scholarly journals Formation of coated vesicles from coated pits in broken A431 cells.

1989 ◽  
Vol 108 (3) ◽  
pp. 843-853 ◽  
Author(s):  
E Smythe ◽  
M Pypaert ◽  
J Lucocq ◽  
G Warren
Keyword(s):  
Horseradish Peroxidase ◽  
Coated Vesicles ◽  
A431 Cells ◽  
Coated Pits ◽  
Intact Cells ◽  
Internalization Process

Biochemical and morphological techniques were used to demonstrate the early steps in the endocytosis of transferrin in broken A431 cells. After binding 125I-transferrin, the cells were broken by scraping and then warmed. 125I-transferrin became inaccessible to exogenous anti-transferrin antibody providing a measure of the internalization process. Parallel morphological experiments using transferrin coupled to horseradish peroxidase confirmed internalization in broken cells. The process was characterized and compared with endocytosis in intact cells and showed many similar features. The system was used to show that both the appearance of new coated pits and the scission of coated pits to form coated vesicles were dependent on the addition of cytosol and ATP whereas invagination of pits was dependent on neither.

scholarly journals Endocytosis occurs independently of annexin VI in human A431 cells

1994 ◽  
Vol 124 (3) ◽  
pp. 301-306 ◽  
Author(s):  
E Smythe ◽  
PD Smith ◽  
SM Jacob ◽  
J Theobald ◽  
SE Moss
Keyword(s):  
Transferrin Receptor ◽  
Squamous Carcinoma ◽  
Cell Types ◽  
Northern Blotting ◽  
Endocytic Pathway ◽  
A431 Cells ◽  
Coated Pits ◽  
Intact Cells ◽  
Calcium Dependent ◽  
Annexin Vi

Annexin VI is one of a family of calcium-dependent phospholipid-binding proteins. Although the function of this protein is not known, various physiological roles have been proposed, including a role in the budding of clathrin-coated pits (Lin et al., 1992. Cell. 70:283-291.). In this study we have investigated a possible endocytotic role for annexin VI in intact cells, using the human squamous carcinoma cell line A431, and report that these cells do not express endogenous annexin VI, as judged by Western and Northern blotting and PCR/Southern blotting. To examine whether endocytosis might in some way be either facilitated or inhibited by the presence of annexin VI, a series of A431 clones were isolated in which annexin VI expression was achieved by stable transfection. These cells expressed annexin VI at similar levels to other human cell types. Using assays for endocytosis and recycling of the transferrin receptor, we report that each of these cellular processes occurs with identical kinetics in both transfected and wild-type A431 cells. In addition, purified annexin VI failed to support the scission of coated pits in permeabilized A431 cells. We conclude that annexin VI is not an essential component of the endocytic pathway, and that in A431 cells, annexin VI fails to exert any influence on internalization and recycling of the transferrin receptor.

scholarly journals Receptor-mediated endocytosis in cultured fibroblasts: cryptic coated pits and the formation of receptosomes.

1981 ◽  
Vol 29 (9) ◽  
pp. 1003-1013 ◽  
Author(s):  
M C Willingham ◽  
A V Rutherford ◽  
M G Gallo ◽  
J Wehland ◽  
R B Dickson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Horseradish Peroxidase ◽  
Cell Surface ◽  
Concanavalin A ◽  
Ruthenium Red ◽  
Coated Vesicles ◽  
Surface Markers ◽  
Coated Pits ◽  
Cultured Fibroblasts ◽  
Receptor Mediated Endocytosis

Concentrative receptor-mediated endocytosis of many specific ligands by cultured fibroblasts occurs through the coated pit-receptosome pathway. The formation of receptosomes was studied using two impermeant electron-dense labels for the cell surface, ruthenium red and concanavalin A-horseradish peroxidase. These studies show that at 4 degrees C, virtually all coated structures near the plasma membrane are in communication with the cell surface, and are not isolated coated vesicles. On warming cells to 37 degrees C for only 1 minute, a major portion of these structures become cryptic, that is, not labeled by these surface markers. However, on cooling cells immediately back to 4 degrees C, virtually all of these structures are again in communication with the surface. Many images showed that membrane of these cryptic pits to be continuous with the cell surface when caught in the appropriate plane of section; often there was a very narrow entrance that excluded extracellular label. At 37 degrees C, receptosomes could be occasionally seen forming as an invagination of membrane adjacent to the coated region. Mechanisms by which receptosomes may form and other evidence demonstrating the failure of coated pits to pinch off to form isolated coated vesicles during endocytosis are discussed.

scholarly journals Assembly and packing of clathrin into coats.

1981 ◽  
Vol 91 (3) ◽  
pp. 790-797 ◽  
Author(s):  
R A Crowther ◽  
B M Pearse
Keyword(s):  
Electron Microscope ◽  
Mechanical Strength ◽  
Coated Vesicles ◽  
Coated Pits ◽  
Assembly Unit

We present a model for the packing of clathrin molecules into the characteristic hexagons and pentagons covering coated pits and vesicles. The assembly unit is a symmetrical trimer with three extended legs. Polymerization of these units occurs in seconds under suitable conditions, giving empty polyhedral cages resembling the structures around coated vesicles. Images of small, negatively stained fragments of cages, assembled directly on electron microscope grids, reveal details of the structure, which correlate well with the predicted features of the model. There is one clathrin trimer at each polyhedral vertex, and each leg of the trimer extends along two neighboring polyhedral edges. Quasi-equivalent packing in pentagons and hexagons in polyhedra of different sizes requires a variable joint at the vertex of the molecule and a hinge in each leg. The construction of clathrin coats is remarkable for the extended fibrous contacts that each molecule makes with many others. Such contacts may confer mechanical strength combined with flexibility needed when a vesicle is pinched off from the membrane.

scholarly journals AP-2/Eps15 Interaction Is Required for Receptor-mediated Endocytosis

1998 ◽  
Vol 140 (5) ◽  
pp. 1055-1062 ◽  
Author(s):  
Alexandre Benmerah ◽  
Christophe Lamaze ◽  
Bernadette Bègue ◽  
Sandra L. Schmid ◽  
Alice Dautry-Varsat ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fusion Protein ◽  
Binding Sites ◽  
Fluorescent Protein ◽  
Coated Vesicle ◽  
Mutant Form ◽  
A431 Cells ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Terminal Domain

We have previously shown that the protein Eps15 is constitutively associated with the plasma membrane adaptor complex, AP-2, suggesting its possible role in endocytosis. To explore the role of Eps15 and the function of AP-2/Eps15 association in endocytosis, the Eps15 binding domain for AP-2 was precisely delineated. The entire COOH-terminal domain of Eps15 or a mutant form lacking all the AP-2–binding sites was fused to the green fluorescent protein (GFP), and these constructs were transiently transfected in HeLa cells. Overexpression of the fusion protein containing the entire COOH-terminal domain of Eps15 strongly inhibited endocytosis of transferrin, whereas the fusion protein in which the AP-2–binding sites had been deleted had no effect. These results were confirmed in a cell-free assay that uses perforated A431 cells to follow the first steps of coated vesicle formation at the plasma membrane. Addition of Eps15-derived glutathione-S-transferase fusion proteins containing the AP-2–binding site in this assay inhibited not only constitutive endocytosis of transferrin but also ligand-induced endocytosis of epidermal growth factor. This inhibition could be ascribed to a competition between the fusion protein and endogenous Eps15 for AP-2 binding. Altogether, these results show that interaction of Eps15 with AP-2 is required for efficient receptor-mediated endocytosis and thus provide the first evidence that Eps15 is involved in the function of plasma membrane–coated pits.

scholarly journals Organelle relationships in cultured 3T3-L1 preadipocytes.

1980 ◽  
Vol 87 (1) ◽  
pp. 180-196 ◽  
Author(s):  
A B Novikoff ◽  
P M Novikoff ◽  
O M Rosen ◽  
C S Rubin
Keyword(s):  
Spatial Relationship ◽  
Morphological Differentiation ◽  
Coated Vesicles ◽  
Coated Pits ◽  
Exogenous Lipid ◽  
Acetate Esterase ◽  
Alpha Naphthyl Acetate Esterase ◽  
High Level ◽  
Relationship Of

In differentiating 3T3-L1 cells, lipid spheres, the endoplasmic reticulum (ER), microperoxisomes, and mitochondria form "constellations" that may reflect the interplay of lipid metabolizing enzymes in these organelles. ER cisternae are also situated very close to "rosettes,"plasmalemmal specializations found in mature adipocytes in vivo. As in hepatocytes and absorptive cells of the intestine, this spatial relationship of ER and plasmalemma suggests a role for rosettes in the uptake of exogenous lipid precursors. The morphological differentiation of 3T3-L1 preadipocytes includes the loss of "stress fibers" and the appearance of microfilament like structures that encase, in a complex manner, the cytosolic lipid spheres that appear during differentiation. Other features described for the first time in 3T3-L1 preadipocytes include: (a) the presence of an extensive acid phosphatase (AcPase) positive GERL from which coated vesicles apparently arise (these coated vesicles display AcPase activity and are much smaller and far more numerous than the coated vesicles that seem to arise from the plasmalemmal coated pits); (b) the abundance of AcPase-positive autophagic vacuoles; and (c) a high level of alpha-naphthyl-acetate-esterase activity which, by light microscopy cytochemistry, appears to be localized in the cytosol.

Intracellular regulation of 17β-hydroxysteroid dehydrogenase type 2 catalytic activity in A431 cells

1997 ◽  
Vol 153 (3) ◽  
pp. 453-464 ◽  
Author(s):  
C H Blomquist ◽  
B S Leung ◽  
C Beaudoin ◽  
D Poirier ◽  
Y Tremblay
Keyword(s):  
Reductase Activity ◽  
Maximum Velocity ◽  
Hydroxysteroid Dehydrogenase ◽  
A431 Cells ◽  
Intact Cells ◽  
Cell Monolayers ◽  
Intracellular Regulation

Abstract There is growing evidence that various isoforms of 17β-hydroxysteroid dehydrogenase (17-HSD) are regulated at the level of catalysis in intact cells. A number of investigators have proposed that the NAD(P)/NAD(P)H ratio may control the direction of reaction. In a previous study, we obtained evidence that A431 cells, derived from an epidermoid carcinoma of the vulva, are enriched in 17-HSD type 2, a membrane-bound isoform reactive with C18 and C19 17β-hydroxysteroids and 17-ketosteroids. The present investigation was undertaken to confirm the presence of 17-HSD type 2 in A431 cells and to assess intracellular regulation of 17-HSD at the level of catalysis by comparing the activity of homogenates and microsomes with that of cell monolayers. Northern blot analysis confirmed the presence of 17-HSD type 2 mRNA. Exposure of cells to epidermal growth factor resulted in an increase in type 2 mRNA and, for microsomes, increases in maximum velocity (Vmax) with no change in Michaelis constant (Km) for testosterone and androstenedione, resulting in equivalent increases in the Vmax/Km ratio consistent with the presence of a single enzyme. Initial velocity data and inhibition patterns were consistent with a highly ordered reaction sequence in vitro in which testosterone and androstenedione bind only to either an enzyme–NAD or an enzyme–NADH complex respectively. Microsomal dehydrogenase activity with testosterone was 2- to 3-fold higher than reductase activity with androstenedione. In contrast, although cell monolayers rapidly converted testosterone to androstenedione, reductase activity with androstenedione or dehydroepiandrosterone (DHEA) was barely detectable. Lactate but not glucose, pyruvate or isocitrate stimulated the conversion of androstenedione to testosterone by monolayers, suggesting that cytoplasmic NADH may be the cofactor for 17-HSD type 2 reductase activity with androstenedione. However, exposure to lactate did not result in a significant change in the NAD/NADH ratio of cell monolayers. It appears that within A431 cells 17-HSD type 2 is regulated at the level of catalysis to function almost exclusively as a dehydrogenase. These findings give further support to the concept that 17-HSD type 2 functions in vivo principally as a dehydrogenase and that its role as a reductase in testosterone formation by either the Δ4 or Δ5 pathway is limited. Journal of Endocrinology (1997) 153, 453–464

Clathrin-Coated Pits and Coated Vesicles: Functional and Structural Studies

Hepatology ◽  
2007 ◽  
Vol 3 (3) ◽  
pp. 437-454 ◽  
Author(s):  
Clifford J. Steer ◽  
Richard D. Klausner
Keyword(s):  
Coated Vesicles ◽  
Structural Studies ◽  
Coated Pits

scholarly journals Phosphorylation of the cation-independent mannose 6-phosphate receptor is closely associated with its exit from the trans-Golgi network.

1993 ◽  
Vol 120 (1) ◽  
pp. 67-75 ◽  
Author(s):  
S Méresse ◽  
B Hoflack
Keyword(s):  
Cell Surface ◽  
Specific Inhibitor ◽  
Cytoplasmic Domain ◽  
Single Step ◽  
Coated Vesicles ◽  
Coated Pits ◽  
Trans Golgi Network ◽  
Mannose 6 Phosphate ◽  
Golgi Network

We have previously shown that two serine residues present in two conserved regions of the bovine cation-independent mannose 6-phosphate receptor (CI-MPR) cytoplasmic domain are phosphorylated in vivo (residues 2421 and 2492 of the full length bovine CI-MPR precursor). In this study, we have used CHO cells to investigate the phosphorylation state of these two serines along the different steps of the CI-MPR exocytic and endocytic recycling pathways. Transport and phosphorylation of the CI-MPR in the biosynthetic pathway were examined using deoxymannojirimycin (dMM), a specific inhibitor of the cis-Golgi processing enzyme alpha-mannosidase I which leads to the accumulation of N-linked high mannose oligosaccharides on glycoproteins. Upon removal of dMM, normal processing to complex-type oligosaccharides (galactosylation and then sialylation) occurs on the newly synthesized glycoproteins, including the CI-MPR which could then be purified and analyzed on lectin affinity columns. Phosphorylation of the newly synthesized CI-MPR was concomitant with the sialylation of its oligosaccharides and appeared as a major albeit transient modification. Phosphorylation of the cell surface CI-MPR was examined during its endocytosis as well as its return to the Golgi using antibody tagging and exogalactosylation. The cell surface CI-MPR was not phosphorylated when it entered clathrin-coated pits or when it moved to the early and late endosomes. In contrast, the surface CI-MPR was phosphorylated when it had been resialylated upon its return to the trans-Golgi network. Subcellular fractionation experiments showed that the phosphorylated CI-MPR and the corresponding kinase were found in clathrin-coated vesicles. Collectively, these results indicate that phosphorylation of the two serines in the CI-MPR cytoplasmic domain is associated with a single step of transport of its recycling pathways and occurs when this receptor is in the trans-Golgi network and/or has left this compartment via clathrin-coated vesicles.

scholarly journals Conversion of epidermal growth factor (EGF) into a stimulatory ligand for A431-cell growth by herbimycin A by decreasing the level of expression of EGF receptor

1994 ◽  
Vol 301 (1) ◽  
pp. 57-62 ◽  
Author(s):  
Y Murakami ◽  
H Fukazawa ◽  
S Mizuno ◽  
Y Uehara
Keyword(s):  
Growth Factor ◽  
Cell Growth ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Affinity Binding ◽  
Receptor Kinase ◽  
A431 Cells ◽  
Intact Cells ◽  
Herbimycin A ◽  
Epidermal Growth

We examined effect of the tyrosine kinase inhibitor herbimycin A on A431 human epidermoid carcinoma cells which over-express epidermal growth factor (EGF) receptor. Herbimycin A inhibited the autophosphorylation of EGF-stimulated receptors in intact cells both time- and dose-dependently. The inhibition was found to be due to a decrease in the level of expression of the receptor amount, because herbimycin A equally decreased the receptor quantity and EGF-stimulated receptor kinase activity in intact cells, but did not exhibit a direct inhibitory effect on EGF receptor kinase activity in vitro. The decrease of the level of EGF receptor was also confirmed by 125I-EGF binding to herbimycin A-treated cells, and Scatchard analysis showed that the decrease in the receptor number occurred in the major population of the low-affinity binding ones, whereas the number with high-affinity binding was unaffected. Interestingly, although the proliferation of A431 cells was inhibited by EGF, herbimycin A converted EGF into a stimulatory ligand for cell growth, as determined by both cell number and DNA synthesis. These findings indicated that herbimycin A decreased the level of expression of EGF receptor by a mechanism other than inactivation of the receptor kinase and reversed the transformed phenotype of A431 cells to a normal one in the proliferative response to EGF.

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