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.

scholarly journals Ultrastructural visualization of selective peanut agglutinin binding sites in rat osteoclasts.

1988 ◽  
Vol 36 (1) ◽  
pp. 95-101 ◽  
Author(s):  
M Takagi ◽  
H Yagasaki ◽  
T Baba ◽  
H Baba
Keyword(s):  
Plasma Membrane ◽  
Cell Membrane ◽  
Cell Surface ◽  
Binding Sites ◽  
Peanut Agglutinin ◽  
Coated Pits ◽  
Cytoplasmic Surface ◽  
Coated Membranes ◽  
Entire Cell ◽  
Ruffled Border

We investigated the distribution of concanavalin A (ConA)-reactive alpha-D-mannosyl and alpha-D-glucosyl groups and peanut agglutinin (PNA)-reactive beta-D-galactose-(1----3)-N-acetyl-D-galactosamine residues on the surface of osteoclasts with pre-embedment ultrastructural lectin cytochemistry after aldehyde fixation of the metaphyses of the rat tibiae. By routine morphology, the plasma membrane of the ruffled border of the osteoclast was distinguished from the rest of the cell membrane, with the exception of the membrane of coated pits, by its characteristic thick coat at its cytoplasmic surface. Cytochemistry, using ConA in combination with horseradish peroxidase (ConA-HRP) and PNA conjugated to HRP, showed that binding of ConA was distributed over the entire cell surface of osteoclasts. In contrast, intense binding of PNA was limited to the membranes of the ruffled border and coated pits, whereas the remainder of the cell membrane stained weakly or not at all. These results demonstrate that preferential PNA binding sites of the cell surface correspond to coated membranes associated with osteoclastic endocytosis.

scholarly journals Surface redistribution of 125I-insulin in cultured human lymphocytes.

1981 ◽  
Vol 91 (1) ◽  
pp. 17-25 ◽  
Author(s):  
J L Carpentier ◽  
E Van Obberghen ◽  
P Gorden ◽  
L Orci
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Human Lymphocytes ◽  
Freeze Fracture ◽  
Total Cell ◽  
Coated Pits ◽  
Villous Surface ◽  
Specific Receptors ◽  
Related Proteins ◽  
Preferential Association

The cultured human lymphocyte (IM-9) binds 125I-insulin by a receptor-mediated process; the receptor, in turn, is regulated by the ligand. In the present study we have examined quantitatively the morphologic events involved in 125I-insulin interaction with the surface of the lymphocyte. At 2 min of incubation of 15 degrees or 37 degrees C, the ligand localizes preferentially at the villous surface of the cell, whereas with longer periods of incubation, the ligand distributes indistinguishably between the villous and nonvillous surface. When rebinding is blocked, 125I-insulin localizes preferentially at the nonvillous surface of the cell. When the total cell surface is considered, there is little preferential association with coated pits; when only the nonvillous surface is considered, a preferential association with coated pits is found and is quantitatively increased in the absence of rebinding of the ligand. This cell has an abundant villous surface (approximately 55% of the total surface); and, as seen on freeze-fracture replicas, the plasma membrane of the villous surface contains a 60% greater density of intramembrane particles than the nonvillous surface. The data suggest an ordered pattern of insulin interaction with the cell surface (i.e., binding to villi followed by redistribution to the nonvillous portion of the cell containing coated pits). These events probably reflect the mechanism by which the cell segregates specific receptors and related proteins in the plane of the membrane so that they can be selectively removed.

scholarly journals Lattices, rafts, and scaffolds: domain regulation of receptor signaling at the plasma membrane

2009 ◽  
Vol 185 (3) ◽  
pp. 381-385 ◽  
Author(s):  
Patrick Lajoie ◽  
Jacky G. Goetz ◽  
James W. Dennis ◽  
Ivan R. Nabi
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Lipid Raft ◽  
Scaffold Proteins ◽  
Cell Junctions ◽  
Cross Link ◽  
Coated Pits ◽  
Receptor Dynamics ◽  
Raft Domains ◽  
Molecular Lattices

The plasma membrane is organized into various subdomains of clustered macromolecules. Such domains include adhesive structures (cellular synapses, substrate adhesions, and cell–cell junctions) and membrane invaginations (clathrin-coated pits and caveolae), as well as less well-defined domains such as lipid rafts and lectin-glycoprotein lattices. Domains are organized by specialized scaffold proteins including the intramembranous caveolins, which stabilize lipid raft domains, and the galectins, a family of animal lectins that cross-link glycoproteins forming molecular lattices. We review evidence that these heterogeneous microdomains interact to regulate substratum adhesion and cytokine receptor dynamics at the cell surface.

scholarly journals p180, a novel recycling transmembrane glycoprotein with restricted cell type expression.

1990 ◽  
Vol 10 (6) ◽  
pp. 2606-2618 ◽  
Author(s):  
C M Isacke ◽  
P van der Geer ◽  
T Hunter ◽  
I S Trowbridge
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Biochemical Characterization ◽  
Surface Proteins ◽  
Sequence Information ◽  
Osteosarcoma Cell ◽  
Binding Studies ◽  
Coated Pits ◽  
Transmembrane Glycoprotein ◽  
Morphological Studies

A 180-kilodalton (kDa) protein (p180) was identified among the antigens for a panel of monoclonal antibodies raised against human fibroblast cell surface proteins. Binding studies with 125I-Fab' fragments of an anti-p180 monoclonal antibody demonstrated that 10 to 30% of p180 was located on the plasma membrane and that the remaining 70 to 90% was on intracellular membranes. p180 was rapidly internalized from the cell surface at 37 degrees C, and kinetic analyses indicated that this was a constitutive process followed by the recycling of p180 back to the plasma membrane. Morphological studies demonstrated that on the cell surface p180 was concentrated in coated pits, whereas inside the cell it was found in endosomes as suggested by its colocalization with the transferrin receptor. Immunoblot analysis with a polyclonal antiserum raised against purified human protein showed that p180 has a restricted distribution with expression at high levels in fibroblast cultures and in tissues containing cells of mesodermal origin. A biochemical characterization of p180 showed it to be a transmembrane glycoprotein with an extracellular domain, which consists of approximately 30 kDa of complex oligosaccharides attached to at least 45 kDa of the protein core. The cytoplasmic domain of p180 was found to contain a serine residue(s) that was phosphorylated both in vivo and in vitro by activated protein kinase C. p180 was purified by subjecting solubilized membrane proteins from a human osteosarcoma cell line to immunoaffinity chromatography and gel filtration. The N-terminal sequence information obtained from the purified protein showed no homology to other known proteins. It was concluded that p180 may be a novel recycling receptor which is highly restricted in its expression to fibroblastlike cells.

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 The paramyxovirus simian virus 5 hemagglutinin-neuraminidase glycoprotein, but not the fusion glycoprotein, is internalized via coated pits and enters the endocytic pathway.

1996 ◽  
Vol 7 (1) ◽  
pp. 155-172 ◽  
Author(s):  
G P Leser ◽  
K J Ector ◽  
R A Lamb
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Simian Virus ◽  
Endocytic Pathway ◽  
Coated Pits ◽  
Immunogold Staining ◽  
Simian Virus 5 ◽  
Fusion Glycoprotein ◽  
Infected Cells ◽  
Endosomal System

The hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of the paramyxovirus simian virus 5 (SV5) are expressed on the surface of virus-infected cells. Although the F protein was found to be expressed stably, the HN protein was internalized from the plasma membrane. HN protein lacks known internalization signals in its cytoplasmic domain that are common to many integral membrane proteins that are internalized via clathrin-coated pits. Thus, the cellular pathway of HN protein internalization was examined. Biochemical analysis indicated that HN was lost from the cell surface with a t1/2 of approximately 45-50 min and turned over with a t1/2 of approximately 2 h. Immunofluorescent analysis showed internalized SV5 HN in vesicle-like structures in a juxtanuclear pattern coincident with the localization of ovalbumin. In contrast the SV5 F glycoprotein and the HN glycoprotein of the highly related parainfluenza virus 3 (hPIV-3) were found only on the cell surface. Immunogold staining of HN on the surface of SV5-infected CV-1 cells and examination using electron microscopy, showed heavy surface labeling that gradually decreased with time. Concomitantly, gold particles were detected in the endosomal system and with increasing time, gold-labeled structures having the morphology of lysosomes were observed. On the plasma membrane approximately 5% of the gold-labeled HN was found in coated pits. The inhibition of the pinching-off of coated pits from the plasma membrane by cytosol acidification significantly reduced HN internalization. Internalized HN was co-localized with gold-conjugated transferrin, a marker for the early endosomal compartments, and with gold-conjugated bovine serum albumin, a marker for late endosomal compartments. Taken together, these data strongly suggest that the HN glycoprotein is internalized via clathrin-coated pits and delivered to the endocytic 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.

Macromolecular Glucosamine-Containing Component of the Surface of Cultivated Mouse Cells

1970 ◽  
Vol 7 (2) ◽  
pp. 337-355
Author(s):  
K. ONODERA ◽  
ROSE SHEININ
Keyword(s):  
Plasma Membrane ◽  
Cell Division ◽  
Cell Surface ◽  
Plating Efficiency ◽  
Transformed Cells ◽  
3T3 Cells ◽  
Vital Stain ◽  
Surface Component ◽  
Mouse Cells ◽  
Macromolecular Component

It has been demonstrated that a glucosamine-containing macromolecular component of the cell surface of 3T3 mouse cells, and SV40-transformed cells, is released from cells by treatment with trypsin under conditions in which the plasma membrane remains functionally intact. This was shown by the fact that the treated cells could be cloned with high plating efficiency and remained impermeable to the vital stain, erythrocin. A method for specifically marking this surface component has been devised based on the finding that in 3T3 cells growing synchronously after subculture by trypsin maximum incorporation of glucosamine into this material occurs 12-13 h thereafter. Of the total radioactive glucosamine incorporated into macro-molecular cell constituents, over 80% was recovered in surface component. Studies on the biosynthesis of surface component revealed that this was periodic during a cycle of cell duplication, with an increased rate of formation immediately after cell division. It was found that the surface component of 3T3 cells differed from that of SV40-transformed cells.

scholarly journals Rings of membrane sterols surround the openings of vesicles and fenestrae, in capillary endothelium.

1983 ◽  
Vol 97 (5) ◽  
pp. 1592-1600 ◽  
Author(s):  
N Simionescu ◽  
F Lupu ◽  
M Simionescu
Keyword(s):  
Plasma Membrane ◽  
Cell Membrane ◽  
Cell Surface ◽  
Freeze Fracture ◽  
Short Exposure ◽  
Capillary Endothelium ◽  
Anionic Sites ◽  
Microvascular Endothelium ◽  
Coated Pits ◽  
Plasmalemmal Vesicles

We investigated the distribution of sterols in the cell membrane of microvascular endothelium (mouse pancreas, diaphragm, brain, heart, lung, kidney, thyroid, adrenal, and liver) with the polyene antibiotic filipin, which reportedly has binding specificity for free 3-beta-hydroxysterols. In some experiments, concomitantly, cell-surface anionic sites were detected with cationized ferritin. Vessels were perfused in situ with PBS, followed by light fixation and filipin administration for 10 to 60 min. Tissues were further processed for thin-section and freeze-fracture electron microscopy. Short exposure (10 min) to filipin-glutaraldehyde solution resulted in the initial appearance, on many areas, of rings of characteristic filipin-sterol complexes within the rim surrounding stomata of most plasmalemmal vesicles, transendothelial channels, and fenestrae. Such rings were absent from the rims of the large openings of the sinusoid endothelium (liver, adrenal), coated pits and phagocytic vacuoles. After longer exposure (30-60 min), filipin-sterol complexes labeled randomly the rest of plasma membrane (except for coated pits, and partially the interstrand areas of junctions), and also marked most plasmalemmal vesicles. These peristomal rings of sterols were displayed mostly on the P face, and, at their full development, consisted of 6-8 units around a vesicle stoma, and 10-12 units around a fenestra. At their level, the intramembranous particles and the cell surface anionic sites were virtually excluded. Peristomal rings of sterols were also detected on the plasma membrane of pericytes and smooth muscle cells of the microvascular wall, which otherwise were poorly labeled with filipin-sterol complexes as compared to endothelial plasmalemma. It is presumed that the peristomal rings of cholesterol may represent important contributors to the local transient stabilization of plasma membrane and to the phase separation between cell membrane and vesicle membrane at a certain stage of their fusion/fission process.

scholarly journals An internalization signal in the simian immunodeficiency virus transmembrane protein cytoplasmic domain modulates expression of envelope glycoproteins on the cell surface.

1996 ◽  
Vol 132 (5) ◽  
pp. 795-811 ◽  
Author(s):  
M M Sauter ◽  
A Pelchen-Matthews ◽  
R Bron ◽  
M Marsh ◽  
C C LaBranche ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Simian Immunodeficiency Virus ◽  
Cytoplasmic Domain ◽  
Viral Envelope ◽  
Envelope Glycoproteins ◽  
Coated Pits ◽  
Immunodeficiency Virus ◽  
Infected Cells

A Tyr to Cys mutation at amino acid position 723 in the cytoplasmic domain of the simian immunodeficiency virus (SIV) transmembrane (TM) molecule has been shown to increase expression of envelope glycoproteins on the surface of infected cells. Here we show that Tyr-723 contributes to a sorting signal that directs the rapid endocytosis of viral glycoproteins from the plasma membrane via coated pits. On cells infected by SIVs with a Tyr at position 723, envelope glycoproteins were transiently expressed on the cell surface and then rapidly endocytosed. Similar findings were noted for envelope molecules expressed in the absence of other viral proteins. Immunoelectron microscopy demonstrated that these molecules were localized in patches on the cell surface and were frequently associated with coated pits. In contrast, envelope glycoproteins containing a Y723C mutation were diffusely distributed over the entire plasma membrane. To determine if an internalization signal was present in the SIV TM, chimeric molecules were constructed that contained the CD4 external and membrane spanning domains and a SIV TM cytoplasmic tail with a Tyr or other amino acids at SIV position 723. In Hela cells stably expressing these molecules, chimeras with a Tyr-723 were rapidly endocytosed, while chimeras containing other amino acids at position 723, including a Phe, were internalized at rates only slightly faster than a CD4 molecule that lacked a cytoplasmic domain. In addition, the biological effects of the internalization signal were evaluated in infectious viruses. A mutation that disrupted the signal and as a result, increased the level of viral envelope glycoprotein on infected cells, was associated with accelerated infection kinetics and increased cell fusion during viral replication. These results demonstrate that a Tyr-dependent motif in the SIV TM cytoplasmic domain can function as an internalization signal that can modulate expression of the viral envelope molecules on the cell surface and affect the biological properties of infectious viruses. The conservation of an analogous Tyr in all human and simian immunodeficiency viruses suggests that this signal may be present in other primate lentiviruses and could be important in the pathogenesis of these viruses in vivo.

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