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M Edidin

Publications and source records attributed to M Edidin.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1988

Constraint of the translational diffusion of a membrane glycoprotein by its external domains.

The translational diffusion of wild-type and underglycosylated molecules of a membrane-integral glycoprotein the Ld class I major histocompatibility complex (MHC) antigen has been measured. The Ld mutant molecules, which lack one or more glycosylation sites, had larger translational diffusion coefficients, D, than did wild-type Ld molecules glycosylated at three sites. The increase in D is linear with loss of glycosylation. The highest value of D approaches that for translational diffusion of molecules constrained only by viscosity of the membrane lipid bilayer. These results indicate that the external portions of cell surface glycoproteins interact significantly with other nearby molecules.

Cell Line↗
PubMed1988

Lateral diffusion measurements give evidence for association of the Tac peptide of the IL-2 receptor with the T27 peptide in the plasma membrane of HUT-102-B2 T cells.

Fluorescence photobleaching recovery measurements show that the F1-IgG-labeled Tac peptide of the IL-2R can diffuse in the plane of the membrane of HUT-102-B2 T lymphocytes, with a mean diffusion coefficient of 2 to 3 x 10(-10) cm2s-1. Although only a fraction (mean 37%) of the Tac peptides is mobile on any given cell, lateral diffusion of the Tac peptide can be measured in 94% of cells examined. In contrast, the 95-kDa peptide, T27, is 90 to 100% immobilized in cells labeled with OKT27. Immobilization of T27 also affects the lateral diffusion of the Tac peptide, because the Tac peptide is immobile in more than 30% of cells pretreated with OKT27 and then labeled with anti-Tac IgG. The effect is specific for OKT27 to the extent that pretreatment with an anti-HLA mAb does not immobilize the Tac peptide. It appears, then, that Tac and T27 peptide not only are in proximity on HUT-102-B2 lymphocyte membranes but also interact physically in situ.

Antibodies, Monoclonal↗
PubMed1988

Modulation of HLA antigens in response to the binding of epidermal growth factor by A431 cells.

In a previous study [(1984) J. Cell Biol. 98, 725-731] we showed that the level of human MHC, HLA antigens on A431 carcinoma cells is reduced after these cells bind epidermal growth factor (EGF). Here we use flow cytometry to determine the effects of various doses and times of EGF treatment on HLA expression. We then show that the reduction in HLA expression is associated with a reduction in the level of phosphorylation of immunoprecipitable surface HLA antigens, although longer exposure of cells with EGF increased both surface HLA expression and their phosphorylation levels. Lateral diffusion of HLA antigens is lower in EGF-treated than in control cells. The lower diffusion coefficients measured may be causally related to the decreased phosphorylation of HLA antigens.

Aged↗
PubMed1988

Specific associations of fluorescent beta-2-microglobulin with cell surfaces. The affinity of different H-2 and HLA antigens for beta-2-microglobulin.

We prepared single-labeled FITC derivatives of beta-2-microglobulin (b2m) and examined their interactions with class I MHC Ag H chains on living cells. Human b2m was reacted with FITC under mild conditions and separated by hydroxylapatite chromatography into three peaks containing single labeled derivatives of b2m peaks A, B, and C, and a peak containing the unmodified protein. The three fluorescent derivatives labeled the surfaces of cells bearing class I MHC Ag. The labeling was specific for class I MHC Ag as indicated by failure to label cells in the presence of excess unlabeled b2m and failure to label the HLA-negative cell lines Daudi and 721.221. Mouse cells labeled with fluorescent human b2m were recognized by mAb to the class I MHC Ag and by virus-restricted cytotoxic T lymphocytes, suggesting that labeling with the fluorescent b2m does not significantly alter the structure of class I MHC Ag or impair their ability to present viral antigens to cytotoxic T lymphocytes. We determined the kinetic and equilibrium binding parameters for the fluorescent b2m derivatives associating with the class I H chains of mouse and human cells. Peaks B and C exhibited biphasic binding to the mouse lymphoma cells EL-4(G-CSA-) (Kd1 = 1 x 10(-9) M; K2 = 1.5 to 3.0 x 10(-8) M whereas peak A bound to a small number of low affinity binding sites. In contrast to the biphasic binding observed with EL-4(G-CSA-), only monophasic binding was observed for peak C binding to RDM4 cells. Biphasic binding was also observed with the human B cell line LCL 721. Analysis of a series of LCL 721 class I MHC loss mutants and gene transferents revealed that the heterogeneity in binding is due to differences in the affinity of different class I encoded H chains for b2m.

Animals↗
PubMed1988

Selective anchoring in the specific plasma membrane domain: a role in epithelial cell polarity.

We have studied the role of restrictions to lateral mobility in the segregation of proteins to apical and basolateral domains of MDCK epithelial cells. Radioimmunoassay and semiquantitative video analysis of immunofluorescence on frozen sections showed that one apical and three basolateral glycoproteins, defined by monoclonal antibodies and binding of beta-2-microglobulin, were incompletely extracted with 0.5% Triton X-100 in a buffer that preserves the cortical cytoskeleton (Fey, E. G., K. M. Wan, and S. Penman. 1984. J. Cell Biol. 98:1973-1984; Nelson, W. T. and P. J. Veshnock. 1986. J. Cell Biol. 103:1751-1766). The marker proteins were preferentially extracted from the "incorrect" domain (i.e., the apical domain for a basolateral marker), indicating that the cytoskeletal anchoring was most effective on the "correct" domain. The two basolateral markers were unpolarized and almost completely extractable in cells prevented from establishing cell-cell contacts by incubation in low Ca++ medium, while an apical marker was only extracted from the basal surface under the same conditions. Procedures were developed to apply fluorescent probes to either the apical or the basolateral surface of live cells grown on native collagen gels. Fluorescence recovery after photobleaching of predominantly basolateral antigens showed a large percent of cells (28-52%) with no recoverable fluorescence on the basal domain but normal fluorescence recovery on the apical surface of most cells (92-100%). Diffusion coefficients in cells with normal fluorescence recovery were in the order of 1.1 x 10(-9) cm2/s in the apical domain and 0.6-0.9 x 10(-9) cm2/s in the basal surface, but the difference was not significant. The data from both techniques indicate (a) the existence of mobile and immobile protein fractions in both plasma membrane domains, and (b) that linkage to a domain specific submembrane cytoskeleton plays an important role in the maintenance of epithelial cell surface polarity.

Antibodies, Monoclonal↗
PubMed1987

Insulin binding to human B lymphoblasts is a function of HLA haplotype.

A variety of genetic and biochemical evidence points to an association between major histocompatibility complex (MHC) haplotype and several types of cell surface receptors including epidermal growth factor and insulin receptors. We report evidence for such associations between human class I MHC antigens, HLA antigens, and specific insulin binding sites on human B lymphoblasts. We have measured insulin binding to cells of an HLA-heterozygous, Epstein-Barr virus-transformed B-cell line, LCL 721, and to derivative mutants from which all or part of the HLA complex had been deleted. The affinity, Ka, of insulin binding sites is approximately 10(8) M-1 in mutants expressing antigen HLA-B5 together with other HLA antigens and in mutants expressing only HLA-C. HLA-A1; HLA-A1,B8; HLA-A2,C; and HLA null mutants (not expressing any HLA antigens) bind insulin to sites with an affinity of approximately 10(9) M-1.

B-Lymphocytes↗
PubMed1987

Micrometer-scale domains in fibroblast plasma membranes.

We have used the technique of fluorescence photobleaching recovery to measure the lateral diffusion coefficients and the mobile fractions of a fluorescent lipid probe, 1-acyl-2-(12-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)aminododecanoyl]) phosphatidylcholine (NBD-PC), and of labeled membrane proteins of human fibroblasts. Values for mobile fractions decrease monotonically with increasing size of the laser spot used for the measurements, over a range of 0.35-5.0 microns. Values for NBD-PC diffusion coefficients increase in part of this range to reach a plateau at larger laser spots. This variation is not an artifact of the measuring system, since the effects are not seen if diffusion of the probe is measured in liposomes. We also find that the distribution of diffusion coefficients measured with small laser spots is heterogeneous indicating that these small spots can sample different regions of the membrane. These regions appear to differ in protein concentration. Our data strongly indicate that fibroblast surface membranes consist of protein-rich domains approximately 1 micron in diameter, embedded in a relatively protein-poor lipid continuum. These features appear in photographs of labeled cell surfaces illuminated by the expanded laser beam.

Cell Membrane↗
PubMed1986

Site-directed labeling of a monoclonal antibody: targeting to a disulfide bond.

We have designed and synthesized crabescein, the first member of a class of fluorescent labels that add across disulfide bonds. Crabescein is a fluorescein derivative that reports the rotational correlation time of the immunoglobulin G (IgG) segment to which it is covalently bound. Chemical analysis of the IgG labeled with crabescein indicates that the fluorophore is inserted into the third disulfide bond (cysteine-229 of mouse IgG2a) in the hinge region. The rotational correlation time of this labeled macromolecule was measured as a single exponential with a decay constant of 26.8 ns. This is in contrast to the double exponential with decay constants of 14.3 and 0.2 ns for the same IgG when labeled with fluorescein via a conventional labeling reagent in which the probe is bound to the macromolecule by one-point attachments. Thus, crabescein is the prototype of a class of fluorescent and phosphorescent probes that, by virtue of their two-point attachments to proteins, faithfully report on the dynamics of the segment of macromolecule to which they are covalently bound.

Animals↗
PubMed1986

The class I MHC antigens of erythrocytes: a serologic and biochemical study.

We have measured the binding of anti-H-2K monoclonal antibodies to purified erythrocytes of a number of mouse strains. The purified erythrocytes specifically bound between 500 and 2000 molecules of labeled anti-H-2K antibodies per cell, whereas lymphocytes bound approximately 10(5) molecules of antibody per cell. Not all of the antibodies that bound to lymphocytes bound to erythrocytes. Antibodies to both public and private specificities that reacted with H-2k lymphocytes failed to react with H-2k erythrocytes. A similar pattern was found when comparing H-2b erythrocytes with lymphocytes. Failure to bind erythrocytes is not a function of antibody isotype or avidity. Isolated H-2K antigens of erythrocytes are integral membrane proteins identical in apparent m.w. and very similar in charge heterogeneity to the H-2K antigens of lymphocytes. Limited peptide maps of exogenously labeled antigens are also nearly identical. We also find that antibodies against epitopes of all three external domains of H-2K may react with erythrocyte H-2K. The altered reactivity of erythrocyte H-2K antigens is unlikely to be due to domain deletion, or to differences in membrane environments in erythrocytes and lymphocytes. The difference in reactivity could be due to subtle post-transcriptional modifications, or to expression of other class I genes in erythroid precursors.

Animals↗
PubMed1986

Hydrophobic adsorption chromatography to reduce nonspecific staining by rhodamine-labeled antibodies.

Rhodamine absorbed to protein was removed from rhodamine-conjugated antibody solutions by adsorption to hydrophobic macroporous beads (Bio-Beads SM-2) following gel permeation chromatography. This simple technique eliminated the contaminating free dye more effectively than gel filtration alone, but neither significantly reduced the level of fluorescently conjugated antibody nor altered the latter's binding characteristics. Passage of the fluorescent antibody solution over the SM-2 beads eliminated the high background of nonspecific staining caused by internalization of residual free dye.

Antibodies↗
PubMed1986

Effect of serum on cell-to-cell associations during in vitro development of preimplantation mouse embryos.

We have identified an activity which alters the morphology and developmental timing of post-compaction mouse embryos. A 15-min exposure of 4- and 8-cell mouse embryos to sera containing this activity induced monolayer formation, changing the normal positions of blastomeres at the 16- to 64-cell stages. Recovered embryos form normal blastocysts, based on morphology and in vitro production of trophectoderm and inner cell mass derivatives. These results suggest that under certain circumstances blastomeres remain developmentally labile as late as the sixth or seventh cleavage cycle.

Animals↗