Structural dynamics of the Ca2(+)-ATPase of sarcoplasmic reticulum. Temperature profiles of fluorescence polarization and intramolecular energy transfer.

The temperature dependence of fluorescence polarization and Förster-type resonance energy transfer (FRET) was analyzed in the Ca2(+)-ATPase of sarcoplasmic reticulum using protein tryptophan and site-specific fluorescence indicators such as 5-[2-[iodoacetyl)amino)ethyl]aminonaphthalene-1-sulfonic acid (IAEDANS), fluorescein 5'-isothiocyanate (FITC), 2',3'-O-(2,4,3-trinitrophenyl)adenosine monophosphate (TNP-AMP) or lanthanides (Pr3+, Nd3+) as probes. The normalized energy transfer efficiency between AEDANS bound at cysteine-670 and -674 and FITC bound at lysine-515 increases with increasing temperature in the range of 10-37 degrees C, indicating the existence of a relatively flexible structure in the region of the ATPase molecule that links the AEDANS to the FITC site. These observations are consistent with the theory of Somogyi, Matko, Papp, Hevessy, Welch and Damjanovich (Biochemistry 23 (1984) 3403-3411) that thermally induced structural fluctuations increase the energy transfer. Structural fluctuations were also evident in the energy transfer between FITC linked to the nucleotide-binding domain and Nd3+ bound at the putative Ca2+ sites. By contrast the normalized energy transfer efficiency between AEDANS and Pr3+ was relatively insensitive to temperature, suggesting that the region between cysteine-670 and the putative Ca2+ site monitored by the AEDANS-Pr3+ pair is relatively rigid. A combination of the energy transfer data with the structural information derived from analysis of Ca2(+)-ATPase crystals yields a structural model, in which the location of the AEDANS-, FITC- and Ca2+ sites are tentatively identified.

Intracellular free calcium concentration in lymphocytes of patients with muscular dystrophies.

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.

Effect of cyclosporin A on the membrane potential and Ca2+ level of human lymphoid cell lines and mouse thymocytes.

The effect of the immunosuppressive cyclosporin A (CsA) on the cytosolic free Ca2+ concentration ([Ca2+]i) and membrane potential of human B and T lymphoblastoid cells and mouse thymocytes was studied in order to reveal some features of the early stage of drug-cell interaction. Cytosolic free Ca2+ concentration of the cells was measured by spectrofluorimetry using indo-1 and quin2 fluorescent calcium indicators. Membrane potential was monitored in a flow cytometer with oxonol dye. CsA applied at 2-20 micrograms/ml final concentrations caused a dose-dependent, rapid, transient rise of [Ca2+]i in all cell types. This effect could be blocked by chelating the extracellular Ca2+ with EGTA but was not sensitive to Ca2+ channel blockers verapamil and nifedipine or K+ channel blocker 4-aminopyridine. A possible explanation for the calcium mobilizing effect of CsA is an ionophore-like mode of action at the cell membrane level. Besides directly interfering with mitogenic signals, the elevation of [Ca2+]i could be responsible for an initial hyperpolarization observed in CsA-treated T lymphocytes. This hyperpolarization, however, was not detectable in B lymphoblastoid cells. A further difference between B and T cells was the diverse pattern of depolarization following CsA treatment. This variance in the behaviour of T and B lymphocytes and the diversity of membrane transport systems in its background could account for the different final outcome of the drug-cell interaction.

The effect of transmembrane potential on the dynamic behavior of cell membranes.

The relationship between transmembrane potential and lipid dynamics in the cytoplasmic membrane of mouse thymus cells has been investigated. Changes of transmembrane potential was followed by measuring the fluorescence emission of the anionic dye, bis-(1,3-dibutylbarbiturate)trimethine oxonol (diBa-C4-(3)). Assessment of lipid fluidity was carried out applying three fluorescent lipid probes, 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), 12-(9-anthroyloxy)stearic acid (12-AS) and 1,6-diphenyl-1,3,5-hexatriene (DPH) used to monitor different structural regions of the bilayer. The fluorescence anisotropy of these probes was measured as a function of temperature at two values of transmembrane potential. In the case of DPH it proved to depend on the membrane potential in the higher temperature range (above 28 degrees C), while no such a dependence could be observed for DPH below this temperature range and for TMA-DPH and 12-AS in between 20 and 37 degrees C. These data suggest that changes in transmembrane potential are accompanied with some local alteration in membrane lipid dynamics and/or structure.

Segmental mobility in glycogen phosphorylase b.

The dynamics and structuredness of the pyridoxal 5'-phosphate-binding region in glycogen phosphorylase b (EC 2.4.1.1) has been investigated with different techniques of fluorescence spectroscopy. Fluorescence polarization data of the thermal Perrin plot indicate some mobility in the cofactor binding site, while the isothermic measurements (at 20 degrees C, in high-viscosity solvents) demonstrate that the mobile unit carrying the emission oscillator is practically insensitive to the external viscosity. Characteristics of the thermal Perrin plots obtained for both native and reduced phosphorylase b can be interpreted either as a freely moving cofactor in a medium of high viscosity (0.3 P) or as the motion of a unit larger than a lysine-bonded pyridoxal 5'-phosphate in a medium with the viscosity of water. Data for acrylamide quenching and time-resolved fluorescence measurements suggest that the latter interpretation should valid. These data also suggest a tightly packed microenvironment around the pyridoxal moiety.

A monoclonal antibody directed against human von Willebrand factor induces type 2B-like alterations.

We have previously described a monoclonal antibody (mAb), 1C1E7, against von Willebrand factor (VWF), that increases ristocetin-induced platelet aggregation (RIPA) and induces a preferential binding of the high-molecular-weight multimers of VWF to platelet GPIb. Further investigations using a rotational viscometer at a shear rate of 4000 s(-1) could now demonstrate that shear-induced platelet aggregation (SIPA) is significantly increased with 1C1E7 and that this could be completely inhibited by the anti-GPIb mAb 6D1. In contrast, platelet adhesion to a collagen surface at a shear rate of 2600 s(-1), using a rectangular perfusion chamber, was significantly inhibited in the presence of 1C1E7. When citrated whole blood was incubated with 1C1E7, a spontaneous binding of VWF to the platelet GPIb could be demonstrated by flow cytometric analysis. Parallel to this, a decrease of the highest molecular weight multimers of VWF in the plasma was found. Platelets with bound VWF on their surface were able to form macroaggregates but were no longer able to adhere. These phenomena are very similar to the alterations described in von Willebrand's disease type 2B. The epitope of this mAb could be localized to the N-terminal part of the subunit; therefore a distant conformational change in the A1 domain of VWF is suggested.

Apoptosis of murine thymocytes induced by extracellular ATP is dose- and cytosolic pH-dependent.

Thymocytes from young Balb/C mice responded to low extracellular ATP (ATPec) doses (< or = 0.3 mM) with a rapid intracellular acidification (mean pH: ca. 0.3 pH unit) that was inhibited by the Ca2+ channel blocker verapamil, or by suramin (50 microM) and TNP-ATP (40 microM), potent P2x (and P2y) purinoreceptor antagonists. ATPec also triggered a remarkable DNA fragmentation and cell shrinkage detectable only at these low doses. DNA fragmentation gradually disappears with increasing [ATPec] above 0.5 mM, with a concomitant dominance of cytosolic alkalinization of the cells. Suramin and TNP-ATP also blocked the ATPec-triggered DNA fragmentation efficiently. oATP, inhibitor of P2z nonspecific ATP-gated membrane pores, and 2 mM extracellular Mg2+ did not influence either the cytosolic acidification or the DNA fragmentation, but almost completely abolished the intracellular alkalinization characteristic of P2z receptor activation at high ATPec doses. Antagonist-sensitivity of the ATPec-induced membrane potential responses indicates that hyperpolarization is associated with intracellular acidification, while rapid depolarization is linked to alkalinization. These data together indicate that the Ca2+-dependent hyperpolarization and cytosolic acidification triggered by low ATPec doses are essential early signals in apoptosis of murine thymocytes and are likely mediated by P2x1 type ATP-gated ion channels. Subset specificity of the early purinergic signals suggests that the double positive thymocytes are most sensitive to ATPec showing both P2z and P2x receptor activation characteristics, the double negative thymocytes preferentially show P2z-type, while single positive (CD4- CD8+ or CD4+ CD8-) thymocytes respond mostly by weaker P2x-type changes, indicating that ATPec, similarly to adenosine may serve as a potential regulator of cell death and differentiation in the thymus.

A photobleaching energy transfer analysis of CD8/MHC-I and LFA-1/ICAM-1 interactions in CTL-target cell conjugates.

The photobleaching energy transfer (pbFRET) technique is a fluorescence method to measure proximity relationships between molecules, especially cell surface proteins, labeled with fluorophore-conjugated monoclonal antibodies, on a pixel-by-pixel base using digital imaging microscopy. This technique enables analysis of inter- and intramolecular proximities at cell surfaces at physiological conditions. We have developed a pbFRET approach to measure intercellular proximities in order to access spatial organization of interacting proteins in the contact region of two 'communicating' cells. Two examples, as possible application areas of this approach, are presented here: interaction between CD8 and MHC-I molecules in point contacts and interaction between LFA-1 and ICAM-1 molecules in focal contacts of CTL-target conjugates. The geometry of these protein contacts based on our resonance energy transfer (RET) data is consistent with the observed blocking effects of monoclonal antibodies (directed against the interacting proteins) on the cytolytic activity of CTLs and suggest a critical role for CD8beta-subunit in signal transmission in peripheral T-lymphocytes.

Modification of membrane cholesterol level affects expression and clustering of class I HLA molecules at the surface of JY human lymphoblasts.

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.

Effect of TSH and anti-TSH receptor antibodies on the plasma membrane potential of polymorphonuclear granulocytes.

Effects of thyrotropin hormone (TSH) and anti-TSH receptor antibodies on the plasma membrane potential of polymorphonuclear granulocytes (PMN) were analyzed by means of flow cytometry. Both TSH and the autoantibody caused a rapid, dose-dependent hyperpolarization of the plasma membrane of PMNs. TSH was also able to mask (revert) the depolarizing effect of a chemotactic peptide, fMLP, on PMNs. No detectable rise in the cytosolic free calcium level accompanied the observed hyperpolarization. Quinine, a blocker of Ca(2+)-activated and voltage-gated K+ channels did not affect the hyperpolarization by TSH and antibodies. Decreasing the [K+] gradient across the plasma membrane by valinomycin, however, blocked the hyperpolarizing effect. Peptide362-376 (derived from the extracellular domain of TSH receptor) also blocked the hyperpolarization induced by both TSH and anti-TSHR antibodies. These data suggest that the observed hyperpolarization is a specific, receptor-mediated early signal during interaction of PMNs with TSH or anti-TSHR antibodies.

Changes in membrane potential of target cells promotes cytotoxic activity of effector T lymphocytes.

The effector function of CD8+ lymphocytes depends on recognition by the TcR-CD3 complex of an oligopeptide presented by an MHC class I molecule on target cells. Recently it has been shown that MHC class I molecules change their conformation upon depolarization of human B lymphoblastoid JY cells. We studied here the effects of changes in membrane potential of target cells on the function of cytotoxic T lymphocytes (CTL). Selective alterations of plasma membrane potential of JY target cells were achieved by treatments with specific ionophore molecules as well as with Na(+)-K(+)-ATPase inhibitor, while the cytotoxic lymphocytes were not influenced. The plasma membrane was depolarized by gramicidin D and ouabain, while hyperpolarization was induced by valinomycin treatment. Alterations of the resting membrane potential of target cells in both direction resulted in an enhanced cytotoxic activity. The observed changes in cytolytic activities of cytotoxic T effectors may have a more general biological significance, namely apoptotic cells become depolarized after a given time, moreover neoplastic and virus infected cells also frequently show decreased membrane potential. A more efficient recognition of these cells by CTL is supposed to enhance the efficiency of their elimination, as well.

Ion-channel activities regulate transmembrane signaling in thymocyte apoptosis and T-cell activation.

Several examples have shown that plasma membrane ion channels (e.g., Ca2+ and K+ channels) make an important contribution to lymphocyte activation or thymocyte apoptosis. Here we report on the importance of these ion channels in the sensitivity or resistance of lymphoid cells to extracellular ATP-induced apoptosis. Thymocytes of Balb/c mice responded to extracellular ATP (ATPex) sensitively, with an immediate increase in the intracellular calcium level and later with an increased membrane permeability to low MW markers. Mature (medullary) thymocytes showed a higher sensitivity than did cortical thymocytes. Three human lymphoma cell lines, including SUPT13, a cell line reported to be sensitive to TcR/CD3 activation-induced apoptosis, showed a high resistance to ATPex action. These observations suggest that maturation/differentiation state-dependent activity or disappearance of early ATP-receptor operated signaling systems (including ion channels) are critical for the cells in developing towards apoptosis. Using the patch-clamp technique we demonstrated that bretylium tosylate (a particular K(+)-channel blocker) known as inhibitor of T-lymphocyte proliferation also influences the single-channel properties of voltage-gated K+ channels through depressing whole-cell K+ currents. This finding is yet another example underlying the importance of K+ channel activity in T-lymphocyte proliferation.

Inhibition of IgE-mediated triggering of mast cells by complement-derived peptides interacting with the Fc epsilon RI.

Mucosal type mast cells, in contrast to the serosal type ones, do not respond to cationic agents, or to the complement-derived peptides C3a and C5a. Earlier we have found that while C3a does not activate the rat mucosal type mast cells (line RBL-2H3), it strongly inhibits the IgE-mediated triggering of these cells, by interfering with the Fc epsilon RI-initiated signaling pathway. In the present study we further investigated the mechanism of this process. It is shown, that C3a interacts with the beta-chain of the Fc epsilon RI complex. Binding of the complement peptide to the cells apparently causes a decrease in the proximity of the IgE-binding Fc epsilon RI. Investigating certain sequences of C3a we found that the inhibition is caused by the C-terminal sequences of the complement-peptide, ranging from positions 56 to 77 and also by a shorter sequence, ranging from positions 56 to 64. The inhibitory effect of these peptides was observed both in the case of RBL-2H3 cells and mouse bone marrow derived mast cells.

Hydrocarbon uptake by Streptomyces.

Oligocarbophilic Streptomyces strains capable of hydrocarbon uptake and utilization were isolated from the polluted desert of Kuwait and used in this study. Transmission electron-microscopy of hyphae revealed that they become enriched with large less electron dense areas in the cytoplasm, when biomass samples were incubated with alkanes. The Streptomyces isolate could utilize n-hexadecane as sole carbon and energy source and their fatty acid content showed an increase in the fatty acids with chain length equivalent to those of the alkane substrates. Fluorescence measurements of diphenylhexatriene dissolved in the representative alkane, n-hexadecane, showed that the kinetics of hydrocarbon uptake are quite different in hydrocarbon-utilizer compared with non-utilizer Streptomyces strain. Microviscosity of the cellular membrane of the utilizer strain was also different from that of the non-utilizer control strain Streptomyces griseus after incubation in the presence of n-hexadecane. Very likely the hydrocarbon utilizer transported these compounds more efficiently across their membranes and accumulated them as inclusions in the cytoplasm.

Two-dimensional receptor patterns in the plasma membrane of cells. A critical evaluation of their identification, origin and information content.

A concise review is presented on the nature, possible origin and functional significance of cell surface receptor patterns in the plasma membrane of lymphoid cells. A special emphasize has been laid on the available methodological approaches, their individual virtues and sources of errors. Fluorescence energy transfer is one of the oldest available means for studying non-randomized co-distribution patterns of cell surface receptors. A detailed and critical description is given on the generation of two-dimensional cell surface receptor patterns based on pair-wise energy transfer measurements. A second hierarchical-level of receptor clusters have been described by electron and scanning force microscopies after immuno-gold-labeling of distinct receptor kinds. The origin of these receptor islands at a nanometer scale and island groups at a higher hierarchical (mum) level, has been explained mostly by detergent insoluble glycolipid-enriched complexes known as rafts, or detergent insoluble glycolipids (DIGs). These rafts are the most-likely organizational forces behind at least some kind of receptor clustering [K. Simons et al., Nature 387 (1997) 569]. These models, which have great significance in trans-membrane signaling and intra-membrane and intracellular trafficking, are accentuating the necessity to revisit the Singer-Nicolson fluid mosaic membrane model and substitute the free protein diffusion with a restricted diffusion concept [S.J. Singer et al., Science 175 (1972) 720].

A continuous assay method for glycogen synthesis from UDPglucose.

A light scattering assay was developed for continuous monitoring of glycogen synthesis from UDPglucose. A close linear correlation between light-scattering signal and the amount of radioactively labelled glucose incorporated into glycogen suggests that the scattering signal is determined by the average molecular weight of the glycogen molecules. The method has the advantage of continuity, and it is suitable to follow the time course of the reaction at different conditions and to determine the initial rate of glycogen synthesis in a relatively simple and fast way. The possibility of measurement of glycogen synthase activity in muscle extracts was also tested.

Fluorescent lipid probes 12-AS and TMA-DPH report on selective, purinergically induced fluidity changes in plasma membranes of lymphoid cells.

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.

Mapping microscopic order in plant and mammalian cells and tissues: novel differential polarization attachment for new generation confocal microscopes (DP-LSM).

Elucidation of the molecular architecture of complex, highly organized molecular macro-assemblies is an important, basic task for biology. Differential polarization (DP) measurements, such as linear (LD) and circular dichroism (CD) or the anisotropy of the fluorescence emission (r), which can be carried out in a dichrograph or spectrofluorimeter, respectively, carry unique, spatially averaged information about the molecular organization of the sample. For inhomogeneous samples-e.g. cells and tissues-measurements on macroscopic scale are not satisfactory, and in some cases not feasible, thus microscopic techniques must be applied. The microscopic DP-imaging technique, when based on confocal laser scanning microscope (LSM), allows the pixel by pixel mapping of anisotropy of a sample in 2D and 3D. The first DP-LSM configuration, which, in fluorescence mode, allowed confocal imaging of different DP quantities in real-time, without interfering with the 'conventional' imaging, was built on a Zeiss LSM410. It was demonstrated to be capable of determining non-confocally the linear birefringence (LB) or LD of a sample and, confocally, its FDLD (fluorescence detected LD), the degree of polarization (P) and the anisotropy of the fluorescence emission (r), following polarized and non-polarized excitation, respectively (Steinbach et al 2009 Acta Histochem.111 316-25). This DP-LSM configuration, however, cannot simply be adopted to new generation microscopes with considerably more compact structures. As shown here, for an Olympus FV500, we designed an easy-to-install DP attachment to determine LB, LD, FDLD and r, in new-generation confocal microscopes, which, in principle, can be complemented with a P-imaging unit, but specifically to the brand and type of LSM.