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 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.

Experimental Dissection of the Cellular Machinery Involved in Receptor Mediated Endocytosis and Translocation

1981 ◽  
Vol 39 ◽  
pp. 528-531
Author(s):  
J.L. Salisbury
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Regulatory Protein ◽  
Fold Increase ◽  
Coated Pits ◽  
Surface Distribution ◽  
Receptor Mediated Endocytosis ◽  
Calcium Dependent ◽  
Receptor Complexes ◽  
Human Lymphoblastoid Cell

The cultured human lymphoblastoid cell line WiL2 is a model system of choice for studies on receptor mediated endocytosis (RME). These cells display antigen receptor immunoglobulin of the IgM class (rIgM) as integral plasma membrane proteins which are present in diffuse cell surface distribution in unstimulated cells. Initially, rIgM occurs over uncoated regions of the plasma membrane. Crosslinking rIgM with multivalent antibody (ligand) results in the entry of ferritin-labelled ligand-rIgM complexes into the RME pathway (Figure 1). Stimulation of RME by ligand challenge results in an approximately three-fold increase in cell surface area displaying clathrin coats on the cytoplasmic face of the membrane. The newly formed coated pits are located directly beneath ferritin-labelled ligand-receptor complexes and their appearance is sensitive to the calmodulin directed drug trifluoperazine dihydrochloride (TFP). Calmodulin is a calcium dependent regulatory protein which recognizes local transient fluxes of cytoplasmic Ca+2 and activates a wide variety of enzymes and other protein systems. In addition, antibodies raised against calf brain calmodulin were used in indirect immunofluorescence studies.

Autoradiographic analysis of formylpeptide chemoattractant binding, uptake and intracellular processing by neutrophils

1989 ◽  
Vol 94 (1) ◽  
pp. 155-168
Author(s):  
A.H. Janeczek ◽  
W.A. Marasco ◽  
P.J. van Alten ◽  
R.J. Walter
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Active Phase ◽  
Coated Pits ◽  
Surface Receptors ◽  
Dense Granules ◽  
Receptor Complexes ◽  
Intracellular Processing ◽  
Autoradiographic Analysis ◽  
Formylpeptide Receptor

Formylpeptide chemoattractant binds specifically to leukocyte cell surface receptors and during chemotactic activation, is internalized and processed by these cells. Using electron microscope autoradiographic techniques, we have examined the binding, uptake and disposition of fNle-Leu-Phe-Nle-[125I]Tyr-Lys by rabbit peritoneal neutrophils. Cells were incubated with ligand for 15 min at 4 degrees C, rinsed and then further incubated in buffer at 4 degrees C, 15 degrees C, 24 degrees C, or 37 degrees C for 0–40 min. For all cells incubated at 4 degrees C, grains were predominantly localized on the plasma membrane. Initially, this formylpeptide was seen in small clusters or microaggregates that were not associated with coated pits. Upon further incubation at 37 degrees C for 1 min, formylpeptide clustering on the plasma membrane increased and extensive internalization of formylpeptide was observed. Endocytosis of formylpeptide-receptor complexes clearly involved uncoated membrane pits and vesicles but did not appear to involve coated pits or coated vesicles. In the following 3 min, peptide proceeded in waves through compartments composed of small uncoated endocytic vesicles, then large vesicles, and then into dense granules. After 4 min at 37 degrees C, the most active phase of intracellular processing subsided. The percentage of grains in the cytoplasm, granule and small vesicle compartments very gradually increased during the remainder of the 40 min incubation. Formylpeptide neither bound at 4 degrees C nor accumulated at 37 degrees C on the cell surface in proximity to the underlying Golgi/centrosome region of the cell. At 24 degrees C, processing was slowed but formylpeptide-receptor complexes proceeded through a similar series of compartments. The t1/2 for formylpeptide uptake at 37 degrees C was 15–20 s, whereas at 24 degrees C the t 1/2 was approximately 10 min. No uptake was observed at 15 degrees C. The distinctive characteristics of formylpeptide binding and receptor-complex uptake seen here may be essential in initiating and maintaining continued chemotactic responsiveness.

scholarly journals A glycolipid-anchored prion protein is endocytosed via clathrin-coated pits.

1994 ◽  
Vol 125 (6) ◽  
pp. 1239-1250 ◽  
Author(s):  
S L Shyng ◽  
J E Heuser ◽  
D A Harris
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Prion Protein ◽  
Transmembrane Protein ◽  
Primary Cultures ◽  
Neuroblastoma Cells ◽  
Cellular Prion Protein ◽  
Coated Vesicles ◽  
Coated Pits ◽  
Hypertonic Medium

The cellular prion protein (PrPc) is a glycolipid-anchored, cell surface protein of unknown function, a posttranslationally modified isoform of which PrPSc is involved in the pathogenesis of Creutzfeldt-Jakob disease, scrapie, and other spongiform encephalopathies. We have shown previously that chPrP, a chicken homologue of mammalian PrPC, constitutively cycles between the cell surface and an endocytic compartment, with a transit time of approximately 60 min in cultured neuroblastoma cells. We now report that endocytosis of chPrP is mediated by clathrin-coated pits. Immunogold labeling of neuroblastoma cells demonstrates that the concentration of chPrP within 0.05 microns of coated pits is 3-5 times higher than over other areas of the plasma membrane. Moreover, gold particles can be seen within coated vesicles and deeply invaginated coated pits that are in the process of pinching off from the plasma membrane. ChPrP is also localized to coated pits in primary cultures of neurons and glia, and is found in coated vesicles purified from chicken brain. Finally, internalization of chPrP is reduced by 70% after neuroblastoma cells are incubated in hypertonic medium, a treatment that inhibits endocytosis by disrupting clathrin lattices. Caveolae, plasmalemmal invaginations in which several other glycolipid-anchored proteins are concentrated, are not seen in neuroblastoma cells analyzed by thin-section or deep-etch electron microscopy. Moreover, these cells do not express detectable levels of caveolin, a caveolar coat protein. Since chPrP lacks a cytoplasmic domain that could interact directly with the intracellular components of clathrin-coated pits, we propose that the polypeptide chain of chPrP associates with the extracellular domain of a transmembrane protein that contains a coated pit internalization signal.

scholarly journals Role of coated vesicles, microfilaments, and calmodulin in receptor-mediated endocytosis by cultured B lymphoblastoid cells.

1980 ◽  
Vol 87 (1) ◽  
pp. 132-141 ◽  
Author(s):  
J L Salisbury ◽  
J S Condeelis ◽  
P Satir
Keyword(s):  
Cell Surface ◽  
Coated Vesicles ◽  
Cell Surface Receptor ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Rabbit Muscle ◽  
Coated Pits ◽  
Receptor Complexes ◽  
Alternate Pathway ◽  
Receptor Clusters

Cell surface receptor IgM molecules of cultured human lymlphoblastoid cells (WiL2) patch and redistribute into a cap over the Golgi region of the cell after treatment with multivalent anti-IgM antibodies. During and after the redistribution, ligand-receptor clusters are endocytosed into coated pits and coated vesicles. Morphometric analysis of the distribution of ferritin-labeled ligand at EM resolution reveals the following sequence of events in the endocytosis of cell surface IgM: (a) binding of the multivalent ligand in a diffuse cell surface distribution, (b) clustering of the ligand-receptor complexes, (c) recruitment of clathrin coats to the cytoplasmic surface of the cell membrane opposite ligand-receptor clusters, (d) assembly and (e) internalization of coated vesicles, and (f) delivery of label into a large vesicular compartment, presumably partly lysosomal. Most of the labeled ligand enters this pathway. The recruitment of clathrin coats to the membrane opposite ligand-receptor clusters is sensitive to the calmodulin-directed drug Stelazine (trifluoperazine dihydrochloride). In addition, Stelazine inhibits an alternate pathway of endocytosis that does not involve coated vesicle formation. The actin-directed drug dihydrocytochalasin B has no effect on the recruitment of clathrin to the ligand-receptor clusters and the formation of coated pits and little effect on the alternate pathway, but this drug does interfere with subsequent coated vesicle formation and it inhibits capping. Cortical microfilaments that decorate with heavy meromyosin with constant polarity are observed in association with the coated regions of the plasma membrane and with coated vesicles. SDS-polyacrylamide gel electrophoresis analysis of a coated vesicle preparation isolated from WiL2 cells demonstrates that the major polypeptides in the fraction are a 175-kdalton component that comigrates with calf brain clathrin, a 42-kdalton component that comigrates with rabbit muscle actin and a 18.5-kdalton minor component that comigrates with calmodulin as well as 110-, 70-, 55-, 36-, 30-, and 17-kdalton components. These results clarify the pathways of endocytosis in this cell and suggest functional roles for calmodulin, especially in the formation of clathrin-coated pits, and for actin microfilaments in coated vesicle formation and in capping.

scholarly journals Isolation of concanavalin a caps during various stages of formation and their association with actin and myosin

1979 ◽  
Vol 80 (3) ◽  
pp. 751-758 ◽  
Author(s):  
J Condeelis
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Concanavalin A ◽  
Specific Binding ◽  
Lectin Binding ◽  
Metabolic Energy ◽  
Con A ◽  
Thin Filaments ◽  
Lectin Receptor ◽  
Receptor Complexes

Regions of plasma membrane of dictyostelium discoideum amoebae that contain concanavalin A (Con A)-receptor complexes are more resistant to disruption by Triton X-100. This resistance makes possible the isolation of Con A-associated membrane fragments in sufficient quantity and homogeneity to permit the direct biochemical and ultrastructural study of receptor-cytoskeletal interactions across the cell membrane. After specific binding of Con A to the cell surface, a large amount of the cell's actin and myosin copurifies with the plasma membrane fragments. Myosin is more loosely bound to the isolated membranes that actin and is efficiently removed by treating membranes with ATP and low ionic strength. If cells are not lysed immediately after lectin binding, all of the Con A that is bound to the cell surface is swept into a cap in a process requiring metabolic energy. When cells are lysed at different stages of cap formation, the amount of actin and myosin that copurifies with the isolated membranes remains the same. Thick and thin filaments that are attached to the protoplasmic surface of the isolated membranes underlie lectin-receptor complexes during all stages of cap formation. Once the cap is complete, the amount of actin and myosin that tightly bound to the plasma membrane is concentrated into the cap along with the Con A-receptor complexes. These results suggest that the ATP-dependent sliding of membrane-associated actin and myosin filaments is responsible for the accumulation of Con A-receptor complexes into a cap on the cell surface.

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 Receptor-mediated endocytosis of alpha 2-macroglobulin in cultured fibroblasts.

1980 ◽  
Vol 28 (8) ◽  
pp. 818-823 ◽  
Author(s):  
M C Willingham ◽  
F R Maxfield ◽  
I Pastan
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Coated Pits ◽  
Cultured Fibroblasts ◽  
Receptor Mediated Endocytosis ◽  
Cytoplasmic Vesicles

Alpha 2-macroglobulin is internalized into cultured fibroblasts by receptor-mediated endocytosis. This ligand binds initially to diffusely distributed receptors on the cell surface which cluster rapidly into bristle-coated pits. Within a few minutes at 37 degrees C, these complexes are internalized into uncoated cytoplasmic vesicles, called receptosomes, which move about in the cell by saltatory motion. These vesicles interact with the Golgi-endoplasmic reticulum-lysosome system in the cell to deliver the ligand to newly formed lysosomes within 30--60 min.

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