysosomal labeling was intermediate between that of RER and Golgi apparatus. This labeling of lysosomes may be a consequence of the high blood plasma concentrations of vitellogenin that occur in the frog model, or to the well-known crinophagy phenomenon present in secretory cells.

scholarly journals Opposing roles for SNAP23 in secretion in exocrine and endocrine pancreatic cells

2016 ◽  
Vol 215 (1) ◽  
pp. 121-138 ◽  
Author(s):  
Masataka Kunii ◽  
Mica Ohara-Imaizumi ◽  
Noriko Takahashi ◽  
Masaki Kobayashi ◽  
Ryosuke Kawakami ◽  
...  
Keyword(s):  
Endocrine Cells ◽  
Plasma Membranes ◽  
Secretory Granules ◽  
Attachment Protein ◽  
Pancreatic Cells ◽  
Protein Receptor ◽  
Pancreatic Exocrine ◽  
Protein Attachment ◽  
Endocrine Pancreatic Cells

The membrane fusion of secretory granules with plasma membranes is crucial for the exocytosis of hormones and enzymes. Secretion disorders can cause various diseases such as diabetes or pancreatitis. Synaptosomal-associated protein 23 (SNAP23), a soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE) molecule, is essential for secretory granule fusion in several cell lines. However, the in vivo functions of SNAP23 in endocrine and exocrine tissues remain unclear. In this study, we show opposing roles for SNAP23 in secretion in pancreatic exocrine and endocrine cells. The loss of SNAP23 in the exocrine and endocrine pancreas resulted in decreased and increased fusion of granules to the plasma membrane after stimulation, respectively. Furthermore, we identified a low molecular weight compound, MF286, that binds specifically to SNAP23 and promotes insulin secretion in mice. Our results demonstrate opposing roles for SNAP23 in the secretion mechanisms of the endocrine and exocrine pancreas and reveal that the SNAP23-binding compound MF286 may be a promising drug for diabetes treatment.

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