scholarly journals Transmembrane orientation of the fibronectin receptor complex (integrin) demonstrated directly by a combination of immunocytochemical approaches.

1988 ◽  
Vol 36 (3) ◽  
pp. 297-306 ◽  
Author(s):  
S C Mueller ◽  
T Hasegawa ◽  
S S Yamada ◽  
K M Yamada ◽  
W T Chen
Keyword(s):  
Plasma Membrane ◽  
Band 3 ◽  
Cell Contact ◽  
Positive Staining ◽  
Double Labeling ◽  
Intact Cells ◽  
Fibronectin Receptor ◽  
Contact Sites ◽  
Attachment Sites ◽  
Membrane Attachment

The avian 140-KD cell adhesion receptor or "integrin," a complex of three glycoproteins with molecular masses averaging 140 KD, interacts with extracellular fibronectin and forms a linkage complex that co-localizes with intracellular actin. To probe the molecular interactions involved in this linkage complex, we used monoclonal antibodies and a combination of immunolocalization approaches to determine whether any component was transmembrane. Immunoadsorption and immunoblotting experiments demonstrated that anti-120-KD Mabs recognized the band 3 component of integrin isolated from chicken embryo fibroblasts (CEF) by JG22E immunoaffinity chromatography, and they co-localize with anti-fibronectin and polyclonal anti-integrin at cell contact sites in double-labeling experiments. Immunofluorescence experiments involved comparisons of double-labeled intact cells or substrate-attached, ventral plasma membrane "rip-off" fragments, using anti-fibronectin and each of the anti-120-KD Mabs. The extracellular faces of living intact cells were strongly labeled by a majority (approximately 70%) of the anti-120-KD Mabs at fibronectin-membrane attachment sites. The remainder (approximately 30%) labeled intact cells weakly or not at all. However, although the anti-120-KD Mab ES186 did not stain living cells, it did demonstrate positive staining above substratum contact sites over entire isolated rip-off membranes. In contrast, Mabs directed against putative extracellular epitopes and anti-fibronectin antibodies did not label these sites at the center of rip-offs unless the membranes were detergent permeabilized. Proteolysis experiments suggested that the ES186 epitope was located at one end of the molecule, since removal of short fragments from integrin band 3 concomitantly removed or destroyed the ES186 epitope, whereas the extracellular epitopes still remained. These experiments directly demonstrate that integrin band 3 is a transmembrane polypeptide with at least one epitope recognized by anti-120-KD Mabs on the cytoplasmic side of the plasma membrane and at least one epitope on the extracellular cell surface.

scholarly journals Localization of MOC-1 cell surface antigen in small-cell lung carcinoma cell lines: an immunohistochemical and immunoelectron microscopic study.

1993 ◽  
Vol 41 (9) ◽  
pp. 1303-1310 ◽  
Author(s):  
V Speirs ◽  
S Eich-Bender ◽  
C R Youngson ◽  
E Cutz
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Western Blot ◽  
Cell Line ◽  
Cell Lines ◽  
Carcinoma Cell ◽  
Small Cell ◽  
Cell Contact ◽  
Positive Staining ◽  
Carcinoma Cell Lines

Expression of cell surface antigens of the neural cell adhesion molecule (N-CAM) class was recently shown to be shared by both fetal and neoplastic neuroendocrine cells, including those of the lung. We investigated the expression and localization of MOC-1 antigen on small-cell (neuroendocrine) lung carcinoma cell lines with immunohistochemical methods at the light (LM) and electron microscopy (EM) level and by Western blot. At LM level, using monoclonal antibody (MAb) MOC-1 with the ABC method and immunofluorescence, positive staining was observed on surfaces of cells from all tumor lines examined. Strongest immunostaining was found on cell surfaces of pulmonary small-cell carcinoma-derived cell line NCI-H69 with the majority of cells showing positive staining. An adherent variant of NCI-H69 cell line, H69V, exhibited positive staining in about 60% of cells, whereas only occasional cells of NCI-H727 cell line derived from pulmonary carcinoid tumor were positive for MOC-1 antigen. Western blot analysis confirmed these findings, showing a strong MOC-1-specific band in cell extracts of NCI-H69, with weaker band densities for H69V and NCI-H727. Immunoelectron microscopy (IEM) revealed that MOC-1 was not uniformly distributed on the outer surface of plasma membrane; immunogold particles appeared concentrated in areas of thick cell surface "fuzz" coating, surface microvilli, and in areas of cell-cell contact. In some cells, areas of plasma membrane invaginations and a few intracytoplasmic vesicles were also labeled, suggesting endocytosis. Surface labeling for SEM confirmed the finding of more dense labeling over the microvilli, cell membrane folds, and in areas of cell-cell contact. The cell lines derived from pulmonary neuroendocrine cell tumors can provide a useful model to study the role and function of neural adhesion molecules in pulmonary neoplasia and during lung development.

scholarly journals Immunofluorescent localization of a 39,000-dalton substrate of tyrosine protein kinases to the cytoplasmic surface of the plasma membrane.

1983 ◽  
Vol 96 (6) ◽  
pp. 1601-1609 ◽  
Author(s):  
E A Nigg ◽  
J A Cooper ◽  
T Hunter
Keyword(s):  
Plasma Membrane ◽  
Protein Kinases ◽  
Cytoskeletal Proteins ◽  
Cell Contact ◽  
Immunofluorescent Localization ◽  
Intact Cells ◽  
Rous Sarcoma ◽  
Indirect Immunofluorescence Microscopy ◽  
Cytoplasmic Surface ◽  
Sarcoma Virus

The intracellular distribution of p39, a 39,000-dalton substrate for a number of tyrosine protein kinases, has been determined by indirect immunofluorescence microscopy. No binding of anti-p39 antibodies to intact cells was observed, indicating that this protein is not accessible to antibody on the cell surface. Following detergent permeabilization of formaldehyde-fixed cells, a reasonably uniform cytoplasmic labeling was observed. This fluorescence was most pronounced in membrane ruffles, especially in the leading lamellae of migrating cells, and in areas of cell-cell contact. Brief permeabilization of cells with detergent prior to formaldehyde fixation resulted in the appearance of a reticular lattice. An identical staining pattern was observed when fluorescently-labeled lectins were used as plasma membrane markers, but not when antibodies to a variety of cytoskeletal proteins were used. Taken together, these results indicate that p39 is, at least in part, located at the cytoplasmic surface of the plasma membrane. Immunolabeling of Rous sarcoma virus-transformed cells with anti-p39 antibodies resulted in fluorescent staining patterns indistinguishable from those observed in untransformed cells. It is conceivable that p39 plays some structural role within a protein network underlying the plasma membrane.

Mapping the Assembly of Band 3 and Rhesus Multi-Protein Complexes During Erythropoiesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 812-812
Author(s):  
Emile van den Akker ◽  
Timothy J. Satchwell ◽  
Geoff Daniels ◽  
Ashley M Toye
Keyword(s):  
Plasma Membrane ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Surface Expression ◽  
Band 3 ◽  
Membrane Expression ◽  
Intact Cells ◽  
Cell Lysates ◽  
Protein 4.2 ◽  
Pronase Treatment

Abstract Abstract 812 Band 3 forms the core of a large multiprotein complex in the erythrocyte membrane, the Band 3 macrocomplex, which also includes proteins of the Rhesus complex (Rh and RhAG). Mutations in genes encoding proteins within this complex can result in hereditary spherocytosis with varying severity. The effect of distinct mutations and deficiencies in proteins of the Band 3 macrocomplex has been studied in detail in mature erythrocytes. This revealed important functional and structural properties of individual proteins and their relationships with other proteins within the Band 3 macrocomplex. Nevertheless, considerably less is know about the spatio-temporal mechanisms that direct the formation of the Band 3 macrocomplex, and that may explain the aberrations in the complex observed in spherocytosis. Therefore, we studied expression and mutual interactions of proteins of the band3 macrocomplex during development of proerythroblasts to reticulocytes. Using confocal microscopy and western blotting, significant pools of intracellular Band 3 and RhAG were found in the basophilic normoblast. These intracellular pools gradually decreased in the polychromatic normoblast and were absent or low in the orthochromatic normoblast and reticulocytes, while surface expression increased. We used pronase treatment of intact cells to remove extracellular epitopes of BRIC 6 (Band 3 antibody) and LA1818 (RhAG antibody) to study the mechanism by which the intracellular pool of Band 3 and RhAG contributes to formation of the Band 3 complex on the cell surface. Pronase treatment of cells incubated with cycloheximide to block protein synthesis resulted in a reduced but still significant reappearance of BRIC6 (Band 3) and LA1818 (RhAG) epitopes on the plasma membrane confirming the presence of intracellular Band 3 and RhAG pools. It also showed that the bulk of Band 3 and RhAG is synthesized and trafficked to the membrane between the early basophilic and polychromatic stage. Immuneprecipitation of Band 3 from cell lysates of pronase treated cells pre-treated with brefeldin A to collapse the Golgi showed no increase in co-immuneprecipitated protein 4.2 albeit an increase in intracellular Band 3 expression. This suggests that protein 4.2 and Band 3 interact in the first Golgi compartment or late ER. In addition, pre-treatment of cells with cycloheximide prior to pronase treatment resulted in depletion of the intracellular Band 3 and co-immuneprecipitated protein 4.2 pool indicating that Band 3 and protein 4.2 traffic as a complex to the plasma-membrane. We were unable to co-immuneprecipitate Rh or Band 3 with intracellular pools of RhAG, whereas Rh was co-immuneprecipitated with RhAG from the plasma-membrane and from total cell lysates. Knockdown of RhAG in differentiating erythroblasts revealed a concomitant drop in membrane expression of Rh, leaving Band 3 unaffected, indicating that plasma-membrane expression of Rh but not Band 3 is dependent on RhAG. In conclusion, despite the described association between the RhAG complex and the Band 3 complex in erythrocytes, the data suggest that the Band 3-protein 4.2 complex traffics and assembles independently from Rh and RhAG during erythroid differentiation. The experiments suggest that Rh and RhAG do not traffic as a complex to the plasma-membrane but probably assemble in the plasma-membrane. The RhAG knockdown experiments suggest that the dependency of Rh on RhAG as observed in Rhnull syndrome erythrocytes (“Rh regulator type”) originates early during erythropoiesis. Band3 surface expression was not affected upon RhAG knock down, which re-produced the unperturbed Band 3 levels seen in these patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Erythrocyte membrane protein band 3: its biosynthesis and incorporation into membranes.

1981 ◽  
Vol 91 (3) ◽  
pp. 637-646 ◽  
Author(s):  
E Sabban ◽  
V Marchesi ◽  
M Adesnik ◽  
D D Sabatini
Keyword(s):  
Plasma Membrane ◽  
Electrophoretic Mobility ◽  
Messenger Rna ◽  
Transmembrane Protein ◽  
Band 3 ◽  
In Vitro Translation ◽  
Intact Cells ◽  
Amino Terminal ◽  
In Cells

Band 3, a transmembrane protein that provides the anion channel of the erythrocyte plasma membrane, crosses the membrane more than once and has a large amino terminal segment exposes on the cytoplasmic side of the membrane. The biosynthesis of band 3 and the process of its incorporation into membranes were studied in vivo in erythroid spleen cells of anemic mice and in vitro in protein synthesizing cell-free systems programmed with polysomes and messenger RNA (mRNA). In intact cells newly synthesized band 3 is rapidly incorporated into intracellular membranes where it is glycosylated and it is subsequently transferred to the plasma membrane where it becomes sensitive to digestion by exogenous chymotrypsin. The appearance of band 3 in the cell surface is not contingent upon its glycosylation because it proceeds efficiently in cells treated with tunicamycin. The site of synthesis of band 3 in bound polysomes was established directly by in vitro translation experiments with purified polysomes or with mRNA extracted from them. The band-3 polypeptide synthesized in an mRNA-dependent system had the same electrophoretic mobility as that synthesized in cells treated with tunicamycin. When microsomal membranes were present during translation, the in vitro synthesized band-3 polypeptide was cotranslationally glycosylated and inserted into the membranes. This was inferred from the facts that when synthesis was carried out in the presence of membranes the product had a lower electrophoretic mobility and showed partial resistance to protease digestion. Our observations indicate that the primary translation product of band-3 mRNA is not proteolytically processed either co- or posttranslationally. It is, therefore, proposed that the incorporation of band 3 into the endoplasmic reticulum (ER) membrane is initiated by a permanent insertion signal. To account for the cytoplasmic exposure of the amino terminus of the polypeptide we suggest that this signal is located within the interior of the polypeptide. a mechanism that explains the final transmembrane disposition of band 3 in the plasma membrane as resulting from the mode of its incorporation into the ER is presented.

scholarly journals A plasma membrane integral sialoglycoprotein (Sgp 130) molecularly distinguishes nonjunctional dense plaque sites of microfilament attachment.

1987 ◽  
Vol 105 (2) ◽  
pp. 819-831 ◽  
Author(s):  
A A Rogalski
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Cardiac Muscle ◽  
Cultured Cells ◽  
Molecular Forms ◽  
Cell Coupling ◽  
Membrane Structures ◽  
Adult Chicken ◽  
Attachment Sites ◽  
Membrane Attachment

An integral sialoglycoprotein with Mr approximately 130,000 (Sgp 130) and highest expression in adult chicken gizzard smooth muscle has been recently identified as an excellent candidate for classification as a plasma membrane protein natively associated (directly or indirectly) with actin microfilaments (Rogalski, A.A., and S.J. Singer, 1985, J. Cell Biol., 101:785-801). In this study, the relative in situ distributions of the Sgp 130 integral species (a designation that also includes non-smooth muscle molecular forms) and the peripheral protein, vinculin, have been simultaneously revealed for the first time in selected cultured cells and tissues abundant in microfilament-membrane attachment sites, particularly, smooth and cardiac muscle. Specific antibody probes against Sgp 130 (mouse mAb 30B6) and vinculin (affinity-purified rabbit antibody) were used in double indirect immunofluorescent and immunoelectron microscopic experiments. In contrast to the widespread distributions of vinculin at microfilament-membrane attachment sites, Sgp 130 has been shown to exhibit striking site-specific variation in its abundancy levels in the plasma membrane. Sgp 130 and vinculin were found coincidentally concentrated at focal contact sites in cultured chick embryo fibroblasts and endothelial cells, membrane dense plaques of smooth muscle, and sarcolemma dense plaque sites overlying the Z line in cardiac muscle. However, at the fascia adherens junctional sites of cardiac muscle where vinculin is sharply confined, Sgp 130 was immunologically undetectable in both intact and EGTA-uncoupled tissue. This latter result was confirmed with immunoblotting experiments using isolated forms of the fascia adherens. The double immunolabeling studies of this report establish Sgp 130 as a major integral protein component of nonjunctional membrane dense plaque structures and raise the possibility that the 130-kD integral sialoglycoprotein (Sgp 130) and vinculin assume stable transmembrane associations at these particular microfilament-membrane attachment sites. Nonjunctional dense plaques are further suggested to be a molecularly distinct class of plasma membrane structures rather than a subgroup of adherens junctions. Our data also support a hypothesis that Sgp 130 is involved in plasma membrane force coupling events but not in junctional-related cell-cell coupling.

scholarly journals Isolation and characterization of gelatinase granules from human neutrophils

Blood ◽  
1994 ◽  
Vol 83 (6) ◽  
pp. 1640-1649 ◽  
Author(s):  
L Kjeldsen ◽  
H Sengelov ◽  
K Lollike ◽  
MH Nielsen ◽  
N Borregaard
Keyword(s):  
Plasma Membrane ◽  
Qualitative Difference ◽  
Human Neutrophils ◽  
Immunogold Electron Microscopy ◽  
Double Labeling ◽  
Intact Cells ◽  
Cytochrome B558 ◽  
Isolation And Characterization ◽  
Specific Granules

We recently confirmed the existence of gelatinase granules as a subpopulation of peroxidase-negative granules by double-labeling immunogold electron microscopy on intact cells and by subcellular fractionation. Further characterization of gelatinase granules has been hampered by poor separation of specific and gelatinase granules on both two-layer Percoll gradients and sucrose gradients. We have developed a three-layer Percoll density gradient that allows separation of the different granules and vesicles from human neutrophils; in particular, it allows separation of specific and gelatinase granules. This allows us to characterize these two granule populations with regard to their content of membrane proteins, which become incorporated into the plasma membrane during exocytosis. We found that gelatinase granules, defined as peroxidase-negative granules containing gelatinase but lacking lactoferrin, contain 50% of total cell gelatinase, with the remaining residing in specific granules. Furthermore, we found that 20% to 25% of both the adhesion protein Mac-1 and the NADPH-oxidase component cytochrome b558 is localized in gelatinase granules. Although no qualitative difference was observed between specific granules and gelatinase granules with respect to cytochrome b558 and Mac-1, stimulation of the neutrophil with FMLP resulted in a selective mobilization of the least dense peroxidase-negative granules, ie, gelatinase granules, which, in concert with secretory vesicles, furnish the plasma membrane with Mac-1 and cytochrome b558. This shows that gelatinase granules are functionally important relative to specific granules in mediating early inflammatory responses.

scholarly journals Targeting of chimeric G alpha i proteins to specific membrane domains

1994 ◽  
Vol 107 (3) ◽  
pp. 507-515 ◽  
Author(s):  
J.B. de Almeida ◽  
E.J. Holtzman ◽  
P. Peters ◽  
L. Ercolani ◽  
D.A. Ausiello ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Eukaryotic Expression ◽  
Chimeric Proteins ◽  
Membrane Domains ◽  
Amino Terminal ◽  
Golgi Membranes ◽  
Attachment Sites ◽  
Membrane Attachment ◽  
Carboxy Terminal ◽  
Plasma Membrane Domain

Heterotrimeric guanine nucleotide-regulatory (G) proteins are associated with a variety of intracellular membranes and specific plasma membrane domains. In polarized epithelial LLC-PK1 cells we have shown previously that endogenous G alpha i-2 is localized on the basolateral plasma membrane, whereas G alpha i-3 is localized on Golgi membranes. The targeting of these highly homologous G alpha i proteins to distinct membrane domains was studied by the transfection and expression of chimeric G alpha i proteins in LLC-PK1 cells. Chimeric cDNAs were constructed from the cDNAs for G alpha i-3 and G alpha i-2 and introduced into a pMXX eukaryotic expression vector containing a mouse metallothionein-I promoter. Stably transfected cell lines were produced that expressed either G alpha i-2/3 or G alpha i-3/2 chimeric proteins. Chimeric and endogenous G alpha i proteins were detected in cells using specific carboxy-terminal peptide antibodies. Immunofluorescence staining was used to localize endogenous and chimeric G alpha i proteins in LLC-PK1 cells. The staining of chimeric proteins was detected as an increased intensity of staining on membranes containing endogenous G alpha i proteins. Using confocal microscopy and image analysis we localized G alpha i-2 to a specific sub-domain of the lateral membrane of polarized cells, the chimeric G alpha i-3/2 protein was then shown to colocalize with endogenous G alpha i-2 in the same lateral plasma membrane domain. The chimeric G alpha i-2/3 protein colocalized with endogenous G alpha i-3 on Golgi membranes in LLC-PK1 cells. These results show that chimeric G alpha i proteins were targeted to the same membrane domains as endogenous G alpha i proteins and the specificity of their membrane targeting was conferred by the carboxy-terminal end of the proteins. These data provide the first evidence for specific targeting information contained in the carboxy termini of G alpha i proteins, which appears to be independent of amino-terminal membrane attachment sites in these proteins.

scholarly journals Ergosterol promotes pheromone signaling and plasma membrane fusion in mating yeast

2008 ◽  
Vol 180 (4) ◽  
pp. 813-826 ◽  
Author(s):  
Hui Jin ◽  
J. Michael McCaffery ◽  
Eric Grote
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Cell Contact ◽  
Mating Pair ◽  
Pheromone Response ◽  
Contact Sites ◽  
Pheromone Signaling ◽  
Independent Functions ◽  
Contact Transmission ◽  
Acyl Chains

Ergosterol depletion independently inhibits two aspects of yeast mating: pheromone signaling and plasma membrane fusion. In signaling, ergosterol participates in the recruitment of Ste5 to a polarized site on the plasma membrane. Ergosterol is thought to form microdomains within the membrane by interacting with the long acyl chains of sphingolipids. We find that although sphingolipid-free ergosterol is concentrated at sites of cell–cell contact, transmission of the pheromone signal at contact sites depends on a balanced ratio of ergosterol to sphingolipids. If a mating pair forms between ergosterol-depleted cells despite the attenuated pheromone response, the subsequent process of membrane fusion is retarded. Prm1 also participates in membrane fusion. However, ergosterol and Prm1 have independent functions and only prm1 mutant mating pairs are susceptible to contact-dependent lysis. In contrast to signaling, plasma membrane fusion is relatively insensitive to sphingolipid depletion. Thus, the sphingolipid-free pool of ergosterol promotes plasma membrane fusion.

scholarly journals Hsp27 inhibits sublethal, Src-mediated renal epithelial cell injury

2009 ◽  
Vol 297 (3) ◽  
pp. F760-F768 ◽  
Author(s):  
Andrea Havasi ◽  
Zhiyong Wang ◽  
Jonathan M. Gall ◽  
Max Spaderna ◽  
Vikram Suri ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Organ Dysfunction ◽  
Cell Injury ◽  
Atp Depletion ◽  
Cell Contact ◽  
Intact Cells ◽  
Renal Epithelial Cells ◽  
Contact Sites ◽  
Junction Protein ◽  
Cell Cell

Disruption of cell contact sites in renal epithelial cells contributes to organ dysfunction after ischemia. We hypothesized that heat shock protein 27 (Hsp27), a known cytoprotectant protein, preserves cell architecture and cell contact site function during ischemic stress. To test this hypothesis, renal epithelial cells were subjected to transient ATP depletion, an in vitro model of ischemia-reperfusion injury. Compared with control, selective Hsp27 overexpression significantly preserved cell-cell junction function during metabolic stress as evidenced by reduced stress-mediated redistribution of the adherens junction protein E-cadherin, higher transepithelial electrical resistance, and lower unidirectional flux of lucifer yellow. Hsp27 overexpression also preserved paxillin staining within focal adhesion complexes and significantly decreased cell detachment during stress. Surprisingly, Hsp27, an F-actin-capping protein, only minimally reduced stress induced actin cytoskeleton collapse. In contrast to Hsp27 overexpression, siRNA-mediated knockdown had the opposite effect on these parameters. Since ischemia activates c-Src, a tyrosine kinase that disrupts both cell-cell and cell-substrate interactions, the relationship between Hsp27 and c-Src was examined. Although Hsp27 and c-Src did not coimmunoprecipitate and Hsp27 overexpression failed to inhibit whole cell c-Src activation during injury, manipulation of Hsp27 altered active c-Src accumulation at cell contact sites. Specifically, Hsp27 overexpression reduced, whereas Hsp27 knockdown increased active p-416Src detected at contact sites in intact cells as well as in a purified cell membrane fraction. Together, this evidence shows that Hsp27 overexpression prevents sublethal REC injury at cell contact sites possibly by a c-Src-dependent mechanism. Further exploration of the biochemical link between Hsp27 and c-Src could yield therapeutic interventions for ameliorating ischemic renal cell injury and organ dysfunction.

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