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.

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 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.

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.

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.

scholarly journals Role of Receptor-Mediated Endocytosis in the Formation of Vaccinia Virus Extracellular Enveloped Particles

2005 ◽  
Vol 79 (7) ◽  
pp. 4080-4089 ◽  
Author(s):  
Matloob Husain ◽  
Bernard Moss
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Vaccinia Virus ◽  
Adaptor Protein ◽  
Viral Proteins ◽  
Envelope Proteins ◽  
Viral Envelope ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Viral Envelope Proteins

ABSTRACT Infectious intracellular mature vaccinia virus particles are wrapped by cisternae, which may arise from trans-Golgi or early endosomal membranes, and are transported along microtubules to the plasma membrane where exocytosis occurs. We used EH21, a dominant-negative form of Eps15 that is an essential component of clathrin-coated pits, to investigate the extent and importance of endocytosis of viral envelope proteins from the cell surface. Several recombinant vaccinia viruses that inducibly or constitutively express an enhanced green fluorescent protein (GFP)-EH21 fusion protein were constructed. Expression of GFP-EH21 blocked uptake of transferrin, a marker for clathrin-mediated endocytosis, as well as association of adaptor protein-2 with clathrin-coated pits. When GFP-EH21 was expressed, there were increased amounts of viral envelope proteins, including A33, A36, B5, and F13, in the plasma membrane, and their internalization was inhibited. Wrapping of virions appeared to be qualitatively unaffected as judged by electron microscopy, a finding consistent with a primary trans-Golgi origin of the cisternae. However, GFP-EH21 expression caused a 50% reduction in released enveloped virions, decreased formation of satellite plaques, and delayed virus spread, indicating an important role for receptor-mediated endocytosis. Due to dynamic interconnection between endocytic and exocytic pathways, viral proteins recovered from the plasma membrane could be used by trans-Golgi or endosomal cisternae to form new viral envelopes. Adherence of enveloped virions to unrecycled viral proteins on the cell surface may also contribute to decreased virus release in the presence of GFP-EH21. In addition to a salvage function, the retrieval of viral proteins from the cell surface may reduce immune recognition.

scholarly journals alpha 2-macroglobulin adsorbed to colloidal gold: a new probe in the study of receptor-mediated endocytosis.

1981 ◽  
Vol 89 (1) ◽  
pp. 29-34 ◽  
Author(s):  
R B Dickson ◽  
M C Willingham ◽  
I Pastan
Keyword(s):  
Electron Microscope ◽  
Cell Surface ◽  
Vesicular Stomatitis Virus ◽  
Golgi Complex ◽  
Colloidal Gold ◽  
Microscope Level ◽  
Coated Pits ◽  
Electron Microscope Level ◽  
Receptor Mediated Endocytosis ◽  
Vesicular Stomatitis

alpha 2-Macroglobulin (alpha 2 M) was adsorbed to colloidal gold and used as a new tool in the study of receptor-mediated endocytosis. alpha 2 M-gold is easy to prepare and is clearly visualized at the electron microscope level. When cells were incubated with alpha 2 M-gold at 0 degrees C, gold was visualized both diffusely over the cell surface and concentrated in coated pits. After cells to which alpha 2 M-gold had been bound at 0 degrees C were warmed, the gold was rapidly internalized into uncoated vesicles, previously termed receptosomes. After 30 min of incubation or longer, gold was found in small lysosomes and, later, in large lysosomes and very small vesicles in the region of the Golgi complex. This pattern of localization is similar to that previously described, using peroxidase-labeled anti-alpha 2 M antibodies. By incubating cells with both alpha 2 M-gold and vesicular stomatitis virus (VSV), we studied the internalization of these two markers simultaneously. VSV and alpha 2 M-gold rapidly clustered in the same coated pits and were internalized in the same receptosomes. Proteins and hormones adsorbed to gold may be useful in the study of receptor-mediated endocytosis.

scholarly journals Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation.

1989 ◽  
Vol 108 (2) ◽  
pp. 389-400 ◽  
Author(s):  
J E Heuser ◽  
R G Anderson
Keyword(s):  
Cell Surface ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Coated Pits ◽  
Normal Medium ◽  
Hypertonic Medium ◽  
Receptor Mediated Endocytosis ◽  
Cell Biol ◽  
Experimental Treatments ◽  
Small Clusters

Two seemingly unrelated experimental treatments inhibit receptor mediated endocytosis: (a) depletion of intracellular K+ (Larkin, J. M., M. S. Brown, J. L. Goldstein, and R. G. W. Anderson. 1983. Cell. 33:273-285); and (b) treatment with hypertonic media (Daukas, G., and S. H. Zigmond. 1985. J. Cell Biol. 101:1673-1679). Since the former inhibits the formation of clathrin-coated pits (Larkin, J. M., W. D. Donzell, and R. G. W. Anderson, 1986. J. Cell Biol. 103:2619-2627), we were interested in determining whether hypertonic treatment has the same effect, and if so, why. Fibroblasts (human or chicken) were incubated in normal saline made hypertonic with 0.45 M sucrose, then broken open by sonication and freeze-etched to generate replicas of their inner membrane surfaces. Whereas untreated cells display typical geodesic lattices of clathrin under each coated pit, hypertonic cells display in addition a number of empty clathrin "microcages". At first, these appear around the edges of normal coated pit lattices. With further time in hypertonic medium, however, normal lattices largely disappear and are replaced by accumulations of microcages. Concomitantly, low density lipoprotein (LDL) receptors lose their normal clustered distribution and become dispersed all over the cell surface, as seen by fluorescence microscopy and freeze-etch electron microscopy of LDL attached to the cell surface. Upon return to normal medium at 37 degrees C, these changes promptly reverse. Within 2 min, small clusters of LDL reappear on the surfaces of cells and normal clathrin lattices begin to reappear inside; the size and number of these receptor/clathrin complexes returns to normal over the next 10 min. Thus, in spite of their seeming unrelatedness, both K+ depletion and hypertonic treatment cause coated pits to disappear, and both induce abnormal clathrin polymerization into empty microcages. This suggests that in both cases, an abnormal formation of microcages inhibits endocytosis by rendering clathrin unavailable for assembly into normal coated pits.

What do nanometer molecular trajectories tell us about heterogeneous cell surface domaines?

1995 ◽  
Vol 53 ◽  
pp. 786-787
Author(s):  
Watt W. Webb
Keyword(s):  
Cell Surface ◽  
Lipid Membranes ◽  
Surface Proteins ◽  
Protein Diffusion ◽  
Coated Pits ◽  
Cell Surface Proteins ◽  
Membrane Heterogeneity ◽  
Fluorescence Photobleaching Recovery ◽  
Photobleaching Recovery ◽  
Plasma Membrane Heterogeneity

Plasma membrane heterogeneity is implicit in the existence of specialized cell surface organelles which are necessary for cellular function; coated pits, post and pre-synaptic terminals, microvillae, caveolae, tight junctions, focal contacts and endothelial polarization are examples. The persistence of these discrete molecular aggregates depends on localized restraint of the constituent molecules within specific domaines in the cell surface by strong intermolecular bonds and/or anchorage to extended cytoskeleton. The observed plasticity of many of organelles and the dynamical modulation of domaines induced by cellular signaling evidence evanescent intermolecular interactions even in conspicuous aggregates. There is also strong evidence that universal restraints on the mobility of cell surface proteins persist virtually everywhere in cell surfaces, not only in the discrete organelles. Diffusion of cell surface proteins is slowed by several orders of magnitude relative to corresponding protein diffusion coefficients in isolated lipid membranes as has been determined by various ensemble average methods of measurement such as fluorescence photobleaching recovery(FPR).

Ultrastructural study of cytoskeletal interactions with endosomes in macrophages by quickfreezing and deep-etching method

1990 ◽  
Vol 48 (3) ◽  
pp. 576-577
Author(s):  
Takeshi Baba ◽  
Nobuki Shiozawa ◽  
Masao Hotch ◽  
Shinichi Ohno
Keyword(s):  
Immune Complex ◽  
Ultrastructural Study ◽  
Fc Receptor ◽  
Coated Pits ◽  
Deep Etching ◽  
Receptor Mediated Endocytosis ◽  
Etching Method ◽  
Quick Freezing

Endosomes are vesicular or tubular organelles that play important roles in transports of receptors and receptor―bound ligands during receptor-mediated endocytosis. The mechanisms of endocytic transports from clathrin-coated pits to lysosomes have been studied by many investigators. However, few studies were reported about the interactions between endosomes and cytoskeletons. In this study, Fc-receptor-mediated endocytosis in macrophages are investigated by quick-freezing and deep-etching (QF-DE) method combined with gold-labeled immune complex and “replica scraping method”.

The immunogenicity of VP7, a rotavirus antigen resident in the endoplasmic reticulum, is enhanced by cell surface expression.

1990 ◽  
Vol 64 (10) ◽  
pp. 4776-4783 ◽  
Author(s):  
M E Andrew ◽  
D B Boyle ◽  
P L Whitfeld ◽  
L J Lockett ◽  
I D Anthony ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression
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