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A theoretical analysis of experimental data is presented in this mini-review on non-random homo- and hetero-associations of cell surface receptors, which can be recruited in the plasma membrane or at the surface of the rough endoplasmic reticulum during the protein synthesis. In the latter case, the likely genetic origin of these supramolecular formations is analyzed, contrasting this concept to the mobility of the cell surface proteins. A model is offered which, on the one hand, allows the mobility in a restricted way even among microdomain-confined receptor proteins through 'swapping partners'. On the other hand, the lack of mixing molecular components of protein clusters will be analyzed, when homo-and hetero-associations are studied through cell fusion experiments. The most frequently studied cell surface patterns have included lipid raft organized HLA class I and II, ICAM-1, tetraspan molecules, IL2 and IL15 and other receptors, as well. On the contrary coated pit-associated transferrin receptors would not mix with the above lipid raft associated receptor patterns, although transferrin receptor would readily oligomerize into homo-associates. The functional consequences of these superstructures are also analyzed. On the 30th anniversary of the Singer-Nicolson fluid mosaic membrane model one has to pay tribute to the authors, because of their deep insight emphasizing also the mosaicism of the membranes in general and that of the plasma membrane, in particular.

Receptors in the plasma membrane of blood cells in general and in that of lymphocytes in particular are supposed to move around in a random walk fashion relatively freely driven by thermal diffusion, as described by the Singer-Nicolson fluid mosaic membrane model. In this article we summarized data and techniques that indicated nonrandom codistribution patterns of receptor superstructures under conditions, where the generation of such molecular colocalizations by the methods themselves were excluded. Application of fluorescence energy transfer in a flow cytometer helped to analyze such codistribution patterns in cell populations. After normalizing energy transfer values for possible differences between labeling ratios of the targeting monoclonal antibodies and using the mean values of energy transfer distribution curves, two-dimensional receptor maps were generated from data obtained in a pair-wise fashion between receptors. Major histocompatibility complex (MHC) class I and II, intercellular adhesion molecule-1 (ICAM-1), TcR-CD3-CD4, tetraspan molecules (CD81, CD82, CD53), and the subunits of the multisubunit IL-2 receptor displayed nonrandom codistribution patterns sometimes with, but very frequently without induction by their ligand. Immunogold-bead "sandwich" labeling analyzed by atomic force microscopy has shown that such receptor "islands" existed also in "receptor-island-groups". This indicated the existence of nonrandom receptor distribution of MHC class I and II molecules also at an elevated hierarchical level. An analysis is given herein concerning a standardized approach. The apparent incompatibility of these supramolecular patterns with the Singer-Nicolson type "free-protein and lipid-mobility paradigm" was resolved by recommending an additional emphasis on the mosaicism of the membrane besides receptor mobility.

The effect of extracellular ATP (ATPex) on the anisotropy of 1-[4-(trimethylamino) phenyl]-6-phenyl-hexa-3,5 triene (TMA-DPH) and 12-anthroyloxi-stearic acid (12-AS) fluorescence was investigated in Balb/C mouse thymocytes and in JY human lymphoblasts. These cells have been shown recently to be sensitive and resistant to ATPex, respectively, in terms of cellular responses. Extracellular ATP (1 mM) induced a time-dependent elevation in the emission anisotropy of both probes (indicating an increased lipid packing density) in the plasma membrane of thymocytes. The maximal effect, at 37 degrees C, was observed between 20 and 60 min after ATPex administration, and followed by a gradual decrease of fluorescence anisotropy at longer times (60-180 min). ATPex did not change membrane fluidity of thymocytes below the phase transition temperature (at 18 degrees C). Oxidized ATP (oATP), a selective antagonist of P2z purinoreceptors, blocked the ATPex-induced decrease in membrane fluidity. Low ATPex concentrations (100-300 microM)--which are known to induce distinct signals (changes in membrane potential and intracellular Ph)--slightly fluidized the plasma membrane of thymocytes. This effect was partially blocked by quinine, a blocker of Ca(2+)-activated K+ channels. Neither 12-AS nor TMA-DPH showed any change in their emission anisotropy upon ATPex-treatment in the plasma membrane of the resistant human JY lymphoblast cells. No other signalling event (membrane potential change, Ca2+ response) is elicited by ATPex in this cell line. These data suggest that the changes in the membrane fluidity are likely consequences of specific, purinoreceptor-mediated signalling events, such as hyper-or depolarization of the plasma membrane or Ca2+ influx. These signals may induce changes in the conformation or lateral organization of membrane proteins, perturbing protein-lipid interactions, as well.

Recently we have found that class I HLA molecules, key elements of the antigen presentation system for CD8 + effector cells, show a clustered lateral distribution (homoassociation) at the surface of activated human T- and B-lymphocytes as well as virus-transformed T- and B-lymphoblasts, in contrast to a disperse distribution on resting human PBLs (Matk6 et al. (1994) J. Immunol. 152, 3353; Bene et al. (1994) Eur. J. Immunol. 24, 2115). Expression of beta2m-free HLA heavy chains and exogenous beta2m have been shown as potential regulation factors of HLA-I clustering, which in turn may affect cytotoxic activity of CD8+ effector cells. Here we report a study on the effect of plasma membrane-modification (by exogenous cholesterol and phosphatidylcholine) on the expression of free HLA heavy chains and beta2m-bound HLA-I molecules on JY human B-lymphoblasts. The modulating effect of these two treatments on the lipid fluidity of cells was demonstrated by fluorescence anisotropy of DPH lipid probe. The lateral clustering (association) of HLA-I molecules was detected by flow cytometric fluorescence resonance energy transfer (FCET) and digital imaging microscopic photobleaching energy transfer (pbFRET) methods, using flourescein-isothiocyanate (FITC) (donor)- and tetramethyl-rhodamine-isothiocyanate (TRITC) (acceptor)-labeled W6/32 or KE2 antibodies directed against intact HLA-I molecules. Cholesterol enrichment of the plasma membrane increased membrane fluidity and reduced the expression of heavy- and light-chain determinants of HLA-I molecules and free heavy chains (FHCs). This was accompanied with a higher degree of HLA-I clustering as shown by the enhanced intermolecular energy transfer efficiency. In contrast, cholesterol depletion resulted in membrane fluidization and increased expression of HLA-I epitopes. Our results suggest that both cholesterol level and lipid structure/fluidity of the plasma membrane in lymphoblastoid cells may also potentially regulate lateral organization and consequently the presentation efficiency of HLA-I molecules.

Major histocompatibility complex (MHC) class I antigens in the plasma membranes of human T (HUT-102B2) and B (JY) lymphoma cells were probed by immunochemical reagents using fluorescence, transmission electron, and scanning force microscopies. Fluorescent labels were attached to monoclonal antibodies W6/32 or KE-2 directed against the heavy chain of HLA class I (A, B, C) and L368 or HB28 against the beta 2-microglobulin light chain. The topological distribution in the nanometer range was studied by photobleaching fluorescence resonance energy transfer (pbFRET) on single cells. A nonrandom codistribution pattern of MHC class I molecules was observed over distances of 2-10 nm. A second, nonrandom, and larger-scale topological organization of the MHC class I antigens was detected by indirect immunogold labeling and imaging by transmission electron microscopy (TEM) and scanning force microscopy (SFM). Although some differences in antigen distribution between the B- and T-cell lines were detected by pbFRET, both cell lines exhibited similar clustering patterns by TEM and SFM. Such defined molecular distributions on the surfaces of cells of the immune system may reflect an underlying specialization of membrane lipid domains and fulfill important functional roles in cell-cell contacts and signal transduction.

The accessibility of cell surface sulfhydryl groups in human peripheral lymphocytes was investigated with 5,5'-dithiobis-(2-nitrobenzoic acid) in the presence and absence of ionophore antibiotics and the monoclonal antibody, OKT-3. Only a few accessible protein thiols have been found on the cells as demonstrated by labeling with a fluorescent non-penetrating thiol-marker, monobromotrimethyl-ammoniobimane and the subsequent gel electrophoretic analysis of the protein pattern. Difference spectrophotometric measurement of thiol-DTNB reaction revealed that ionophores altering the transmembrane potential induced an enhanced cell surface thiol-exposure on the minute time scale. The effect showed a dependence on the external concentration of the cations. The binding of monoclonal antibody, OKT-3, directed against T3 complexes, resulted in a similar, concentration-dependent increase of thiol-accessibility. These data are interpreted as early membrane-effects of ionophores and the specific antibody including changes in the conformational equilibrium or vertical displacements of certain membrane proteins. These events are likely to be coupled to the changes in the transmembrane potential of the lymphocytes.

Intracellular free calcium concentration [( Ca2+]i) of human peripheral blood lymphocytes was determined by fluorescence spectroscopic measurements with quin2 in patients with different types of muscular dystrophy and in controls. The [Ca2+]i level in lymphocytes showed a significant increase in adult type (facioscapulohumeral and limb-girdle) muscular dystrophies, while it showed a decrease in Duchenne dystrophy as compared to the values of age- and sex-matched controls. The data obtained suggest an alteration in the effectiveness of the calcium pump in lymphocytes and may represent a sign of generalized membrane damage in these hereditary muscle diseases.