scholarly journals Endocytic vesicles and surface invaginations in cultured vascular endothelium: a morphometric comparison.

1981 ◽  
Vol 29 (12) ◽  
pp. 1437-1441 ◽  
Author(s):  
P F Davies ◽  
L Kuczera
Keyword(s):  
Cell Surface ◽  
Vascular Endothelium ◽  
Ruthenium Red ◽  
Coated Vesicles ◽  
Membrane Glycoproteins ◽  
Coated Pits ◽  
Ruthenium Red Staining ◽  
Almost All ◽  
Endocytic Vesicles

Ruthenium red staining of plasma membrane glycoproteins of confluent cultured arterial endothelial cells revealed that the limiting membrane of many apparently discrete cytoplasmic vesicles was continuous with the plasmalemma. Surface invaginations accessible to ruthenium red appeared as vesicles when sectioned out of the plane of attachment to the cell surface, Morphometric analysis of ruthenium red-positive (RR+) and ruthenium red-negative vesicles (RR-) indicated that 47.2% of the total apparent vesicle population was RR+ and that those infoldings accounted for 19.6 +/- 1.4% of the cell surface in transverse sections. Whereas 14.9% of the true vesicles (ruthenium red-negative) were coated vesicles, only 1.1% of RR+ "vesicles" were coated pits. These studies show that although many deep infoldings of the cell surface may be misinterpreted as vesicles, almost all are uncoated. The existence of discrete coated vesicles (independent of coated pits) in vascular endothelium in vitro is readily apparent.

Expression of auxilin or AP180 inhibits endocytosis by mislocalizing clathrin: evidence for formation of nascent pits containing AP1 or AP2 but not clathrin

2001 ◽  
Vol 114 (2) ◽  
pp. 353-365 ◽  
Author(s):  
X. Zhao ◽  
T. Greener ◽  
H. Al-Hasani ◽  
S.W. Cushman ◽  
E. Eisenberg ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Coated Vesicles ◽  
Coated Pits ◽  
Trans Golgi Network ◽  
J Domain ◽  
Almost All ◽  
Golgi Network ◽  
Assembly Proteins

Although uncoating of clathrin-coated vesicles is a key event in clathrin-mediated endocytosis it is unclear what prevents uncoating of clathrin-coated pits before they pinch off to become clathrin-coated vesicles. We have shown that the J-domain proteins auxilin and GAK are required for uncoating by Hsc70 in vitro. In the present study, we expressed auxilin in cultured cells to determine if this would block endocytosis by causing premature uncoating of clathrin-coated pits. We found that expression of auxilin indeed inhibited endocytosis. However, expression of auxilin with its J-domain mutated so that it no longer interacted with Hsc70 also inhibited endocytosis as did expression of the clathrin-assembly protein, AP180, or its clathrin-binding domain. Accompanying this inhibition, we observed a marked decrease in clathrin associated with the plasma membrane and the trans-Golgi network, which provided us with an opportunity to determine whether the absence of clathrin from clathrin-coated pits affected the distribution of the clathrin assembly proteins AP1 and AP2. Surprisingly we found almost no change in the association of AP2 and AP1 with the plasma membrane and the trans-Golgi network, respectively. This was particularly obvious when auxilin or GAK was expressed with functional J-domains since, in these cases, almost all of the clathrin was sequestered in granules that also contained Hsc70 and auxilin or GAK. We conclude that expression of clathrin-binding proteins inhibits clathrin-mediated endocytosis by sequestering clathrin so that it is no longer available to bind to nascent pits but that assembly proteins bind to these pits independently of clathrin.

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 The preendosomal compartment comprises distinct coated and noncoated endocytic vesicle populations.

1991 ◽  
Vol 113 (4) ◽  
pp. 731-741 ◽  
Author(s):  
S H Hansen ◽  
K Sandvig ◽  
B van Deurs
Keyword(s):  
Cell Surface ◽  
Coated Vesicles ◽  
Minor Role ◽  
Specific Marker ◽  
Con A ◽  
Early Endosomes ◽  
Gold Conjugate ◽  
Entire Cell ◽  
A Minor ◽  
Endocytic Vesicles

The transfer of molecules from the cell surface to the early endosomes is mediated by preendosomal vesicles. These vesicles, which have pinched off completely from the plasma membrane but not yet fused with endosomes, form the earliest compartment along the endocytic route. Using a new assay to distinguish between free and cell surface connected vesicle profiles, we have characterized the preedosomal compartment ultrastructurally. Our basic experimental setup was labeling of the entire cell surface at 4 degrees C with Con A-gold, warming of the cells to 37 degrees C to allow endocytosis, followed by replacing incubation medium with fixative, all within either 30 or 60 s. Then the fixed cells were incubated with anti-Con A-HRP to distinguish truly free (gold labeled) endocytic vesicles from surface-connected structures. Finally, analysis of thin (20-30 nm) serial sections and quantification of vesicle diameters were carried out. Based on this approach it is shown that the preendosomal compartment comprises both clathrin-coated and non-coated endocytic vesicles with approximately the same frequency but with distinct diameter distributions, the average noncoated vesicle being smaller (95 nm) than the average coated one (110 nm). In parallel experiments, using an anti-transferrin receptor gold-conjugate as a specific marker for clathrin-dependent endocytosis it is also shown that uncoating of coated vesicles plays only a minor role for the total frequency of noncoated vesicles. Furthermore, after perturbation of clathrin-dependent endocytosis by potassium depletion where uptake of transferrin is blocked, noncoated endocytic vesicles with Con A-gold, but not coated vesicles, exist already after 30 and 60 s. Finally, it is shown that the existence of small, free vesicles in the short-time experiments cannot be ascribed to recycling from the early endosomes.

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 alpha 2 Macroglobulin binding to the plasma membrane of cultured fibroblasts. Diffuse binding followed by clustering in coated regions.

1979 ◽  
Vol 82 (3) ◽  
pp. 614-625 ◽  
Author(s):  
M C Willingham ◽  
F R Maxfield ◽  
I H Pastan
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Coated Vesicles ◽  
The Other ◽  
Con A ◽  
Coated Pits ◽  
Cultured Fibroblasts ◽  
Receptor Complexes ◽  
Transmission Electron ◽  
Epidermal Growth

Using transmission electron microscopy, we have studied the interaction of alpha 2 macroglobulin (alpha 2 M) with the surface of cultured fibroblasts. When cells were incubated for 2 h at 4 degrees C with ferritin-conjugated alpha 2 M, approximately 90% of the alpha 2 M was diffusely distributed on the cell surface, and the other 10% was concentrated in "coated" pits. A pattern of diffuse labeling with some clustering in "coated" pits was also obtained when cells were incubated for 5 min at 4 degrees C with alpha 2 M, fixed with glutaraldehyde, and the alpha 2 M was localized with affinity-purified, peroxidase-labeled antibody to alpha 2 M. Experiments in which cells were fixed with 0.2% paraformaldehyde before incubation with alpha 2 M showed that the native distribution of alpha 2 M receptors was entirely diffuse without significant clustering in "coated" pits. This indicates that some redistribution of the alpha 2 M-receptor complexes into clusters occurred even at 4 degrees C. In experiments with concanavalin A(Con A), we found that some of the Con A clustered in coated regions of the membrane and was internalized in coated vesicles, but much of the Con A was directly internalized in uncoated vesicles or pinosomes. We conclude that unoccupied alpha 2 M receptors are diffusely distributed on the cell surface. When alpha 2 M-receptor complexes are formed, they rapidly cluster in coated regions or pits in the plasma membrane and subsequently are internalized in coated vesicles. Because insulin and epidermal growth factor are internalized in the same structures as alpha 2 M (Maxfield, F.R., J. Schlessinger, Y. Schechter, I. Pastan, and M.C. Willingham. 1978. Cell, 14: 805--810.), we suggest that all peptide hormones, as well as other proteins that enter the cell by receptor-mediated endocytosis, follow this same pathway.

scholarly journals Glycoproteins of coated pits, cell junctions, and the entire cell surface revealed by monoclonal antibodies and immunoelectron microscopy.

1983 ◽  
Vol 97 (2) ◽  
pp. 533-541 ◽  
Author(s):  
T L Murphy ◽  
G Decker ◽  
J T August
Keyword(s):  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Immunoelectron Microscopy ◽  
Total Cell ◽  
Cell Junctions ◽  
Membrane Glycoproteins ◽  
3T3 Cells ◽  
Coated Pits ◽  
Entire Cell

Topographical descriptions of three major plasma membrane glycoproteins of murine 3T3 cells were obtained by immunoelectron microscopy with monoclonal antibodies. A glycoprotein of Mr 80,000 was distributed throughout the total cell surface. A second of Mr 90,000 was concentrated in coated pits, and a third of Mr 100,000 was localized at cell junctions.

THE BEHAVIOUR OF RABBIT PLASMINOGEN AT THE LUMINAL SURFACE OF RABBIT AORTA IN VIVO BEFORE AND AFTER BALLOON-CATHETER INJURY

1987 ◽  
Author(s):  
Mark W C Hatton ◽  
Susan Moar ◽  
Mary Richardson
Keyword(s):  
White Male ◽  
Balloon Catheter ◽  
Rabbit Aorta ◽  
Ruthenium Red ◽  
Transmission Electron ◽  
Before And After ◽  
Ruthenium Red Staining ◽  
Balloon Catheter Injury

A previous study from this laboratory has identified the susceptibility of the de-endothelialised aorta, particularly the proteoglycan (PG) components of the extracellular matrix (ECM), to proteolytic damage if exposed to plasmin in vitro. To explore the possiblity that this occurs in vivo, a possible association between 125I-plasminogen (PLG) binding to the arterial wall, its activation to plasmin (PLN) and, subsequently, proteolytic damage to the intimal ECM has been studied. Intravenous injection of 125I-PLG in healthy N.Z. white male rabbits showed that PLG associated minimally (<0.01% of circulating PLG/cm2 /ml blood at 1 h) with the thoracic aorta endothelium, measured after Hautchen preparation from 1-cm vessel segments. Trans endothelial passage, measured as 125I-PLG associated with thg subendothelium (intima-media), progressed to 0.015%/cm2 /ml blood at 1 h. In contrast, the process of de-endothelialisation by balloon catheter led to a rapid uptake of bI-PLG by the denuded vessel surface. At saturation (approx. 10 min after injury), 0.7 - 0.8% of circulating PLG/cm2/ml blood was adsorbed by the entire de-endothelialised intima-media: Of the adsorbed PLG, 2-3% was associated with the platelet layer. Uptake was not inhibited by eACA (dose: 200 mg/kg) given i.v. before I-PLG. Adsorbed PLG was not released significantly from segments incubated in MEM containing 4% (w/v) RSA in vitro PLN activity was not detected. Furthermore, assessment of the ECM by transmission electron microscopy, after ruthenium red staining, showed that uptake of PLG by the de-endothelialised vessel in vivo was not associated with obvious damage to the PG components. Supported by the Heart and Stroke Foundation of Ontario.

scholarly journals Ultrastructural immunocytochemical localization of the transferrin receptor using a monoclonal antibody in human KB cells.

1985 ◽  
Vol 33 (1) ◽  
pp. 59-64 ◽  
Author(s):  
M C Willingham ◽  
I Pastan
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Transferrin Receptor ◽  
Immunocytochemical Localization ◽  
Kb Cells ◽  
Coated Pits ◽  
Human Carcinoma ◽  
Human Transferrin Receptor ◽  
Endocytic Vesicles ◽  
Extracellular Environment

Using a monoclonal antibody (HB21) against the human transferrin receptor, we have localized this receptor in cultured KB human carcinoma cells by fluorescence and ultrastructural immunocytochemistry. The receptor was found diffusely distributed on the cell surface, concentrated in clathrin-coated pits of the cell surface, in intracellular endocytic vesicles (receptosomes) derived from coated pits, in tubular elements of the trans-reticular Golgi system, and in microtubule-associated membranous elements thought to be part of the constitutive exocytic system. This distribution is the same as that previously shown for labeled transferrin in these same cells (Willingham MC, Hanover JA, Dickson BB, Pastan J: Proc Natl Acad Sci USA 81:175, 1984). No significant amounts of receptor were found in lysosomes. An aggregation of membranous elements containing this receptor was found in the pericentriolar region of cells during mitosis. Together with the previous data on the immunocytochemical localization of transferrin, these results suggest that the transferrin receptor may constitutively enter and exit KB cells by endocytosis and exocytosis, carrying bound transferrin into and out of the cell for the purpose of supplying iron from the extracellular environment for cell growth.

scholarly journals Coordinated regulation of AP2 uncoating from clathrin-coated vesicles by rab5 and hRME-6

2008 ◽  
Vol 183 (3) ◽  
pp. 499-511 ◽  
Author(s):  
Sophia Semerdjieva ◽  
Barry Shortt ◽  
Emma Maxwell ◽  
Sukhdeep Singh ◽  
Paul Fonarev ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Coated Vesicles ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Steady State Levels ◽  
Endocytic Vesicles ◽  
Interfering Rna

Here we investigate the role of rab5 and its cognate exchange factors rabex-5 and hRME-6 in the regulation of AP2 uncoating from endocytic clathrin-coated vesicles (CCVs). In vitro, we show that the rate of AP2 uncoating from CCVs is dependent on the level of functional rab5. In vivo, overexpression of dominant-negative rab5S34N, or small interfering RNA (siRNA)–mediated depletion of hRME-6, but not rabex-5, resulted in increased steady-state levels of AP2 associated with endocytic vesicles, which is consistent with reduced uncoating efficiency. hRME-6 guanine nucleotide exchange factor activity requires hRME-6 binding to α-adaptin ear, which displaces the ear-associated μ2 kinase AAK1. siRNA-mediated depletion of hRME-6 increases phospho-μ2 levels, and expression of a phosphomimetic μ2 mutant increases levels of endocytic vesicle-associated AP2. Depletion of hRME-6 or rab5S35N expression also increases the levels of phosphoinositide 4,5-bisphosphate (PtdIns(4,5)P2) associated with endocytic vesicles. These data are consistent with a model in which hRME-6 and rab5 regulate AP2 uncoating in vivo by coordinately regulating μ2 dephosphorylation and PtdIns(4,5)P2 levels in CCVs.

scholarly journals Intracellular transport of transferrin- and asialoorosomucoid-colloidal gold conjugates to lysosomes after receptor-mediated endocytosis.

1985 ◽  
Vol 33 (11) ◽  
pp. 1134-1144 ◽  
Author(s):  
M R Neutra ◽  
A Ciechanover ◽  
L S Owen ◽  
H F Lodish
Keyword(s):  
Acid Phosphatase ◽  
Cell Surface ◽  
Colloidal Gold ◽  
Intracellular Transport ◽  
Ruthenium Red ◽  
Endocytic Pathway ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Gold Probe ◽  
20 Nm

Proteins coupled to colloidal gold particles have been widely used to visualize the uptake and intracellular transport of specific ligands by receptor-mediated endocytosis. The intracellular route of lysosome-directed ligands such as asialoglycoproteins (ASGP) are apparently unaltered by conjugation to gold, but the pathway of transferrin, a ligand that normally recycles to the cell surface, was reported to be altered by conjugation to 15-20 nm gold. In this study, we sought to determine whether a smaller transferrin-gold probe would recycle, and whether it might enter the same endosomal and lysosomal compartments as does a larger, lysosome-directed ASGP gold probe by visualizing their simultaneous uptake in human hepatoma (HepG2) cells. In the same cells, endocytosis of fluid-phase protein was followed using the soluble tracer native ferritin; lysosomal compartments were identified by acid phosphatase cytochemistry; and cell surfaces were labeled with ruthenium red or cationized ferritin. During the first 10 min of uptake at 37 degrees C, specific receptor-bound ferrotransferrin (FeTf)-8 nm gold and asialoorosomucoid (ASOR)-20 nm gold were clustered together in coated pits and entered the same coated vesicles, smooth vesicles, and tubules in the peripheral cytoplasm. At later times, however, transferrin-gold did not return to the cell surface; unlike native transferrin, this gold probe accompanied ASOR-gold into multivesicular bodies (MVB). The MVBs that contained probes were at first devoid of acid phosphatase activity, but at 30 min, enzyme activity was detected in a few MVBs. Native ferritin was present, along with gold probes, in all compartments of the endocytic pathway. We conclude that the normal intracellular pathway of transferrin is altered by its association with a colloidal gold particle.

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