RÉSUMÉ
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.
Sujet(s)
Canaux potassiques voltage-dépendants , Canaux potassiques/métabolisme , Synapses/métabolisme , Lymphocytes T cytotoxiques/métabolisme , Cytotoxicité immunologique , Antigène HLA-A2/physiologie , Humains , Canal potassique Kv1.3 , Activation des lymphocytes , Microdomaines membranaires/métabolisme , Oligopeptides , Peptides/analyse , Lymphocytes T cytotoxiques/immunologieRÉSUMÉ
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.
Sujet(s)
Membrane cellulaire/physiologie , Fluidité membranaire , Modèles biologiques , Animaux , Membrane cellulaire/composition chimique , Phénomènes chimiques , Chimie physique , Diffusion , Transfert d'énergie par résonance de fluorescence , Double couche lipidique , Lipides membranaires/physiologie , Microdomaines membranaires/physiologie , Protéines membranaires/physiologie , Microscopie électronique , Transduction du signalRÉSUMÉ
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.
Sujet(s)
Antigènes CD3/biosynthèse , Membrane cellulaire/métabolisme , Canaux potassiques voltage-dépendants , Canaux potassiques/biosynthèse , Canaux potassiques/composition chimique , Lymphocytes T/métabolisme , Animaux , Membrane cellulaire/immunologie , Électrophysiologie , Épitopes , Transfert d'énergie par résonance de fluorescence , Humains , Immunohistochimie , Cellules Jurkat , Canal potassique Kv1.3 , Souris , Microscopie confocale , Microscopie électronique , Modèles statistiques , TransfectionRÉSUMÉ
Thirty years ago Singer and Nicolson constructed the "fluid mosaic model" of the membrane, which described the structural and functional characteristics of the plasma membrane of non-polarized cells like circulating blood lymphocytes as a fluid lipid phase accommodating proteins with a relatively free mobility. It is a rare phenomenon in biology that such a model could survive 30 years and even today it has a high degree of validity. However, in the light of new data it demands some modifications. In this minireview we present a new concept, which revives the SN model, by shifting the emphasis from fluidity to mosaicism, i.e. to lipid microdomains and rafts. A concise summary of data and key methods is given, proving the existence of non-random co-distribution patterns of different receptor kinds in the microdomain system of the plasma membrane. Furthermore we present evidence that proteins are not only accommodated by the lipid phase, but they are integral structural elements of it. Novel suggestions to the SN model help to develop a modernized version of the old paradigm in the light of new data.
Sujet(s)
Membrane cellulaire/métabolisme , Membrane cellulaire/ultrastructure , Modèles biologiques , Animaux , Humains , Métabolisme lipidique , Lymphocytes/métabolisme , Microscopie électronique à balayage , Récepteurs de surface cellulaire/métabolismeRÉSUMÉ
The glycosylphosphatidylinositol-anchored receptor CD14 plays a major role in the inflammatory response of monocytes to lipopolysaccharide. Here, we describe that ceramide, a constituent of atherogenic lipoproteins, binds to CD14 and induces clustering of CD14 to co-receptors in rafts. In resting cells, CD14 was associated with CD55, the Fcgamma-receptors CD32 and CD64 and the pentaspan CD47. Ceramide further recruited the complement receptor 3 (CD11b/CD18) and CD36 into proximity of CD14. Lipopolysaccharide, in addition, induced co-clustering with Toll-like receptor 4, Fcgamma-RIIIa (CD16a) and the tetraspanin CD81 while CD47 was dissociated. The different receptor complexes may be linked to ligand-specific cellular responses initiated by CD14.
Sujet(s)
Céramides/métabolisme , Protéines de Drosophila , Antigènes CD14/métabolisme , Lipopolysaccharides/pharmacologie , Microdomaines membranaires/métabolisme , Protéines membranaires , Monocytes/métabolisme , Antigènes CD/métabolisme , Antigènes CD47 , Protéines de transport/métabolisme , Humains , Inflammation/métabolisme , Ligands , Antigène macrophage 1/métabolisme , Glycoprotéines membranaires/métabolisme , Récepteurs de surface cellulaire/métabolisme , Antigène CD81 , Récepteur de type Toll-4 , Récepteurs de type TollRÉSUMÉ
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.
Sujet(s)
Plaquettes/métabolisme , Complexe glycoprotéique IIb-IIIa de la membrane plaquettaire/métabolisme , Animaux , Anticorps monoclonaux/métabolisme , Dimérisation , Transfert d'énergie , Cytométrie en flux , Humains , SourisRÉSUMÉ
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.
Sujet(s)
Fusion cellulaire , Antigènes d'histocompatibilité de classe II/métabolisme , Antigènes d'histocompatibilité de classe I/métabolisme , Agrégation des récepteurs/physiologie , Récepteurs de surface cellulaire/métabolisme , Lignée cellulaire , Membrane cellulaire/composition chimique , Membrane cellulaire/métabolisme , Transfert d'énergie , Colorants fluorescents/métabolisme , Or colloïdal/métabolisme , Humains , Microdomaines membranaires , Microscopie , Microscopie de fluorescence/méthodes , Récepteurs à l'interleukine-2RÉSUMÉ
Gerontological research has some past and sporadically also some highlights in Hungary, but its present state can be easily deduced from the following data. During the last 12 years and more, well over 10,000 Hungarian scientific papers have been published in well-recognized national or international journals. Altogether approximately 1% of them have been classified as gerontological publications from Hungary. This low figure shows that gerontology has low priority and--unfortunately low support--in Hungary. This statement does not intend to downgrade Hungarian gerontologists, however points out that the Hungarian trends are not far from those of European or world wide interest in aging. Despite the recognition that we have to accept the inevitable fact that industrial societies will have (they already have) an aging population with all the social and medical problems arising, the focus of interest is wide from this significant and interesting (sub)population, which is neglected (sometimes even despised); yet everybody is absolutely eager to join this club. The average of the Hungarian research achievements and publication activities are among the better European achievements. There are some highlights and new trends even initiated by some outstanding Hungarian scientists, yet the overall weight of gerontology research is still an orphan in the Hungarian scientific life. We deal in this short and far from complete summary almost exclusively with experimental gerontology. We have to apologize if we have not included everybody, who also contributed even significantly to this field because the time for the preparation of this overview was short.
Sujet(s)
Vieillissement , Recherche/tendances , Animaux , Gériatrie , Humains , HongrieRÉSUMÉ
In this paper we report on a hypoosmolality induced current, I(osmo), in embryonic chicken osteoclasts, which could only be studied when blocking a simultaneously active, unidentified slow outward current, I(slo). I(slo) was observed in all of the examined cells when both the intracellular and extracellular solutions contained sodium as the major cation and no potassium. The current was outwardly rectifying and activated at membrane potentials more positive than -44 +/- 12 mV (n = 31). The time to half activation of the current was also voltage dependent and was 350 ms at Vm = +80 mV, and 78 ms at Vm = +120 mV. The current did not inactivate during periods up to 5 s. Extracellular 4-AP (5 mM), TEA (5 mM) and Ba2+ (1 mM), blockers of K+ conductances in chicken osteoclasts, did not influence I(slo). However, I(slo) was inhibited by 50 microM extracellular verapamil, which allowed us to study I(osmo) in isolation. Exposure of the osteoclasts to hypotonic solution resulted in the development of a depolarization activated I(osmo). It developed after a 1-min delay and reached its maximum within 10 minutes. Half-maximal activation occurred after 4.4 +/- 0.9 min (n = 9). The current activated within a few ms upon depolarization and did not inactivate during at least 5 sec. I(osmo) reversed around the calculated Nernst potential for Cl- (E(Cl) = +7.3 mV and V(rev) = +5.4 +/- 3.6 mV, n = 9). The underlying conductance, G(osmo) exhibited moderate outward rectification around 0 mV in symmetrical Cl- solutions. Ion substitution experiments showed that G(osmo) is an anion conductance with P(Cl) approximately = P(F) > P(gluc) >> P(Na). I(osmo) was blocked by 0.5 mM SITS but 50 microM verapamil, 5 mM TEA, 5 mM 4-AP, 1 mM Ba2+, 50 microM cytochalasin D and 0.5 mM alendronate did not have any effect on the current. Cl- currents have been implicated in charge neutralization during osteoclastic acid secretion for bone resorption. The present results imply that osmolality may be a factor controlling this charge neutralization.
Sujet(s)
Ostéoclastes/physiologie , Animaux , Anions , Inhibiteurs des canaux calciques/pharmacologie , Embryon de poulet , Cytochalasine D/pharmacologie , Transport des ions , Potentiels de membrane/effets des médicaments et des substances chimiques , Concentration osmolaire , Ostéoclastes/effets des médicaments et des substances chimiques , Ostéoclastes/métabolisme , Potassium/métabolisme , Sodium/métabolisme , Vérapamil/pharmacologieRÉSUMÉ
Fluorescence resonance energy transfer (FRET) data, in accordance with lateral mobility measurements, suggested the existence of class I HLA dimers and oligomers at the surface of live human cells, including the B lymphoblast cell line (JY) used in the present study. Intra- and intermolecular class I HLA epitope distances were measured on JY B cells by FRET using fluorophore-conjugated Ag-binding fragments of mAbs W6/32 and L368 directed against structurally well-characterized heavy and light chain epitopes, respectively. Out-of-plane location of these epitopes relative to the membrane-bound BODIPY-PC (2-(4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine) was also determined by FRET. Computer-simulated docking of crystallographic structures of class I HLA and epitope-specific Ag-binding fragments, with experimentally determined interepitope and epitope to cell surface distances as constraints, revealed several sterically allowed and FRET-compatible class I HLA dimeric and tetrameric arrangements. Extension of the tetrameric class I HLA model with interacting TCR and CD8 resulted in a model of a supramolecular cluster that may exist physiologically and serve as a functionally significant unit for a network of CD8-HLA-I complexes providing enhanced signaling efficiency even at low MHC-peptide concentrations at the interface of effector and APCs.
Sujet(s)
Antigènes CD8/composition chimique , Transfert d'énergie/immunologie , Antigènes HLA/composition chimique , Antigènes d'histocompatibilité de classe I/composition chimique , Modèles moléculaires , Récepteurs aux antigènes des cellules T/composition chimique , Cellules présentatrices d'antigène/composition chimique , Cellules présentatrices d'antigène/immunologie , Lymphocytes B/composition chimique , Lymphocytes B/immunologie , Lignée de cellules transformées , Membrane cellulaire/composition chimique , Membrane cellulaire/immunologie , Simulation numérique , Cristallographie aux rayons X/méthodes , Déterminants antigéniques des lymphocytes B/composition chimique , Antigène HLA-A2/composition chimique , Humains , Fragments Fab d'immunoglobuline/composition chimique , Cartographie peptidique , Spectrométrie de fluorescence/méthodes , bêta-2-Microglobuline/composition chimiqueRÉSUMÉ
Melatonin is a small amino acid derivative hormone of the pineal gland. Melatonin quickly and reversibly blocked Kv1.3 channels, the predominant voltage-gated potassium channel in human T-lymphocytes, acting from the extracellular side. The block did not show state or voltage dependence and was associated with an increased inactivation rate of the current. A half-blocking concentration of 1.5 mM was obtained from the reduction of the peak current. We explored several models to describe the stoichiometry of melatonin-Kv1.3 interaction considering one or four independent binding sites per channel. The model in which the occupancy of one of four binding sites by melatonin is sufficient to block the channels gives the best fit to the dose-response relationship, although all four binding sites can be occupied by the drug. The dissociation constant for the individual binding sites is 8.11 mM. Parallel application of charybdotoxin and melatonin showed that both compounds can simultaneously bind to the channels, thereby localizing the melatonin binding site out of the pore region. However, binding of tetraethylammonium to its receptor decreases the melatonin affinity, and vice versa. Thus, the occupancy of the two separate receptor sites allosterically modulates each other.
Sujet(s)
Mélatonine/pharmacologie , Canaux potassiques voltage-dépendants , Canaux potassiques/composition chimique , Canaux potassiques/physiologie , Lymphocytes T/physiologie , Anticorps monoclonaux/pharmacologie , Antigènes CD/sang , Antigènes CD/immunologie , Sites de fixation , Antigènes CD2/sang , Antigènes CD2/immunologie , Antigènes CD4/sang , Antigènes CD4/immunologie , Charybdotoxine/pharmacocinétique , Charybdotoxine/pharmacologie , Humains , Techniques in vitro , Cinétique , Canal potassique Kv1.3 , Mélatonine/composition chimique , Mélatonine/métabolisme , Potentiels de membrane/effets des médicaments et des substances chimiques , Potentiels de membrane/physiologie , Inhibiteurs des canaux potassiques , Tétraéthyl-ammonium/pharmacologieRÉSUMÉ
Pandinus imperator scorpion toxins Pi2 and Pi3 differ only by a single amino acid residue (neutral Pro7 in Pi2 vs. acidic Glu7 in Pi3). The binding kinetics of these toxins to human Kv1.3 showed that the decreased ON rate (k(ON) = 2.18 x 10(8) m(-1)sec(-1) for Pi2 and 1.28 x 10(7) m(-1)sec(-1) for Pi3) was almost entirely responsible for the increased dissociation constant (K(d)) of Pi3 (K(d) = 795 pm) as compared to Pi2 (K(d) = 44 pm). The ionic strength dependence of the association rates was exactly the same for the two toxins indicating that through-space electrostatic interactions can not account for the different ON rates. Results were further analyzed on the basis of the three-dimensional structural models of the toxins. A 3D structure of Pi3 was generated from the NMR spectroscopy coordinates of Pi2 by computer modeling. The Pi3 model resulted in a salt bridge between Glu7 and Lys24 in Pi3. Based on this finding our interpretation of the reduced ON rate of Pi3 is that the intramolecular salt bridge reduces the local positive electrostatic potential around Lys24 resulting in decreased short-range electrostatic interactions during the binding step. To support our finding, we constructed a 3D model of the Ser-10-Asp Charybdotoxin mutant displaying distinctly reduced affinity for Shaker channels. The mutant Charybdotoxin structure also displayed a salt bridge between residues Asp10 and Lys27 equivalent to the one between Glu7 and Lys24 in Pi3.
Sujet(s)
Inhibiteurs des canaux potassiques , Canaux potassiques voltage-dépendants , Venins de scorpion/pharmacologie , Séquence d'acides aminés , Animaux , Charybdotoxine/composition chimique , Charybdotoxine/génétique , Charybdotoxine/métabolisme , Charybdotoxine/pharmacologie , Acide glutamique/composition chimique , Humains , Techniques in vitro , Cinétique , Canal potassique Kv1.3 , Lysine/composition chimique , Potentiels de membrane , Modèles moléculaires , Données de séquences moléculaires , Mutation ponctuelle , Canaux potassiques/métabolisme , Conformation des protéines , Venins de scorpion/composition chimique , Venins de scorpion/génétique , Venins de scorpion/métabolisme , Similitude de séquences d'acides aminés , Lymphocytes T/effets des médicaments et des substances chimiques , Lymphocytes T/métabolismeRÉSUMÉ
Streptomyces strains isolated from the Kuwait Burgan oil field were defined as S. griseoflavus, S. parvus, and S. plicatus utilised n-hexadecane, n-octadecane (purified fractions of mineral oil), kerosene, and crude oil as sole carbon and energy sources. The strains were incubated with n-alkanes and increase of the fatty acid content with chain length equivalent to the employed n-alkanes was observed. Signal transducing GTP-binding proteins (GBPs) play an important role in n-alkane uptake in streptomycetes. Specific activators of GBPs increased the uptake of hydrocarbons. Using the hydrophobic fluorescent dye diphenylhexatrien (DPH) as a probe, it was found that the microviscosity of the hydrophobic inner region of the cellular membrane is significantly lower in hydrocarbon utilisers than in non-utilisers. This difference probably reflects differences in the fatty acid composition of the strains. When cultures were grown in n-alkane containing media, electron microscopy revealed that the hydrocarbon utilisers showed less-electron dense areas as inclusions in the cytoplasm. Soil samples inoculated with Streptomyces strains eliminated hydrocarbons much faster than those not containing these strains, serving as control. When inorganic medium was supplied with n-hexadecane-1-14C as sole carbon and energy source, radioactive CO2 was detected. Since streptomycetes have not been used until now for oil elimination, though they are known as abundant soil bacteria tolerating extreme conditions, their possible use for bioremediation of hydrocarbon contaminated soils is discussed.
Sujet(s)
Alcanes/métabolisme , 1,6-Diphényl-hexa-1,3,5-triène/analogues et dérivés , Streptomyces/métabolisme , Dépollution biologique de l'environnement , Membrane cellulaire/composition chimique , Membrane cellulaire/physiologie , Perméabilité des membranes cellulaires , 1,6-Diphényl-hexa-1,3,5-triène/métabolisme , Acides gras/analyse , Colorants fluorescents/métabolisme , Protéines G/métabolisme , Hydrocarbures/métabolisme , Streptomyces/ultrastructureRÉSUMÉ
The field of research considering the working mechanism of anesthetic agents is a complex one and the site or sites of action of general anesthetics are yet to be elucidated. Through the years, on the molecular level, the discussion has shifted from the lipid theories to the more specific interaction with the proteins responsible for the signal transduction. While this approach led to several models, they offer, at best, partial explanations for the observed phenomena. Anesthetic agents interact with many systems, of which the neuronal is best studied, leaving interaction with the immune defense system relatively unexplored. In this study we focus on the interaction of ethanol and halothane with the co-localization on the membrane of HLA I and II molecules. We show that ethanol tends to randomize the distribution of HLA I and II molecules, while halothane increases the clustering of HLA I proteins. The notion that anesthetics modulate cell function by disrupting clustering and thereby promoting a random distribution is a novel approach that may explain the general involvement of many systems during exposition to anesthetic drugs. In this study we show the disturbance of co-localization of molecules that may form a functional network. The relevance of this finding depends on the importance of these networks for extracellular and intracellular processes.
Sujet(s)
Éthanol/pharmacologie , Halothane/pharmacologie , Antigènes d'histocompatibilité de classe II/composition chimique , Antigènes d'histocompatibilité de classe I/composition chimique , Anesthésiques par inhalation/pharmacologie , Anticorps monoclonaux , Lymphocytes B , Lignée cellulaire , Transfert d'énergie , Antigènes d'histocompatibilité de classe I/effets des médicaments et des substances chimiques , Antigènes d'histocompatibilité de classe II/effets des médicaments et des substances chimiques , Humains , Structures macromoléculaires , Spectrométrie de fluorescence/méthodesRÉSUMÉ
Using the patch-clamp technique we determined that Pandinus imperator toxin Pi1, a recently described peptide toxin having four disulfide bridges instead of the usual three in scorpion toxins, blocked Kv1.3 channels of human T lymphocytes from the extracellular side with a 1:1 stoichiometry. Kv1.3 block was instantaneous and removable with toxin-free extracellular solution. The toxin did not influence activation or inactivation of the channels. We found that Pi1 blocked Kv1.3 with less affinity (K(d) = 11.4 nM) than the structurally related three disulfide bridge containing toxins Pi2 (50 pM) and Pi3 (0.5 nM). The fourth disulfide bridge in Pi1 had no influence on the channel binding ability of the toxin; the less effective block was due to differences in amino acid side chain properties at positions 11 and 35.
Sujet(s)
Canaux potassiques voltage-dépendants , Canaux potassiques/effets des médicaments et des substances chimiques , Venins de scorpion/composition chimique , Venins de scorpion/pharmacologie , Lymphocytes T/effets des médicaments et des substances chimiques , Séquence d'acides aminés , Acides aminés/composition chimique , Animaux , Disulfures , Humains , Cinétique , Canal potassique Kv1.3 , Données de séquences moléculaires , Techniques de patch-clamp , Potassium/métabolisme , Liaison aux protéines , Scorpions , Similitude de séquences d'acides aminés , Facteurs tempsRÉSUMÉ
BACKGROUND: Perrin equation suggests an alternative way for the accurate energy transfer determination on a cell-by-cell basis by measuring polarized donor intensities in a conventional flow cytometer. METHODS: The relationship between energy transfer and fluorescence anisotropy of the donor was investigated by flow cytometric generation of Perrin-lifetime plots of fluorescent antibody-labeled MHC class I and class II molecules on the surface of living cells. The energy transfer reduced the fluorescence lifetime of the donor. RESULTS: Perrin plots have proven to be sensitive to the segmental mobility of the labeling dye and that of antibodies of different isotypes, and homo-transfer due to the multiple labeling of antibodies. A method demonstrating the feasibility of energy transfer determination by measuring anisotropy enhancement of the donor is presented. Flow cytometric histograms of the donor anisotropy and of the deduced energy transfer efficiency are shown, indicating clustering of MHC class I and class II molecules on the surface of human T lymphoblasts. In the Appendix, a method for the simultaneous determination of both energy transfer efficiency and donor fluorescence anisotropy, without need for G-factor measurement, is also presented. CONCLUSIONS: We demonstrate that energy transfer efficiency, i.e., proximity, between suitably selected donor and acceptor, and the rotational relaxation of the donor, i.e., donor mobility, can be simultaneously measured in a flow cytometer.
Sujet(s)
Cytométrie en flux/méthodes , Polarisation de fluorescence/méthodes , Récepteurs de surface cellulaire/analyse , Adulte , Anticorps monoclonaux/immunologie , Cellules cultivées , Transfert d'énergie , Colorants fluorescents , Antigènes d'histocompatibilité de classe I/immunologie , Antigènes d'histocompatibilité de classe II/immunologie , Humains , Immunoglobuline G/analyse , Lymphocytes T/immunologie , bêta-2-Microglobuline/immunologieRÉSUMÉ
Immunogold staining and electron microscopy show that IL-2 receptor alpha-subunits exhibit nonrandom surface distribution on human T lymphoma cells. Analysis of interparticle distances reveals that this clustering on the scale of a few hundred nanometers is independent of the presence of IL-2 and of the expression of the IL-2R beta-subunit. Clustering of IL-2Ralpha is confirmed by confocal microscopy, yielding the same average cluster size, approximately 600-800 nm, as electron microscopy. HLA class I and II and CD48 molecules also form clusters of the same size. Disruption of cholesterol-rich lipid rafts with filipin or depletion of membrane cholesterol with methyl-beta-cyclodextrin results in the blurring of cluster boundaries and an apparent dispersion of clusters for all four proteins. Interestingly, the transferrin receptor, which is thought to be located outside lipid rafts, exhibits clusters that are only 300 nm in size and are less affected by modifying the membrane cholesterol content. Furthermore, transferrin receptor clusters hardly colocalize with IL-2Ralpha, HLA, and CD48 molecules (crosscorrelation coefficient is 0.05), whereas IL-2Ralpha colocalizes with both HLA and CD48 (crosscorrelation coefficient is between 0.37 and 0.46). This coclustering is confirmed by electron microscopy. The submicron clusters of IL-2Ralpha chains and their coclustering with HLA and CD48, presumably associated with lipid rafts, could underlie the efficiency of signaling in lymphoid cells.
Sujet(s)
Antigènes CD/analyse , Cholestérol/physiologie , Antigènes HLA/analyse , Lymphome T/anatomopathologie , Lipides membranaires/physiologie , Protéines tumorales/analyse , Récepteurs à l'interleukine-2/analyse , Lymphocytes T/métabolisme , Antigène CD48 , Humains , Immunohistochimie , Fluidité membranaire , Microscopie confocale , Microscopie immunoélectronique , Cellules cancéreuses en cultureRÉSUMÉ
Campath-1H, a humanized mAb undergoing clinical trials for treatment of leukemia, transplantation and autoimmune diseases, produces substantial lymphocyte depletion in vivo. The antibody binds to CD52, a highly glycosylated molecule attached to the membrane by a glycosylphosphatidylinositol anchor. Cross-linked Campath-1H is known to activate T cells in vitro. We have investigated the molecular basis for these effects by comparing the protein tyrosine phosphorylation signals induced by Campath-1H and the CD3 mAb OKT3 in primary T cells, and in CD45(+)TCR(+), CD45(-)TCR(+) and CD45(+)TCR(-) Jurkat subclones transfected with CD52. Our results show that Campath-1H triggers similar tyrosine phosphorylation events as OKT3 in both primary T cells and in the CD45(+)TCR(+) Jurkat sub-clone, albeit at quantitatively lower levels. However, no phospholipase C gamma 1 activation nor calcium signals were detected in response to CD52 ligation. The CD52-mediated induction of protein tyrosine phosphorylation was absolutely dependent upon the expression of both the TCR and the CD45 phosphotyrosine phosphatase at the cell surface. Cross-linking of Campath-1H was essential for signal transduction in all cells investigated. Fluorescence resonance energy transfer was used to demonstrate CD52 homo-association at the cell surface in Jurkat T cells in a TCR- and CD45-independent manner, and CD52-TCR association in CD45(+)TCR(+) cells. We propose a model to explain the activating effects of Campath-1H in which CD52 mAb cross-linking causes the trapping of TCR polypeptides within molecular complexes at the cell surface, thereby inducing signals via the TCR by a process which depends on the CD45-mediated regulation of the p56(lck) and p59(fyn) tyrosine kinases.
Sujet(s)
Anticorps monoclonaux/physiologie , Anticorps antitumoraux/physiologie , Antigènes CD/physiologie , Antigènes néoplasiques , Glycoprotéines/physiologie , Antigènes CD45/physiologie , Récepteurs aux antigènes des cellules T/physiologie , Transduction du signal/immunologie , Lymphocytes T/enzymologie , Lymphocytes T/immunologie , Alemtuzumab , Anticorps monoclonaux humanisés , Antigènes CD/métabolisme , Antigène CD52 , Calcium/physiologie , Signalisation calcique/immunologie , Cellules cultivées , Glycoprotéines/métabolisme , Humains , Inositol 1,4,5-trisphosphate/immunologie , Isoenzymes/immunologie , Cellules Jurkat , Phospholipase C gamma , Récepteurs aux antigènes des cellules T/métabolisme , Transduction du signal/génétique , Lymphocytes T/métabolisme , Transfection , Type C Phospholipases/immunologieRÉSUMÉ
Change of osmolality surrounding spawned sperm from isotonic to hypotonic causes the initiation of sperm motility in the common carp. Here we show that membrane-permeable cAMP does not initiate motility of carp sperm that is quiescent in isotonic solution, and that motility of the demembranated sperm can be reactivated without cAMP. Furthermore, the cAMP level does not change during the initiation of sperm motility, and inhibitors of protein kinase do not affect sperm motility, suggesting that no cAMP-dependent system is necessary for the regulation of sperm motility. Sperm motility could not be initiated in Ca(2+)-free hypoosmotic solutions, and significant increase in the intracellular Ca(2+) level was observed by a Ca-sensitive fluorescence dye during hypoosmolality-induced active motion period. The demembranated sperm cells were fully reactivated in the solutions containing 10(-7) to 10(-5) M Ca(2+). Ca(2+) channel blockers such as verapamil and omega-conotoxin reversibly inhibited the initiation of sperm motility, suggesting that Ca(2+) influx is the prerequisite for the initiation of carp sperm motility. Motility of intact sperm was completely blocked; however, that of the demembranated sperm was not inhibited by the calmodulin inhibitor W7, suggesting that the calmodulin bound close to the plasma membrane participated in the initiation of sperm motility. Flow cytometric membrane potential measurements and spectrophotometric measurements by using fluorescence dyes showed transient membrane hyperpolarization on hypoosmolality-induced motility. This article discusses the role of membrane hyperpolarization on removal of inactivation of Ca(2+) channels, leading to Ca(2+) influx at the initiation of carp sperm motility.
Sujet(s)
Canaux calciques/métabolisme , Mobilité des spermatozoïdes/physiologie , Animaux , Dibutyryl AMP cyclique/métabolisme , Dibutyryl AMP cyclique/pharmacologie , A-23187/pharmacologie , Calcium/métabolisme , Inhibiteurs des canaux calciques/pharmacologie , Carpes (poisson) , Cyclic AMP-Dependent Protein Kinases/antagonistes et inhibiteurs , Cyclic GMP-Dependent Protein Kinases , Dibutyryl GMP cyclique/métabolisme , Dibutyryl GMP cyclique/pharmacologie , Ionophores/pharmacologie , Mâle , Protéine kinase C/antagonistes et inhibiteurs , Inhibiteurs de protéines kinases , Mobilité des spermatozoïdes/effets des médicaments et des substances chimiques , Valinomycine/pharmacologieRÉSUMÉ
The erbB2 oncogene belongs to the type I trans-membrane tyrosine kinase family of receptors. Its medical importance stems from its widespread over-expression in breast cancer. This review will focus on the signal transduction through this protein, and explains how the overexpression of erbB2 may result in poor prognosis of breast cancer, and finally it will summerize our current understanding about the therapeutic potential of receptor-targeted therapy in breast cancer. ErbB2 does not have any known ligand which is able to bind to it with high affinity. However the kinase activity of erbB2 can be activated without any ligand, if it is overexpressed, and by heteroassociation with other members of the erbB family (erbB1 or epidermal growth factor receptor, erbB3 and erbB4). This interaction substantially increases the efficiency and diversity of signal transduction through these receptor complexes. In addition, erbB2 forms large scale receptor clusters containing hundreds of proteins. These receptor islands may take part in recruiting cytosolic factors which relay the signal towards the nucleus or the cytoplasm. Overexpression of erbB2 was linked to higher transforming activity, increased metastatic potential, angiogenesis and drug resistence of breast tumor in laboratory experiments. As a corollary of these properties, erbB2 amplification is generally thought to be associated with a poor prognosis in breast cancer patients. These early findings lead to the development of antibodies that down-regulate erbB2. Such a therapeutic approach has already been found effective in experimental tumor models and in clinical trials as well. Further understanding of the importance of erbB2 and growth factor receptors in the transformation of normal cells to malignant ones may once give us a chance to cure erbB2 over-expressing breast cancer.