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M Edidin

Publications and source records attributed to M Edidin.

At least 19 recordsLinked to original sources
PubMed1992

Self-association of class I major histocompatibility complex molecules in liposome and cell surface membranes.

Fluorescent derivatives of a human MHC class I glycoprotein, HLA-A2, were reconstituted into dimyristoylphosphatidylcholine (DMPC) liposomes. Measurements of lateral diffusion of fluorescein-(Fl-) labeled HLA-A2 by fluorescence photobleaching recovery (FPR), of rotational diffusion of erythrosin-(Er-) labeled HLA-A2 by time-resolved phosphorescence anisotropy (TPA), and of molecular proximity by flow cytometric fluorescence resonance energy transfer (FCET) showed that these class I MHC molecules self-associate in liposome membranes, forming small aggregates even at low surface concentrations. The lateral diffusion coefficient (Dlat) of Fl-HLA-A2 decreases with increasing surface protein concentration over a range of lipid:protein molar ratios (L/P) between 8000:1 and 2000:1. The reduction in Dlat of HLA molecules in DMPC liposomes is found to be sensitive to time and temperature. The rotational correlation time for Er-HLA-A2 in DMPC liposomes at 30 degrees C is 87 +/- 0.8 microseconds, at least 10 times larger than that expected for an HLA monomer. There is also significant quenching of donor (Fl-HLA) fluorescence at 37 degrees C in the presence of acceptor-labeled (sulforhodamine-labeled HLA) protein indicating proximity between HLA molecules even at L/P = 4000:1. FPR and FCET measurements with another membrane glycoprotein, glycophorin, give no evidence for its self-association. HLA aggregation measured by FPR, FCET, and TPA was blocked by beta 2-microglobulin, b2m, added to the liposomes. The aggregation of HLA-A2 molecules is not an artifact of their reconstitution into liposomes. HLA aggregates, defined by FCET, were readily detected on the surface of human lymphoblastoid (JY) cells.(ABSTRACT TRUNCATED AT 250 WORDS)

B-Lymphocytes↗
PubMed1992

Luminescence quenching by nitroxide spin labels in aqueous solution: studies on the mechanism of quenching.

The mechanism of luminescence quenching by spin labels was investigated in aqueous solution by steady-state and time-resolved luminescence techniques. Water-soluble nitroxide radicals strongly quenched the luminescence emitted by Tb3+ chelates and by fluorescein, either free or conjugated to proteins. The following features of the quenching reaction were established: (I) the rate constant for quenching of triplet-state Tb3+ by nitroxides was about 4 orders of magnitude smaller (ca. 10(5) M-1 s-1) than those of the singlet-state probes; (II) the quenchers reduced the excited-state lifetime of both probes; (III) the rate constants for quenching of both probes were found to be apparently independent of the temperature (between 6 and 42 degrees C) and viscosity (up to 60 mPa.s) of the solutions; (IV) both singlet and triplet quenching rates were sensitive to solvent polarity; (V) there is a small but significant spectral overlap between the absorption band of weekly absorbing nitroxide radicals and the emission spectra of luminophores, the extent of which, however, does not correlate with the extent of quenching; (VI) the quenching rate declines sharply with an increasing luminophore to nitroxide distance. The distance dependence of the quenching rate showed a satisfactory fit to an exponential function. These findings indicate that the quenching reaction is dominated by an electron exchange between the excited singlet- or triplet-state luminophore and the nitroxide radical rather than controlled by diffusional properties of the reactants.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic N-Oxides↗
PubMed1992

Cell-specific constraints to the lateral diffusion of a membrane glycoprotein.

We have previously shown that the lateral diffusion, D, of the class I Major Histocompatibility Complex (MHC) glycoprotein H-2Ld is constrained by its glycosylation, when expressed in mouse L-cells. Removal of one or more of the 3 N-linked oligosaccharides of H-2Ld glycoproteins results in an increase in D. In order to further examine the influence of glycosylation on D, we compared lateral diffusion of H-2Ld expressed in wild-type CHO cells with lateral diffusion of the same molecule expressed in mutant CHO cells with aberrant surface glycosylation. In addition, we compared lateral diffusion of wild-type and unglycosylated H-2Ld antigens in these cells. In contrast to the large effect of glycosylation state on lateral diffusion of H-2Ld in mouse L-cells, there was little effect of glycosylation on lateral diffusion of H-2Ld in any of the CHO cells. This, together with similar results on hamster class I antigens, indicates that the constraints to D of H-2Ld and other class I MHC molecules are different in CHO cells than in L-cells. Measurements of lateral diffusion after treatment of cells with cytochalasin D make it clear that interactions between MHC class I molecules and a cytoskeleton are important in reducing the mobile fraction of diffusing molecules, R, though they cannot be shown to directly affect the diffusion coefficient, D.

Animals↗
PubMed1992

Glycolipid depletion using a ceramide analogue (PDMP) alters growth, adhesion, and membrane lipid organization in human A431 cells.

Glycolipids were depleted from the membranes of human A431 cells using 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor of glucosylceramide synthetase. After 6 days of culture in the presence of 5 microM D-threo-PDMP, glycolipid content was reduced to approximately 5% of control levels. By contrast, synthesis per cell of phosphatidylcholine, sphingomyelin, triglycerides, and glycoprotein was relatively unchanged in PDMP-treated cells. In parallel with glycolipid depletion, PDMP-treated cells exhibited a rapid loss of epithelial cell morphology, a reduced rate of cell growth, and inhibition of cell-substrate adhesion. The effects of D-threo-PDMP on cell morphology and substrate adhesion were blocked by exogenous GM3 addition and were not observed with L-threo-PDMP (a relatively inactive enantiomer). Fluorescence photobleaching and recovery (FPR) was used to investigate the hypothesis that glycolipids influence cell behavior, in part, by changing the diffusion characteristics of membrane proteins and lipids. Diffusion coefficients and mobile fractions of two integral membrane proteins, the EGF receptor and a class I MHC antigen, did not differ significantly between control and PDMP-treated cells. Diffusion coefficients of lipid probes, NBD-PC and fluorescent GM1 ganglioside, were similarly unaffected by glycolipid depletion. However, lipid probes did show a significant increase in mobile fraction (the fraction of lipids that are free to diffuse) in PDMP-treated cells. This increase was blocked by culturing cells in the presence of exogenous GM3 ganglioside. The results suggest that glycolipids play a role in the formation of lipid domains in A431 cell membranes. Glycolipid-mediated changes in membrane lipid organization may influence receptor activation and transmembrane signaling, leading to changes in cell growth, morphology, and adhesion.

Carcinoma, Squamous Cell↗
PubMed1991

Lateral movements of membrane glycoproteins restricted by dynamic cytoplasmic barriers.

Cell membranes often are patchy, composed of lateral domains. These domains may be formed by barriers within or on either side of the membrane bilayer. Major histocompatibility complex (MHC) class 1 molecules that were either transmembrane- (H-2Db) or glycosylphosphatidylinositol (GPI)-anchored (Qa2) were labeled with antibody-coated gold particles and moved across the cell surface with a laser optical tweezers until they encountered a barrier, the barrier-free path length (BFP). At room temperature, the BFPs of Qa2 and H-2Db were 1.7 +/- 0.2 and 0.6 +/- 0.1 (micrometers +/- SEM), respectively. Barriers persisted at 34 degrees C, although the BFP for both MHC molecules was fivefold greater at 34 degrees C than at 23 degrees C. This indicates that barriers to lateral movement are primarily on the cytoplasmic half of the membrane and are dynamic.

Animals↗
PubMed1991

Endocytosis and dissociation of class I MHC molecules labeled with fluorescent beta-2 microglobulin.

Membrane class I MHC molecules of Con-A activated and lymphoma murine cells have been labeled by exchange of the cell's beta 2m with soluble fl-beta 2m. It has previously been shown that this method of labeling is specific and does not affect the biologic properties of class I MHC Ag. With this labeling it has been possible to demonstrate the constitutive endocytosis of class I MHC by fluorescence microscopy and by measuring the resistance to quenching by crystal violet of the internalized fl-beta 2m molecules. We could also follow the kinetics of beta 2m dissociation from the class I molecules at different pH. At pH 5.5, that is the average pH of endosomes, there is considerable dissociation within 15 to 20 min, that is the average recycling half time of class I MHC containing endosomes in activated T cells. Inasmuch as the process is reversible it is likely that, in the recycling endosomes of T cells, class I MHC molecules undergo conformational changes with beta 2m going off and on and with consequent changes of the peptide binding site. This process might be involved in Ag presentation, but, because it is apparently limited to T cells, it would play a role in the presentation of the cell's own TCR in idiotypic interactions between T cells.

Endocytosis↗
PubMed1991

Immobilization and internalization of mutated IgE receptors in transfected cells.

Earlier studies have shown that the mast cell receptor IgE (Fc epsilon RI) for is expressed on COS-7 cells transfected with the cDNA for each of the three types of subunits that form the tetrameric, alpha beta gamma 2, receptor. Although such transfected COS cells fail to exhibit some of the early biochemical perturbations initiated by aggregation of the receptor on normal mast cells and related tumor lines, we show here that other characteristics of the endogenous Fc epsilon RI are retained. Thus, the unaggregated transfected wild-type receptors were found to have a restricted translational diffusion similar to that observed for endogenous receptors on mast cells as assessed by fluorescence photobleaching and recovery. Similarly, as with endogenous receptors the mobility of transfected receptors was sharply reduced when the receptors were aggregated by reaction with small oligomers of IgE. In addition, aggregation of the transfected Fc epsilon RI caused them to be internalized by the COS cells by a cytochalasin-sensitive mechanism, albeit at a considerably slower rate than was seen with endogenous receptors on mast cells or with transfected receptors in a line of receptor-deficient mast cells. We also examined the mobility and internalization before and after aggregation, of some 13 different combinations of receptor subunit mutants in which one or more of the five cytoplasmic domains of the receptor had been truncated. Our results show that whatever interactions between the receptor and cellular components may account for the phenomena we studied, such interactions do not critically depend upon the bulk of the cytoplasmic domains of the receptor.

Amino Acid Sequence↗
PubMed1991

Lateral diffusion of CD4 on the surface of a human neoplastic T-cell line probed with a fluorescent derivative of the envelope glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV-1).

The envelope glycoprotein (gp120) of HIV-1 was labeled with fluorescein by using 6-[4,6-dichlorotriazinyl]aminofluorescein. The labeled glycoprotein was found to bind to CD4-positive CEM cells. Monoclonal antibody OKT4a but not OKT4 blocked this binding. Similar specific binding of fluorescein-labeled gp120 with CD4 was observed in a solid-phase ELISA where sCD4 was attached to a polystyrene plate. The syncytium formation induced by HIV-1-infected cells on CEM cells was significantly inhibited in the presence of fluorescein-labeled gp120. Fluorescence photobleaching recovery measurements showed that the diffusion coefficient (D) of CD4 molecules complexed with fluorescein-labeled gp120 was approximately 5 x 10(-10) cm2sec-1, with nearly 61% of the receptor molecules being mobile. Binding of anti-gp120 monoclonal antibody to the CD4-gp120 complex reduced the mobile fraction significantly. Diffusion of CD4 labeled with OKT4 IgG was markedly inhibited with reductions in both D and the mobile fraction, but such inhibition was not observed with OKT4 Fab. It appears that crosslinking of multiple molecules of CD4 by OKT4 antibody is required to reduce CD4 mobility. This suggests that the receptor might be present on the membrane plane as molecular clusters containing at least two molecules of CD4.

Antibodies, Monoclonal↗
PubMed1991

Differences between the lateral organization of conventional and inositol phospholipid-anchored membrane proteins. A further definition of micrometer scale membrane domains.

Plasma membranes of many cells appear to be divided into domains, areas whose composition and function differ from the average for an entire membrane. We have previously used fluorescence photo-bleaching and recovery to demonstrate one type of membrane domain, with dimensions of micrometers (Yechiel, E., and M. Edidin. 1987, J. Cell Biol. 105: 755-760). The presence of membrane domains is inferred from the dependence of the apparent mobile fraction of labeled molecules on the size of the membrane area probed. We now find that by this definition classical class I MHC molecules, H-2Db, are concentrated in domains in the membranes of K78-2 hepatoma cells, while the nonclassical class I-related molecules, Qa-2, are free to pass the boundaries of these domains. The two proteins are highly homologous but differ in their mode of anchorage to the membrane lipid bilayer. H-2Db is anchored by a transmembrane peptide, while Qa-2 is anchored by a glycosylphosphatidylinositol (GPI) anchor. A mutant class I protein with its external portion derived from Qa-2 but with transmembrane and cytoplasmic sequences from a classical class I molecule shows a dependence of its mobile fraction on the area of membrane probed, while a mutant whose external portions are a mixture of classical and nonclassical class I sequences, GPI-linked to the bilayer, does not show this dependence and hence by our definition is not restricted to membrane domains.

Animals↗
PubMed1991

Endothelial cells transformed by SV40 T antigen cause Kaposi's sarcomalike tumors in nude mice.

Injection into nude mice of a well-differentiated SV40 T-antigen-transformed murine endothelial cell (EC) line results in widespread invasive tumors confined to connective tissues. The tumors, which do not metastasize, consist of both host-derived cells and transformed EC, displaying histologic features typical of Kaposi's sarcoma (KS). Although the EC is believed to be the cell of origin in KS, this has not been proven and is the subject of debate. The unusual tumorigenicity of this transformed cell suggests that EC-specific gene products induced by SV40 T antigen may contribute to tumorigenesis by autocrine growth stimulation and recruitment of host cells. KS-like tumors may be the result of EC alteration by any virus that induces relevant EC-derived cytokines.

Animals↗
PubMed1990

Immunogenic Ia-binding peptides immobilize the Ia molecule and facilitate its aggregation on the B cell membrane. Control by the M1s-1 gene.

Aggregation (e.g., through cross-linkage) of cell surface molecules is in various biologic systems a necessary event in cellular activation. Examining the Ia molecule on B cells we found that aggregation is a function of the surface Ag mobility; the higher the fraction of immobile molecules on the plane of the membrane, the better Ia forms aggregates and patches. We identify two factors that control Ia mobility and aggregability. One factor is the M1s-1a gene product; the other factor is an Ia-reactive immunogenic peptide. Both factors increase Ia aggregability and reduce the MHC Ag mobility.

Animals↗
PubMed1990

A mouse lymphoid endothelial cell line immortalized by simian virus 40 binds lymphocytes and retains functional characteristics of normal endothelial cells.

Leukocyte-endothelial cell (EC) interactions regulate the entry of immune effectors into the tissues. This interaction occurs in lymphoid tissues and inflammatory sites at post-capillary high endothelial venules, as opposed to large capacitance vessels lined with flat EC. Transient SV40 infection of mouse EC derived from lymph node stroma has resulted in a cell line, SVEC4-10, that retains morphological and functional characteristics of normal EC. SVEC4-10 cells grow efficiently on plastic as a monolayer with a characteristic epithelioid morphology. They require as little as 2% FCS and are independent of other exogenous growth factors or matrix components. When grown on a synthetic basement membrane, SVEC4-10 forms branching tube-like networks. SVEC4-10 expresses Factor VIII related Ag as measured by indirect immunofluorescence using a rabbit antiserum to human FVIII-associated protein and incorporates acetylated low density lipoprotein. SVEC4-10 specifically binds mouse lymphocytes in vitro. IFN-gamma induces expression of MHC class II Ag in a time course identical to normal EC and the cell line is susceptible to lysis by anti SV40 H-2k CTL clones. Thus, this SV40 immortalized line retains much of the normal cellular physiology of EC.

Animals↗
PubMed1989

Exogenous ATP and other nucleoside phosphates modulate epidermal growth factor receptors of A-431 epidermoid carcinoma cells.

The binding of epidermal growth factor (EGF) by A-431 human epidermoid carcinoma cells was reduced after exposure of the cells to low concentrations (0.01-1 mM) of ATP and other nucleoside 5'-triphosphates at 37 degrees C, but not at 0 degree C. This was due to loss of high-affinity EGF binding sites. The modulation was associated with transient increases in inositol phosphate synthesis and intracellular Ca2+ and with phosphorylation of the EGF receptor on serine and threonine. There was no evidence for entry of labeled ATP into the cells. ATP appeared to bind to specific cell surface receptors. Such binding was demonstrated directly with the nonmetabolizable ATP analogue adenosine 5'-[beta,gamma-imido]triphosphate.

Adenosine Triphosphate↗
PubMed1989

Na+,K+-adenosine triphosphatase polarity in retinal photoreceptors: a role for cytoskeletal attachments.

We have used isolated embryonic photoreceptor cells as a model system with which to examine the mechanisms responsible for the development and maintenance of asymmetric Na+,K+-ATPase (ATPase) distribution. Photoreceptor precursors, which appear round and process free at culture onset, develop structural and molecular properties similar to those of photoreceptor cells in vivo. ATPase, recognized by an anti-ATPase antibody, is distributed over the entire surface of round photoreceptor precursors. As the cells develop, ATPase becomes progressively concentrated in the inner segment (where it is found in cells of the intact retina). This phenomenon occurs in cells developing in the absence of intercellular contacts. The development of ATPase polarity correlates with a decrease in the fraction of ATPase molecules that are mobile in the membrane (as determined by fluorescence photobleaching recovery), as well as with an increase in the fraction of ATPase that remains associated with the cells after detergent extraction. The magnitudes of the mobile ATPase fractions agree well with those of the detergent-extractable fractions in both the immature and developed photoreceptors. The distribution of alpha spectrin and ATPase-immunoreactive materials appeared qualitatively similar, and quantitative image analysis showed similar gradients of spectrin and Na+,K+-ATPase immunofluorescence along the long axis of elongated photoreceptors. Moreover, detergent extractability of alpha spectrin and the ATPase showed similar modifications in response to changes in pH or KCl concentration. ATPase detergent-extractable and mobile fractions were not changed in cultures treated with cytoskeletal inhibitors such as nocodazole. These data are consistent with a role for an asymmetrically distributed, spectrin-containing subcortical cytoskeleton in the preferential accumulation of Na+,K+-ATPase in the photoreceptor inner segment.

Animals↗
PubMed1989

Translational diffusion of class II major histocompatibility complex molecules is constrained by their cytoplasmic domains.

Site-directed mutagenesis in vitro was used to introduce stop codons in the genomic DNA of the alpha and beta chains of the murine class II major histocompatibility complex antigen, I-Ak. Mutated DNA was transfected into B lymphoma cells that were then selected by neomycin resistance and for their ability to express I-Ak molecules on their plasma membrane. The translational diffusion coefficient (Dlat) of I-Ak molecules composed of a wild-type beta chain paired with an alpha chain missing either 6 or 12 amino acids from the cytoplasmic domain is on the average threefold higher than the Dlat of wild-type I-Ak molecules as measured by fluorescence photobleaching and recovery. The removal of 12 amino acids from the cytoplasmic domain of the beta chain did not change the Dlat value from that of wild-type I-Ak if the truncated beta chain was paired with a wild-type alpha chain. Removing all amino acids of the cytoplasmic domains of both the alpha and beta chains resulted in a 10-fold increase in the Dlat, the highest value for any of the truncated I-Ak molecules tested. These data indicate that the carboxy-terminal six amino acids of the cytoplasmic domain of the alpha chain and the six plasma membrane-proximal amino acids of the beta chain are important in constraining the translational diffusion of I-Ak molecules in the plasma membrane.

Amino Acid Sequence↗