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1.
Proc Natl Acad Sci U S A ; 101(5): 1285-90, 2004 Feb 03.
Article in English | MEDLINE | ID: mdl-14745040

ABSTRACT

Membrane proteins of cytotoxic T cells specifically reorganize to form an immunological synapse (IS) on interaction with their specific target. In this paper, we investigated the redistribution of Kv1.3 channels, which are the dominant voltage-gated potassium channels, in the plasma membrane of allogen-activated human cytotoxic T lymphocytes (CTLs) on interacting with their specific target cells. Kv1.3 channels bearing a FLAG epitope were expressed in the CTLs and the cell-surface distribution of fluorescently labeled ion channels was determined from confocal laser-scanning microscopy images. FLAG epitope-tagged Kv1.3 channels showed a patchy distribution in CTLs not engaged with target cells, whereas the channels were accumulated in the IS formed between CTLs and specific target lymphocytes. Localization of Kv1.3 channels in the IS might open an unrevealed possibility in the regulation of ion channel activity by signaling molecules accumulated in the IS.


Subject(s)
Potassium Channels, Voltage-Gated , Potassium Channels/metabolism , Synapses/metabolism , T-Lymphocytes, Cytotoxic/metabolism , Cytotoxicity, Immunologic , HLA-A2 Antigen/physiology , Humans , Kv1.3 Potassium Channel , Lymphocyte Activation , Membrane Microdomains/metabolism , Oligopeptides , Peptides/analysis , T-Lymphocytes, Cytotoxic/immunology
2.
Proc Natl Acad Sci U S A ; 100(14): 8053-8, 2003 Jul 08.
Article in English | MEDLINE | ID: mdl-12832616

ABSTRACT

The fluid mosaic membrane model proved to be a very useful hypothesis in explaining many, but certainly not all, phenomena taking place in biological membranes. New experimental data show that the compartmentalization of membrane components can be as important for effective signal transduction as is the fluidity of the membrane. In this work, we pay tribute to the Singer-Nicolson model, which is near its 30th anniversary, honoring its basic features, "mosaicism" and "diffusion," which predict the interspersion of proteins and lipids and their ability to undergo dynamic rearrangement via Brownian motion. At the same time, modifications based on quantitative data are proposed, highlighting the often genetically predestined, yet flexible, multilevel structure implementing a vast complexity of cellular functions. This new "dynamically structured mosaic model" bears the following characteristics: emphasis is shifted from fluidity to mosaicism, which, in our interpretation, means nonrandom codistribution patterns of specific kinds of membrane proteins forming small-scale clusters at the molecular level and large-scale clusters (groups of clusters, islands) at the submicrometer level. The cohesive forces, which maintain these assemblies as principal elements of the membranes, originate from within a microdomain structure, where lipid-lipid, protein-protein, and protein-lipid interactions, as well as sub- and supramembrane (cytoskeletal, extracellular matrix, other cell) effectors, many of them genetically predestined, play equally important roles. The concept of fluidity in the original model now is interpreted as permissiveness of the architecture to continuous, dynamic restructuring of the molecular- and higher-level clusters according to the needs of the cell and as evoked by the environment.


Subject(s)
Cell Membrane/physiology , Membrane Fluidity , Models, Biological , Animals , Cell Membrane/chemistry , Chemical Phenomena , Chemistry, Physical , Diffusion , Fluorescence Resonance Energy Transfer , Lipid Bilayers , Membrane Lipids/physiology , Membrane Microdomains/physiology , Membrane Proteins/physiology , Microscopy, Electron , Signal Transduction
3.
Proc Natl Acad Sci U S A ; 100(5): 2592-7, 2003 Mar 04.
Article in English | MEDLINE | ID: mdl-12604782

ABSTRACT

Distribution and lateral organization of Kv1.3 potassium channels and CD3 molecules were studied by using electron microscopy, confocal laser scanning microscopy, and fluorescence resonance energy transfer. Immunogold labeling and electron microscopy showed that the distribution of FLAG epitope-tagged Kv1.3 channels (Kv1.3/FLAG) significantly differs from the stochastic Poisson distribution in the plasma membrane of human T lymphoma cells. Confocal laser scanning microscopy images showed that Kv1.3/FLAG channels and CD3 molecules accumulated in largely overlapping membrane areas. The numerical analysis of crosscorrelation of the spatial intensity distributions yielded a high correlation coefficient (C = 0.64). A different hierarchical level of molecular proximity between Kv1.3/FLAG and CD3 proteins was reported by a high fluorescence resonance energy transfer efficiency (E = 51%). These findings implicate that reciprocal regulation of ion-channel activity, membrane potential, and the function of receptor complexes may contribute to the proper functioning of the immunological synapse.


Subject(s)
CD3 Complex/biosynthesis , Cell Membrane/metabolism , Potassium Channels, Voltage-Gated , Potassium Channels/biosynthesis , Potassium Channels/chemistry , T-Lymphocytes/metabolism , Animals , Cell Membrane/immunology , Electrophysiology , Epitopes , Fluorescence Resonance Energy Transfer , Humans , Immunohistochemistry , Jurkat Cells , Kv1.3 Potassium Channel , Mice , Microscopy, Confocal , Microscopy, Electron , Models, Statistical , Transfection
4.
J Photochem Photobiol B ; 65(1): 47-58, 2001 Dec 01.
Article in English | MEDLINE | ID: mdl-11748005

ABSTRACT

Glycoprotein IIb/IIIa is a heterodimer of glycoproteins IIb and IIIa which serves as the inducible receptor for fibrinogen and other adhesive proteins at the surface of platelets. Although a model of the quaternary structure of the GPIIb/IIIa molecule has been constructed in solution by Calvete et al. [Biochem. J. 282 (1992) 523], a corresponding model at the surface of intact platelets is still missing. In the present work conformation and lateral distribution of the GPIIb/IIIa heterodimer were studied at a nanometer resolution on the surface of resting human platelets under physiological conditions. The experiments were based on dual wavelength flow cytometric detection of fluorescence resonance energy transfer and application of a panel of monoclonal antibodies raised against well described binding sites. Monodisperse distribution of the GPIIb/IIIa heterodimer has been observed and a detailed three-dimensional proximity map of antibody binding sites was constructed on the platelet membrane, under physiological conditions, for the first time. Our data support the view that the GPIIb subunit is in a bent conformation. A detailed analysis of the K(d)-values and the number of binding sites for a set of monoclonal antibodies was also carried out giving supplementary data for the topology of the binding sites. Our results provide a refinement of the membrane-topology of the GPIIb/IIIa heterodimer.


Subject(s)
Blood Platelets/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Animals , Antibodies, Monoclonal/metabolism , Dimerization , Energy Transfer , Flow Cytometry , Humans , Mice
5.
J Cell Sci ; 114(Pt 22): 4063-71, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11739638

ABSTRACT

The existence of small- and large-scale membrane protein clusters, containing dimers, oligomers and hundreds of proteins, respectively, has become widely accepted. However, it is largely unknown whether the internal structure of these formations is dynamic or static. Cell fusion was used to perturb the distribution of existing membrane protein clusters, and to investigate their mobility and associations. Scanning near-field optical microscopy, confocal and electron microscopy were applied to detect the exchange of proteins between large-scale protein clusters, whereas photobleaching fluorescence energy transfer was used to image the redistribution of existing small-scale membrane protein clusters. Large-scale clusters of major histocompatibility complex (MHC)-I exchanged proteins with each other and with MHC-II clusters. Similarly to MHC-I, large-scale MHC-II clusters were also dynamic. Exchange of components between small-scale protein clusters was not universal: intermixing did not take place in the case of MHC-II homoclusters; however, it was observed for homoclusters of MHC-I and for heteroclusters of MHC-I and MHC-II. These processes required a fluid state of the plasma membrane, and did not depend on endocytosis-mediated recycling of proteins. The redistribution of large-scale MHC-I clusters precedes the intermixing of small-scale clusters of MHC-I indicating a hierarchy in protein association. Investigation of a set of other proteins (alpha subunit of the interleukin 2 receptor, CD48 and transferrin receptor) suggested that a large-scale protein cluster usually exchanges components with the same type of clusters. These results offer new insight into processes requiring time-dependent changes in membrane protein interactions.


Subject(s)
Cell Fusion , Histocompatibility Antigens Class II/metabolism , Histocompatibility Antigens Class I/metabolism , Receptor Aggregation/physiology , Receptors, Cell Surface/metabolism , Cell Line , Cell Membrane/chemistry , Cell Membrane/metabolism , Energy Transfer , Fluorescent Dyes/metabolism , Gold Colloid/metabolism , Humans , Membrane Microdomains , Microscopy , Microscopy, Fluorescence/methods , Receptors, Interleukin-2
6.
Biophys Chem ; 82(2-3): 99-108, 1999 Dec 13.
Article in English | MEDLINE | ID: mdl-17030342

ABSTRACT

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].

7.
Cytometry ; 33(2): 225-33, 1998 Oct 01.
Article in English | MEDLINE | ID: mdl-9773884

ABSTRACT

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.


Subject(s)
Flow Cytometry , Lymphocytes/chemistry , Microscopy, Atomic Force , Microscopy, Electron , Receptors, Cell Surface/analysis , Antibodies, Monoclonal/immunology , Antigens, CD/analysis , Diffusion , Energy Transfer , Fluorescence , Fluorescent Dyes , HLA Antigens/analysis , Humans , Immunoglobulin Fab Fragments/immunology , Immunohistochemistry , Intercellular Adhesion Molecule-1/analysis , Macromolecular Substances , Membrane Fluidity , Microspheres , Models, Biological , Motion , Receptor-CD3 Complex, Antigen, T-Cell/analysis , Receptors, Interleukin-2/analysis , Tumor Cells, Cultured
8.
Cytometry ; 34(4): 159-79, 1998 Aug 15.
Article in English | MEDLINE | ID: mdl-9725457

ABSTRACT

Fluorescence resonance energy transfer (FRET) phenomenon has been applied to a variety of scientific challenges in the past. The potential utility of this biophysical tool will be revisited in the 21st century. The rapid digital signal processing in conjunction with personal computers and the wide use of multicolor laser technology in clinical flow cytometry opened an opportunity for multiplexed assay systems. The concept is very simple. Color-coded microspheres are used as solid-phase matrix for the detection of fluorescent labeled molecules. It is the homogeneous assay methodology in which solid-phase particles behave similarly to the dynamics of a liquid environment. This approach offers a rapid cost-effective technology that harnesses a wide variety of fluorochromes and lasers. With this microsphere technology, the potential applications for clinical flow cytometry in the future are enormous. This new approach of well-established clinically proven methods sets the stage to briefly review the theoretical and practical aspects of FRET technology. The review shows various applications of FRET in research and clinical laboratories. Combination of FRET with monoclonal antibodies resulted in a boom of structural analysis of proteins in solutions and also in biological membranes. Cell surface mapping of cluster of differentiation molecules on immunocompetent cells has gained more and more interest in the last decade. Several examples for biological applications are discussed in detail. FRET can also be used to improve the spectral characteristics of fluorescent dyes and dye combinations, such as the tandem dyes in flow and image cytometry and the FRET primers in DNA sequencing and polymerase chain reactions. The advantages and disadvantages of donor-acceptor dye combinations are evaluated. In addition, the sensitivity of FRET provides the basis for establishing fast, robust, and accurate enzyme assays and immunoassays. Benefits and limitations of FRET-based assays are thoroughly scrutinized. At the end of the paper we review the future of FRET methodology.


Subject(s)
Spectrometry, Fluorescence , Diagnostic Tests, Routine , Energy Transfer , Humans , Image Cytometry , Research
9.
Proc Natl Acad Sci U S A ; 94(14): 7269-74, 1997 Jul 08.
Article in English | MEDLINE | ID: mdl-9207080

ABSTRACT

Major histocompatibility complex (MHC) class II molecules displayed clustered patterns at the surfaces of T (HUT-102B2) and B (JY) lymphoma cells characterized by interreceptor distances in the micrometer range as detected by scanning force microscopy of immunogold-labeled antigens. Electron microscopy revealed that a fraction of the MHC class II molecules was also heteroclustered with MHC class I antigens at the same hierarchical level as described by the scanning force microscopy data, after specifically and sequentially labeling the antigens with 30- and 15-nm immunogold beads. On JY cells the estimated fraction of co-clustered HLA II was 0.61, whereas that of the HLA I was 0.24. Clusterization of the antigens was detected by the deviation of their spatial distribution from the Poissonian distribution representing the random case. Fluorescence resonance energy transfer measurements also confirmed partial co-clustering of the HLA class I and II molecules at another hierarchical level characterized by the 2- to 10-nm Förster distance range and providing fine details of the molecular organization of receptors. The larger-scale topological organization of the MHC class I and II antigens may reflect underlying membrane lipid domains and may fulfill significant functions in cell-to-cell contacts and signal transduction.


Subject(s)
Cell Membrane/immunology , Histocompatibility Antigens Class II/analysis , Histocompatibility Antigens Class I/analysis , Lymphocytes/immunology , Cell Membrane/ultrastructure , Histocompatibility Antigens Class I/ultrastructure , Histocompatibility Antigens Class II/ultrastructure , Humans , Lymphocytes/ultrastructure , Microscopy, Electron , Tumor Cells, Cultured
10.
Cytometry ; 27(4): 353-7, 1997 Apr 01.
Article in English | MEDLINE | ID: mdl-9098627

ABSTRACT

The nature of charge distributions in membrane-bound macromolecular structures renders them susceptible to interaction with transmembrane potential fields. As a result, conformational changes in such species may be expected to occur when this potential is altered. We have detected reversible conformational change in the major histocompatibility complex (MHC) class I antigen in the plasma membrane of human JY cells, as monitored by flow-cytometric resonance energy-transfer, upon reduction of the transmembrane potential (depolarization). This change increased the intramolecular energy-transfer efficiency between fluorescent donor- and acceptor-labeled monoclonal antibodies directed, respectively, to epitopes on the light (beta 2-microglobulin) and the heavy chains of the MHC class I antigen. Repolarization of the depolarized samples restored the energy-transfer efficiency to the original values measured before depolarization. Depolarization caused similar relative changes in fluorescence resonance energy-transfer efficiency when Fab fragments were used for labeling MHC class I complex, suggesting that the observed phenomenon is not restricted to whole monoclonal antibodies.


Subject(s)
Histocompatibility Antigens Class I/chemistry , beta 2-Microglobulin/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antigen Presentation/immunology , Antigen Presentation/physiology , B-Lymphocytes/cytology , B-Lymphocytes/immunology , B-Lymphocytes/physiology , Cell Membrane/physiology , Energy Transfer/physiology , Enzyme Activation , Flow Cytometry/methods , Fluorescein-5-isothiocyanate , Fluorescent Dyes , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/physiology , Humans , Membrane Potentials , Patch-Clamp Techniques , Protein Conformation , Rhodamines , Sodium-Potassium-Exchanging ATPase/metabolism , Surface Properties , beta 2-Microglobulin/immunology , beta 2-Microglobulin/physiology
11.
Cytometry ; 24(1): 64-73, 1996 May 01.
Article in English | MEDLINE | ID: mdl-8723904

ABSTRACT

Calcium signaling in non-excitable cells is the consequence of calcium release from intracellular stores, at times followed by entry of extracellular calcium through the plasma membrane. To study whether entry of calcium depends upon the level of saturation of intracellular stores, we measured calcium channel opening in the plasma membrane of single confluent A172 glioblastoma cells stimulated with platelet derived growth factor (PDGF) and/or bradykinin (BK). We monitored the entry of extracellular calcium by measuring manganese quenching of Indo-1 fluorescence. PDGF raised intracellular calcium concentration ([Ca2+]i) after a dose-dependent delay (tdel) and then opened calcium channels after a dose-independent delay (tch). At higher doses (> 3 nM), BK increased [Ca2+]i after a tdel approximately 0 s, and tch decreased inversely with both dose and peak [Ca2+]i. Experiments with thapsigargin (TG), BK, and PDGF indicated that BK and PDGF share intracellular Ca2+ pools that are sensitive to TG. When these stores were depleted by treatment with BK and intracellular BAPTA, tdel did not change, but tch fell to almost 0 s in PDGF stimulated cells, indicating that depletion of calcium stores affects calcium channel opening in the plasma membrane. Our data support the capacitative model for calcium channel opening and the steady-state model describing quantal Ca2+ release from intracellular stores.


Subject(s)
Calcium/metabolism , Neuroglia/metabolism , Platelet-Derived Growth Factor/pharmacology , Signal Transduction , Becaplermin , Bradykinin/pharmacology , Calcium Channel Blockers/pharmacology , Calcium-Transporting ATPases/antagonists & inhibitors , Dose-Response Relationship, Drug , Glioblastoma , Humans , Lanthanum/pharmacology , Neuroglia/drug effects , Proto-Oncogene Proteins c-sis , Terpenes/pharmacology , Thapsigargin , Tumor Cells, Cultured
12.
J Mol Recognit ; 8(4): 237-46, 1995.
Article in English | MEDLINE | ID: mdl-8588941

ABSTRACT

Molecular recognition processes between cell surface elements are discussed with special reference to cell surface pattern formation of membrane-bound integral proteins. The existence, as detected by flow cytometric resonance energy transfer (Appendix), and significance of cell surface patterns involving the interleukin-2 receptor, the T-cell receptor-CD3 system, the intercellular adhesion molecule ICAM-1, and the major histocompatibility complex class I and class II molecules in the plasma membrane of lymphocytes are described. The modulation of antigen presentation by transmembrane potential changes is discussed, and a general role of transmembrane potential changes, and therefore of ion channel activities, adduced as one of the major regulatory mechanisms of cell-cell communication. A general role in the mediation and regulation of intercellular interactions is suggested for cell-surface macromolecular patterns. The dynamic pattern of protein and lipid molecules in the plasma membrane is generated by the genetic code, but has a remarkable flexibility and may be one of the major instruments of accommodation and recognition processes at the cellular level.


Subject(s)
Antigens, Surface/physiology , Cell Communication , Membrane Proteins/physiology , Animals , CD3 Complex , Energy Transfer , Flow Cytometry , Humans , Intercellular Adhesion Molecule-1/physiology , Major Histocompatibility Complex , Membrane Potentials , Receptors, Antigen, T-Cell/physiology , Receptors, Cell Surface , Receptors, Interleukin-2/physiology
13.
Immunol Lett ; 44(2-3): 203-8, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7797252

ABSTRACT

The topological relationship of transferrin receptor (TfR) has been studied relative to the heavy and light chains of the HLA class I molecules, class II molecules, interleukin-2 receptor alpha-chain and ICAM-1 molecule in the plasma membrane of HUT-102B2 T and JY B lymphoblastoid cell lines using the flow cytometric fluorescence energy transfer technique (FCET). The effect of different growing conditions (logarithmic and plateau phases) on the relative surface density of the receptors and the lateral organization of the TfR was also studied. The TfR showed a high degree of self-association on the surface of both cell lines regardless of the growing phase. TfR was in close vicinity to HLA class I heavy and light chains on HUT-102B cells in both plateau and logarithmic phases, while it was not associated with HLA class I on the surface of JY cells. HLA class II molecules form a cluster with TfR on HUT-102B cells, while only a modest association was found on JY cells, and only in the logarithmic phase. The possible explanation of this distinct association and a two dimensional model of the antigen and receptor distributions are presented in this paper.


Subject(s)
Cell Membrane/chemistry , Hematopoietic Stem Cells/chemistry , Histocompatibility Antigens Class I/analysis , Lymphocytes/chemistry , Receptors, Transferrin/analysis , B-Lymphocytes/metabolism , Flow Cytometry/methods , Humans , Models, Biological , Protein Binding , Protein Conformation , T-Lymphocytes/metabolism , Tumor Cells, Cultured
14.
J Fluoresc ; 4(4): 303-14, 1994 Dec.
Article in English | MEDLINE | ID: mdl-24233604

ABSTRACT

Flow cytometry is a fast analysis and separation method for large cell populations, based on collection and processing of optical signals gained on a cell-by-cell basis. These optical signals are scattered light and fluorescence. Owing to its unique potential ofStatistical data analysis and sensitive monitoring of (micro)heterogeneities in large cell populations, flow cytometry-in combination with microscopic imaging techniques-is a powerful tool to study molecular details of cellular signal transduction processes as well. The method also has a widespread clinical application, mostly in analysis of lymphocyte subpopulations for diagnostic (or research) purposes in diseases related to the immune system. A special application of flow cytometry is the mapping of molecular interactions (proximity relationships between membrane proteins) at the cell surface, on a cell-by-cell basis. We developed two approaches to study such questions; both are based ondistance-dependent quenching of excited state fluorophores (donors) by fluorescent or dark (nitroxide radical) acceptors via Förstertype dipole-dipole resonance energy transfer (FRET) and long-range electron transfer (LRET) mechanisms, respectively. A critical evaluation of these methods using donor- or acceptor-conjugated monoclonal antibodies (or their Fab fragments) to select the appropriate cell surface receptor or antigen will be presented in comparison with other approaches for similar purposes. The applicability of FRET and LRET for two-dimensional antigen mapping as well as for detection of conformational changes in extracellular domains of membrane-bound proteins is discussed and illustrated by examples of several lymphoma cell lines. Another special application area of flow cytometry is the analysis of different aspects of cellular signal transduction, e.g., changes of intracellular ion (Ca(2+), H(+), Na(+)) concentrations, regulation of ion channel activities, or more complex physiological responses of cell to external stimuli via correlated fluorescence and scatter signal analysis, on a cell-by-cell basis. This way different signaling events such as changes in membrane permeability, membrane potential, cell size and shape, ion distribution, cell density, chromatin structure, etc., can be easily and quickly monitored over large cell populations with the advantage of revealing microheterogeneities in the cellular responses. Flow cytometry also offers the possibility to follow the kinetics of slow (minute- and hour-scale) biological processes in cell populations. These applications are illustrated by the example of complex flow cytometric analysis of signaling in extracellular ATP-triggered apoptosis (programmed cell death) of murine thymic lymphocytes.

16.
Biochem Biophys Res Commun ; 191(2): 378-84, 1993 Mar 15.
Article in English | MEDLINE | ID: mdl-8460997

ABSTRACT

Extracellular ATP induced changes in the membrane potential of thymocytes from BALB/c mice were analyzed. At concentrations below 0.1 mM, ATP hyperpolarizes the cell membrane on the time scale of development of the Ca(2+)-signal. After a longer time hyperpolarization turns to depolarization. ATP concentrations higher than 0.5 mM caused rapid depolarization without previous hyperpolarization. Verapamil, quinine or the absence of extracellular Ca2+ blocked the hyperpolarization by ATP. In Na(+)-free medium the magnitude of depolarization decreased. Our data suggest a contribution of Ca(2+)-activated K+ channels to the hyperpolarizing effect of ATP at lower concentrations. The direction of membrane potential changes is determined presumably by a sensitive balance of ATP-receptor mediated Ca(2+)- and Na(+)-influx and the Ca(2+)-activated K(+)-channel activity.


Subject(s)
Adenosine Triphosphate/pharmacology , Membrane Potentials/drug effects , Thymus Gland/drug effects , Animals , Kinetics , Mice , Mice, Inbred BALB C , Quinine/pharmacology , Thymus Gland/cytology , Thymus Gland/physiology , Verapamil/pharmacology
17.
Immunobiology ; 185(2-4): 337-49, 1992 Aug.
Article in English | MEDLINE | ID: mdl-1452209

ABSTRACT

Molecular interaction and transmembrane signal transducing events generate a very dynamic and ever changing "pattern" in the plasma membranes. Lymphocytes, the key functional elements of the immune system, are eminently suited to be the primary targets to investigate these proximity, mobility, or other physical-chemical changes in their plasma membranes. Recently, a number of experiments suggested that processed peptides from antigens can bind specific components of MHC molecules (Elliott et al., 1991). This is certainly a way to alter their structure. Cell surface patterns of topological nature, assembly and disassembly of oligomeric receptor structure like the IL-2 receptor have been investigated by sophisticated biophysical techniques. The dynamic changes in the two-dimensional cell surface pattern and intramolecular conformational changes within this "larger" macro-pattern may have a strong regulatory role in signal transducing and intercellular recognition processes. Recent data on these problems are presented together with brief and critical discussions.


Subject(s)
Cell Membrane/immunology , Lymphocyte Activation/immunology , Membrane Proteins/immunology , Signal Transduction/immunology , Animals , Humans
18.
Mol Immunol ; 29(4): 517-24, 1992 Apr.
Article in English | MEDLINE | ID: mdl-1565099

ABSTRACT

The role of membrane potential changes in T cell activation was studied on human peripheral blood lymphocytes stimulated with phytohemagglutinin. Addition of bretylium tosylate, a sodium channels opener, to PHA treated lymphocytes modified the membrane potential and consequently blocked cell activation in a dose-dependent fashion. BT was non-toxic even in long-term (72 hr) incubations. It was reversibly removable, and the removal restored the stimulatory effect of PHA. 3H-thymidine incorporation was blocked if BT was present during the first 20-24 hr of the mitogenic activation. The later BT was added after PHA, the less inhibition of proliferation was observed. BT hyperpolarized the lymphocytes also in the presence of PHA. BT hindered the depolarizing effect of high extracellular potassium concns. The sustained polarized state of the lymphocytes did not influence the intracellular calcium increase upon PHA treatment. IL-2 and transferrin receptor expression was not hindered by BT during PHA stimulation of lymphocytes. Addition of rIL-2 did not abolish the inhibitory effect of BT. According to cell-cycle analysis BT arrested the majority of the cells in G1 phase. It is suggested that cell activation demands the flexible maintenance of a relatively narrow membrane potential "window". Any sustained and significant hyper-, or depolarization, may dramatically decrease the effectivity of transmembrane signalling.


Subject(s)
Bretylium Tosylate/pharmacology , Lymphocyte Activation/drug effects , T-Lymphocytes/drug effects , Calcium/analysis , Cell Cycle/drug effects , Dose-Response Relationship, Drug , Flow Cytometry , Humans , In Vitro Techniques , Interleukin-2/analysis , Lymphocyte Activation/physiology , Membrane Potentials/drug effects , Phytohemagglutinins , Receptors, Transferrin/analysis
19.
J Photochem Photobiol B ; 12(4): 323-37, 1992 Mar 13.
Article in English | MEDLINE | ID: mdl-1578295

ABSTRACT

The interaction of cell surface components may influence several events during the process of transmembrane signalling. Receptor clustering, conformational changes and altered molecular interactions often play essential roles in the final outcome of ligand receptor interactions. Fluorescence resonance energy transfer (FRET) is an excellent tool which can be used to determine distance relationships and supramolecular structure on cell surfaces. This paper reviews the theoretical basis of fluorescence resonance energy transfer, its spectrofluorometric and flow cytometric applications, and provides a critical evaluation of the methods. Finally, examples are given to illustrate the use of the method of fluorescence resonance energy transfer in solving biological problems.


Subject(s)
Cell Membrane/metabolism , Membrane Proteins/metabolism , Animals , Flow Cytometry/methods , Fluorescent Dyes , Mathematics , Models, Biological , Spectrometry, Fluorescence/methods
20.
J Immunol ; 146(6): 1862-7, 1991 Mar 15.
Article in English | MEDLINE | ID: mdl-2005381

ABSTRACT

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.


Subject(s)
Histocompatibility Antigens Class I/metabolism , T-Lymphocytes/metabolism , Endocytosis , Fluoresceins , Fluorescent Antibody Technique , Humans , Hydrogen-Ion Concentration , Lymphocyte Activation/physiology , Tumor Cells, Cultured , beta 2-Microglobulin
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