Intracellular Localization Of F.VIII R:Ag In Platelet And Endothelial Cells Using Peroxidase Fab Conjugates In Electron Microscopy

1981 ◽  
Author(s):  
C Jeanneau ◽  
Y Sultan
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Cellular Localization ◽  
Culture Medium ◽  
Intracellular Localization ◽  
Fab Fragments ◽  
Thrombin Activation ◽  
Storage Pool Disease ◽  
Papain Digestion ◽  
Specific Rabbit

In previous studies (Jeanneau et al Thromb. Haemost.1977, 38, 42) specific rabbit antibodies against human F.VIII R:Ag isolated by immuno absorption on insolubilized polymers from monospecific rabbit antisera against the F.VIII/ VW factor molecule and coupled to peroxidase showed the labelling of platelet and endothelial cell membranes but did not allow to visualize intra cellular localization of this antigen. Preparation of the peroxidase coupled Fab fragments of the same specific antibodies showed the intra cellular localization of F.VIII R:Ag in platelet and endothelial cells. Fab antibody fragments were prepared using papain digestion. Fab and Fc fragments were separated from the non digested antibody by filtration through Sephedex G 100. The Fab fragments were then separated from the Fc using CM 52 carboxymethyl cellulose.In human washed platelets, staining was observed on the plasma membrane, the canalicular system and some granules. After thrombin activation the release of granules containing F.VIII R:Ag better visualized in the canalicular system. In patients with storage pool disease only some platelets showed normal SCS labelling, however no stained granules were observed.Native human endothelial cells were obtained from umbilical cord veins, washed and resuspended in culture medium prior to incubation with peroxidase Fab conjugates. After fixation in glutaraldehyde and exposure to diaminobenzidine, the peroxidase staining was seen on the plasma membrane and in a large number of vacuoles. In some endothelial cells the Golgi apparatus appeared labelled demonstrating evidence of F.VIII R:Ag synthesis by endothelial cells.

Endothelial Cell Thiol Isomerases: Potential Role in Thrombus Formation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3709-3709 ◽  
Author(s):  
Reema Jasuja ◽  
Bruce Furie ◽  
Barbara C. Furie
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Endothelial Cell ◽  
Disulfide Bonds ◽  
Cellular Localization ◽  
Cell Activation ◽  
Potential Role ◽  
Ionophore A23187 ◽  
Platelet Surface

Abstract Thiol isomerases are multifunctional enzymes that contain a variable number of thioredoxin-like domains and catalyze the formation and isomerization of disulfide bonds. Members of the thiol isomerase family, including PDI, ERp5, ERp46, ERp57 and ERp72, are found in the endoplasmic reticulum (ER) where they play important roles during protein synthesis. Despite having an ER retention sequence some members of this family have been identified at cellular locations outside the ER with the potential to regulate the activity of proteins with labile disulfide bonds. Some of these proteins are involved in hemostasis, and include tissue factor and platelet surface receptors. Here, we describe the cellular localization and secretion of PDI and the first identification of two additional thiol isomerases in human umbilical vein endothelial cells (HUVEC). Our studies show a rapid secretion of PDI from HUVECs upon activation with thrombin or the calcium ionophore A23187 as detected by immunoblotting of proteins in the culture medium. We have estimated approximately 48000 ± 3600 molecules of PDI per cell. Approximately 8% of HUVEC PDI is secreted over 5 minutes upon cell activation. This secreted PDI is functionally active as measured by the reduction of disulfide bonds in the insulin transhydrogenase assay. Subcellular fractionation studies demonstrated PDI localized in different cellular compartments in activated and quiescent HUVECs. PDI was detected in the ER colocalized with sarcoplasmic and endoplasmic reticulum calcium ATPase (SERCA2) in both resting and activated cells. However PDI was detected in plasma membrane-containing fractions colocalized with alkaline phosphatase in thrombin-activated HUVECs while it was predominantly microsomal in buffer-treated control cells. PDI does not appear colocalized with the Weibel-Palade body marker von Willebrand factor (vWF) in subcellular fractions of activated or resting HUVECs nor is it detected in preparations of isolated Weibel-Palade bodies. Immunocytochemistry confirms localization of PDI in the ER but also shows punctate localization in the cytoplasm. Immunostaining for PDI in human aortic endothelial cells (HAEC) showed a similar staining pattern as HUVEC. Since PDI is secreted following cell activation, it is likely that the punctate PDI staining observed in the cytoplasm indicates PDI localization in secretory granules distinct from Weibel Palade bodies. We have also demonstrated the presence of two thiol isomerases, ERp57 and Erp72, in endothelial cells. Both ERp57 and ERp72 are constitutively secreted from HUVECs with no difference in secretory pattern pre and post activation. In contrast to PDI, ERp57 and ERp72 are detected in the microsomal as well as the plasma membrane fractions regardless of the activation state of the HUVECs. ERp57 was also detected in nuclear fractions consistent with previous immunocytochemical observations for ERp57. The regulated secretion of active PDI from endothelial cells and its rapid kinetics of release along with its subcellular localization suggest a potential role for endothelial cell PDI in modulation of protein structure and function in proteins regulated by endothelial cell injury, such as inflammation and thrombosis.

scholarly journals Intracellular localization of the P21rho proteins.

1992 ◽  
Vol 119 (3) ◽  
pp. 617-627 ◽  
Author(s):  
P Adamson ◽  
H F Paterson ◽  
A Hall
Keyword(s):  
Plasma Membrane ◽  
Cellular Localization ◽  
Biological Function ◽  
Intracellular Localization ◽  
Heterologous Proteins ◽  
Ras Proteins ◽  
Rho Proteins ◽  
Early Endosomes ◽  
Chimeric Molecules ◽  
Palmitoylation Site

The three mammalian ras proteins associated specifically with the plasma membrane and this is essential for their biological activity. Two signals encoded within the extreme COOH terminus of the proteins specify this cellular localization; a CAAX box in combination with either a polybasic domain (p21K-rasB) or a palmitoylation site (p21Ha-ras and p21N-ras). All members of the ras-like and rho-like subfamilies of the ras superfamily of small GTP-binding proteins also have CAAX boxes with potential second site sequences resembling either p21K-rasB or P21N-ras/Ha-ras. However it is not at all clear that they are each located at the plasma membrane, and in fact one of the ras-like proteins, rap1, has been localized to the Golgi (Beranger et al., 1991). None of the mammalian rho-like subfamily has yet been localized. Three forms (A, B, and C) of p21rho, the prototype of this family are known; the COOH termini of p21rhoA and p21rhoC resemble p21K-rasB with a polybasic domain, whereas p21rhoB resembles p21N-ras/Ha-ras with two cysteine residues as potential palmitoylation sites. Despite this similarity to the p21ras proteins, rho proteins have been purified from both particulate and cytosolic fractions of a variety of tissues. In order to localize definitively the three rho proteins we have used an epitope tagging approach coupled to microinjection of living cells. We show that a small fraction of all three proteins is localized to the plasma membrane but the majority of p21rhoA and p21rhoC is cytosolic whereas p21rhoB is associated with early endosomes and a pre-lysosomal compartment. Along with the results obtained with chimeric molecules using heterologous proteins attached to rho COOH termini, this suggests that the p21rho proteins cycle on and off the plasma membrane and this may have important implications for their biological function.

The Immuno-Electron Microscopic Localization of the Mo-Fe Protein Component of Nitrogenase in Cells of Azotobacter Vinelandii

1973 ◽  
Vol 31 ◽  
pp. 574-575
Author(s):  
J. T. Stasny ◽  
R. C. Burns ◽  
R. W. F. Hardy
Keyword(s):  
Cellular Localization ◽  
Direct Evidence ◽  
Azotobacter Vinelandii ◽  
Intracellular Localization ◽  
Protein Component ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Fe Protein ◽  
Intracytoplasmic Membranes

Structure-functlon studies of biological N2-fixation have correlated the presence of the enzyme nitrogenase with increased numbers of intracytoplasmic membranes in Azotobacter. However no direct evidence has been provided for the internal cellular localization of any nitrogenase. Recent advances concerned with the crystallizatiorTand the electron microscopic characterization of the Mo-Fe protein component of Azotobacter nitrogenase, prompted the use of this purified protein to obtain antibodies (Ab) to be conjugated to electron dense markers for the intracellular localization of the protein by electron microscopy. The present study describes the use of ferritin conjugated to goat antitMo-Fe protein immunoglobulin (IgG) and the observations following its topical application to thin sections of N2-grown Azotobacter.

Thrombin Augments Vascular Cell-dependent Migration of Human Mast Cells: Role of MGF

1997 ◽  
Vol 77 (03) ◽  
pp. 577-584 ◽  
Author(s):  
Mehrdad Baghestanian ◽  
Roland Hofbauer ◽  
Hans G Kress ◽  
Johann Wojta ◽  
Astrid Fabry ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Mast Cells ◽  
Umbilical Vein ◽  
Vascular Cells ◽  
Migratory Response ◽  
Thrombin Activation ◽  
Umbilical Vein Endothelial Cells ◽  
Primary Tissue ◽  
Local Expression

SummaryRecent data suggest that auricular thrombosis is associated with accumulation of mast cells (MC) in the upper endocardium (where usually no MC reside) and local expression of MGF (mast cell growth factor) (25). In this study, the role of vascular cells, thrombin-activation and MGF, in MC-migration was analyzed. For this purpose, cultured human auricular endocardial cells (HAUEC), umbilical vein endothelial cells (HUVEC) and uterine-(HUTMEC) and skin-derived (HSMEC) microvascular endothelial cells were exposed to thrombin or control medium, and the migration of primary tissue MC (lung, n = 6) and HMC-1 cells (human MC-line) against vascular cells (supernatants) measured. Supernatants (24 h) of unstimulated vascular cells (monolayers of endocardium or endothelium) as well as recombinant (rh) MGF induced a significant migratory response in HMC-1 (control: 3025 ± 344 cells [100 ± 11.4%] vs. MGF, 100 ng/ml: 8806 ± 1019 [291 ± 34%] vs. HAUEC: 9703 ± 1506 [320.8 ± 49.8%] vs. HUTMEC: 8950 ± 1857 [295.9 ± 61.4%] vs. HSMEC: 9965 ± 2018 [329.4 ± 66.7%] vs. HUVEC: 9487 ± 1402 [313.6 ± 46.4%], p <0.05) as well as in primary lung MC. Thrombin-activation (5 U/ml, 12 h) of vascular cells led to an augmentation of the directed migration of MC as well as to a hirudin-sensitive increase in MGF synthesis and release. Moreover, a blocking anti-MGF antibody was found to inhibit MC-migration induced by unstimulated or thrombin-activated vascular cells. Together, these data show that endocardial and other vascular cells can induce migration of human MC. This MC-chemotactic signal of the vasculature is associated with expression and release of MGF, augmentable by thrombin, and may play a role in the pathophysiology of (auricular) thrombosis.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

Mechanism of cystine reaccumulation by cystinotic fibroblasts in vitro

1990 ◽  
Vol 10 (2) ◽  
pp. 225-229 ◽  
Author(s):  
Susan Forster ◽  
Lynne Scarlett ◽  
John B. Lloyd
Keyword(s):  
Plasma Membrane ◽  
Culture Medium ◽  
Human Fibroblasts ◽  
Competitive Inhibitor ◽  
Free Medium ◽  
Lysosome Membrane ◽  
Putative Mechanism

It is well established that when cystine-depleted cystinotic cells are cultured in cystine-containing medium, they reaccumulate cystine within their lysosomes more rapidly than when cultured in cystine-free medium. This has been a puzzling result, since the lysosome membrane of cystinotic cells is impermeable to cystine. To probe the mechanism of cystine reaccumulation, we have measured reaccumulation in the presence of colchicine, an inhibitor of pinocytosis, or of glutamate, a competitive inhibitor of cystine transport into human fibroblasts. Colchicine had no effect, thus eliminating pinocytosis as a putative mechanism for cystine translocation from the culture medium to the lysosomes. Glutamate, however, strongly inhibited cystine reaccumulation. It is concluded that the true mechanism is as follows. 1. Exogenous cystine crosses the plasma membrane on the cystine-glutamate porter. 2. Cystine is reduced in the cytoplasm by GSH. 3. The cysteine that is generated enters the lysosome, where it becomes cystine by participating in the reduction of cystine residues during intralysosomal proteolysis, or by autoxidation.

scholarly journals Proprotein Convertase 5/6 Is Critical for Embryo Implantation in Women: Regulating Receptivity by Cleaving EBP50, Modulating Ezrin Binding, and Membrane-Cytoskeletal Interactions

Endocrinology ◽  
2011 ◽  
Vol 152 (12) ◽  
pp. 5041-5052 ◽  
Author(s):  
Sophea Heng ◽  
Ana Cervero ◽  
Carlos Simon ◽  
Andrew N. Stephens ◽  
Ying Li ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cellular Localization ◽  
Structural Changes ◽  
Current Knowledge ◽  
Embryo Implantation ◽  
Critical Role ◽  
Human Endometrium ◽  
Scaffolding Protein ◽  
Proprotein Convertase ◽  
Proteomic Approach

Establishment of endometrial receptivity is vital for successful embryo implantation; its failure causes infertility. Epithelial receptivity acquisition involves dramatic structural changes in the plasma membrane and cytoskeleton. Proprotein convertase 5/6 (PC6), a serine protease of the proprotein convertase (PC) family, is up-regulated in the human endometrium specifically at the time of epithelial receptivity and stromal cell decidualization. PC6 is the only PC member tightly regulated in this manner. The current study addressed the importance and mechanisms of PC6 action in regulating receptivity in women. PC6 was dysregulated in the endometrial epithelium during the window of implantation in infertile women of three demographically different cohorts. Its critical role in receptivity was evidenced by a significant reduction in mouse blastocyst attachment of endometrial epithelial cells after PC6 knockdown by small interfering RNA. Using a proteomic approach, we discovered that PC6 cleaved the key scaffolding protein, ezrin-radixin-moesin binding phosphoprotein 50 (EBP50), thereby profoundly affecting its interaction with binding protein ezrin (a key protein bridging actin filaments and plasma membrane), EBP50/ezrin cellular localization, and cytoskeleton-membrane connections. We further validated this novel PC6 regulation of receptivity in human endometrium in vivo in fertile vs. infertile patients. These results strongly indicate that PC6 plays a key role in regulating fundamental cellular remodeling processes, such as plasma membrane transformation and membrane-cytoskeletal interface reorganization. PC6 cleavage of a crucial scaffolding protein EBP50, thereby profoundly regulating membrane-cytoskeletal reorganization, greatly extends the current knowledge of PC biology and provides substantial new mechanistic insight into the fields of reproduction, basic cellular biology, and PC biochemistry.

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