RESUMEN
Multi electrode arrays (MEAs) are increasingly used to detect external field potentials in electrically active cells. Recently, in combination with cardiomyocytes derived from human (induced) pluripotent stem cells they have started to become a preferred tool to examine newly developed drugs for potential cardiac toxicity in pre-clinical safety pharmacology. The most important risk parameter is proarrhythmic activity in cardiomyocytes which can cause sudden cardiac death. Whilst MEAs can provide medium- to high- throughput noninvasive assay platform, the translation of a field potential to cardiac action potential (normally measured by low-throughput patch clamp) is complex so that accurate assessment of drug risk to the heart is in practice still challenging. To address this, we used computational simulation to study the theoretical relationship between aspects of the field potential and the underlying cardiac action potential. We then validated the model in both primary mouse- and human pluripotent (embryonic) stem cell-derived cardiomyocytes showing that field potentials measured in MEAs could be converted to action potentials that were essentially identical to those determined directly by electrophysiological patch clamp. The method significantly increased the amount of information that could be extracted from MEA measurements and thus combined the advantages of medium/high throughput with more informative readouts. We believe that this will benefit the analysis of drug toxicity screening of cardiomyocytes using in time and accuracy.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Miocitos Cardíacos/efectos de los fármacos , Potenciales de Acción , Animales , Simulación por Computador , Humanos , Células Madre Pluripotentes Inducidas/citología , Ratones , Microelectrodos , Modelos Teóricos , Células Madre Pluripotentes/citologíaRESUMEN
The polymer polydimethylsiloxane (PDMS) is widely used to build microfluidic devices compatible with cell culture. Whilst convenient in manufacture, PDMS has the disadvantage that it can absorb small molecules such as drugs. In microfluidic devices like "Organs-on-Chip", designed to examine cell behavior and test the effects of drugs, this might impact drug bioavailability. Here we developed an assay to compare the absorption of a test set of four cardiac drugs by PDMS based on measuring the residual non-absorbed compound by High Pressure Liquid Chromatography (HPLC). We showed that absorption was variable and time dependent and not determined exclusively by hydrophobicity as claimed previously. We demonstrated that two commercially available lipophilic coatings and the presence of cells affected absorption. The use of lipophilic coatings may be useful in preventing small molecule absorption by PDMS.
Asunto(s)
Bioensayo/métodos , Fármacos Cardiovasculares/química , Cromatografía Líquida de Alta Presión/instrumentación , Dimetilpolisiloxanos/química , Evaluación Preclínica de Medicamentos/métodos , Dispositivos Laboratorio en un Chip , Nylons/química , Absorción Fisicoquímica , Fármacos Cardiovasculares/aislamiento & purificación , Cromatografía Líquida de Alta Presión/métodos , Materiales Biocompatibles Revestidos/química , Diseño de Equipo , Análisis de Falla de Equipo , Lípidos/química , Ensayo de Materiales , Preparaciones FarmacéuticasRESUMEN
Histamine receptors are present on the surface of various normal and tumor-derived cell types, where their biological function is incompletely understood. Here we report that histamine not only stimulates cell proliferation under serum-free conditions, but also is chemotactic for human carcinoma (Hela and A431) and melanoma (A875) cells expressing H1 type receptors. Histamine was found to be a potent activator of phospholipase C, leading to polyphosphoinositide hydrolysis and subsequent intracellular Ca2+ mobilization. In addition, histamine also causes the protein kinase C-mediated activation of Na+/H+ exchange, as evidenced by an amiloride-sensitive rise in cytoplasmic pH. All histamine-induced responses, including chemotaxis and DNA synthesis, are completely inhibited by the H1 receptor antagonist pyrilamine, but not by cimetidine, an inhibitor of histamine H2 type receptors. Our results suggest that histamine may have a previously unrecognized role in the migration and proliferation of cells expressing H1 receptors.
Asunto(s)
Quimiotaxis , Sustancias de Crecimiento , Histamina/farmacología , Receptores Histamínicos H1/fisiología , Células Tumorales Cultivadas/fisiología , División Celular/efectos de los fármacos , ADN de Neoplasias/análisis , Factor de Crecimiento Epidérmico/farmacología , Células HeLa/citología , Células HeLa/efectos de los fármacos , Células HeLa/fisiología , Humanos , Fosfatos de Inositol/metabolismo , Insulina/farmacología , Cinética , Melanoma , Receptores Histamínicos H1/efectos de los fármacos , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacos , Fosfolipasas de Tipo C/metabolismoRESUMEN
We investigated intercellular communication during the seventh and tenth cell cycles of Xenopus laevis development using microinjection of Lucifer yellow and FITC-dextran as well as freeze-fracture electron microscopy. We found that gap junction-mediated dye coupling visualized using Lucifer yellow was strongly cell cycle modulated in the tenth cell cycle. Cytoplasmic bridge-mediated dye coupling visualized via FITC-dextran was also, of course, cell cycle modulated. The basis of cell cycle-modulated gap junctional coupling was investigated by measuring the abundance of morphologically detectable gap junctions through the tenth cell cycle. These proved to be six times more abundant at the beginning than at the end of this cell cycle.
Asunto(s)
Comunicación Celular , Ciclo Celular , Cigoto/fisiología , Animales , Femenino , Colorantes Fluorescentes , Técnica de Fractura por Congelación , Técnicas In Vitro , Isoquinolinas , Microscopía Electrónica , Xenopus laevis , Cigoto/citología , Cigoto/ultraestructuraRESUMEN
Modulations in the internal structure of the plasma membrane during the cell cycle of mouse C1300 neuroblastoma cells (clone Neuro-2A) have been studied by freeze-fracture electron microscopy. Both the numerical and lateral distributions of the intramembrane particles (IMP) of the P face of the medium-exposed plasma membrane were determined as a function of the IMP diameter. The lateral IMP-distribution was quantified by a differential density distribution analysis, that could distinguish between random, aggregated, and dispersed distributions of IMP-subpopulations at various levels of spatial organization. Nonrandom lateral IMP-distribution was considered to indicate significant directional constraints on the lateral mobility of the represented molecules. The analysis demonstrated that the density, the size distribution, and the lateral distribution of the IMP are modulated during the cell cycle, such that characteristic structural and dynamic membrane properties can be attributed to the various cell cycle phases (M, G1, S, and G2). The results are interpreted in terms of asynchronous assembly of different membrane components and dynamic reorganizations within the plasma membrane during the cell cycle. Furthermore, they provide a structural manifestation of earlier observed changes in the dynamic properties of membrane proteins and lipids, and functional membrane transport properties in these neuroblastoma cells.
Asunto(s)
Ciclo Celular , Proteínas de la Membrana/análisis , Neuroblastoma/ultraestructura , Animales , Células Cultivadas , Computadores , Técnica de Fractura por Congelación , Lípidos de la Membrana/análisis , Ratones , Microscopía ElectrónicaRESUMEN
Many cell types display two classes of epidermal growth factor receptor (EGFR) as judged from EGF binding studies; i.e., a major class of low affinity EGFR and a minor class of high affinity EGFR. We have studied their respective contribution to the cascade of events elicited by EGF in human A431 carcinoma cells, using anti-EGFR mAb 2E9. This antibody specifically blocks EGF binding to low affinity EGFR, without activating receptors in intact cells, and thus enables us to study the effects of exclusive EGF binding to high affinity EGFR. We show that blocking of low affinity EGFR by mAb 2E9 has almost no effect on the activation of the receptor protein-tyrosine kinase by EGF, suggesting that EGFR kinase activation occurs exclusively through the subclass of high affinity EGFR (5-10%). In addition, we provide evidence that high affinity EGFR exists both in monomeric and dimeric forms, and that cross-phosphorylation of low affinity EGFR by high affinity EGFR may take place in dimers of both receptor types. We demonstrate that the following early cellular response to EGF are also unimpaired in the presence of mAb 2E9: (a) inositol phosphate production, (b) release of Ca2+ from intracellular stores, (c) rise in intracellular pH, (d) phosphorylation of EGF on threonine residue 654, (e) induction of c-fos gene expression, and (f) alteration in cell morphology. As possible nonspecific side effects, we observed that the EGF induced Ca2+ influx and fluid-phase pinocytosis were inhibited in A431 cells in the presence of mAb 2E9. We conclude, therefore, that the activation of the EGFR signal transduction cascade can occur completely through exclusive binding of EGF to the subclass of high affinity EGFR.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/fisiología , Transducción de Señal , Línea Celular , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/fisiología , Receptores ErbB/metabolismo , Humanos , Cinética , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/fisiologíaRESUMEN
Expression of p21rasAsn-17, a dominant negative mutant of p21ras that blocks p21ras activation by growth factors, inhibits activation of extracellular signal-regulated kinase 2 (ERK2) by insulin and platelet-derived growth factor in rat-1 cells [A. M. M. de Vries-Smits, B. M. T. Burgering, S. J. Leevers, C. J. Marshall, and J. L. Bos, Nature (London) 357:602-604, 1992]. Here we report that expression of p21rasAsn-17 does not abolish epidermal growth factor (EGF)-induced phosphorylation of ERK2 in fibroblasts. Since EGF activates p21ras in these cells, this indicates that EGF induces a p21ras-independent pathway for the phosphorylation of ERK2 as well. We investigated whether activation of protein kinase C (PKC) or increase in intracellular calcium could be involved in p21ras-independent signaling. In rat-1 cells, inhibition of either PKC, by prolonged 12-O-tetradecanoylphorbol-13-acetate (TPA) pretreatment, or calcium influx, by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) pretreatment, did not abolish EGF-induced ERK2 phosphorylation. However, a combined inhibition of both p21ras and calcium influx, but not PKC, resulted in a complete inhibition of EGF-induced ERK2 phosphorylation. In contrast, in Swiss 3T3 cells, inhibition of both p21ras activation and TPA-sensitive PKC, but not calcium influx, inhibited EGF-induced ERK2 phosphorylation. These results demonstrate that in fibroblasts, EGF induces alternative pathways of ERK2 phosphorylation in a cell-type-specific manner.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Animales , Calcio/metabolismo , Línea Celular , Ácido Egtácico/farmacología , Ratones , Proteína Quinasa 1 Activada por Mitógenos , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Hypo-osmotic stimulation of human Intestine 407 cells rapidly activated compensatory CL- and K+ conductances that limited excessive cell swelling and, finally, restored the original cell volume. Osmotic cell swelling was accompanied by a rapid and transient reorganization of the F-actin cytoskeleton, affecting both stress fibers as well as apical ruffles. In addition, an increase in total cellular F-actin was observed. Pretreatment of the cells with recombinant Clostridium botulinum C3 exoenzyme, but not with mutant enzyme (C3-E173Q) devoid of ADP-ribosyltransferase activity, greatly reduced the activation of the osmo-sensitive anion efflux, suggesting a role for the ras-related GTPase p21rho. In contrast, introducing dominant negative N17-p21rac into the cells did not affect the volume-sensitive efflux. Cell swelling-induced reorganization of F-actin coincided with a transient, C3 exoenzyme-sensitive tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) as well as with an increase in phosphatidylinositol-3-kinase (PtdIns-3-kinase) activity. Pretreatment of the cells with wortmannin, a specific inhibitor of PtdIns-3-kinase, largely inhibited the volume-sensitive ion efflux. Taken together, our results indicate the involvement of a p21rho signaling cascade and actin filaments in the activation of volume-sensitive chloride channels.
Asunto(s)
Actinas/fisiología , Toxinas Botulínicas , Moléculas de Adhesión Celular/metabolismo , Canales de Cloruro/fisiología , Citoesqueleto/fisiología , Proteínas Tirosina Quinasas/metabolismo , ADP Ribosa Transferasas/farmacología , Actinas/ultraestructura , Aniones/metabolismo , Moléculas de Adhesión Celular/efectos de los fármacos , Células Cultivadas , Canales de Cloruro/efectos de los fármacos , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Proteínas de Unión al GTP/efectos de los fármacos , Proteínas de Unión al GTP/metabolismo , Humanos , Soluciones Hipotónicas/farmacología , Intestinos/citología , Intestinos/efectos de los fármacos , Intestinos/fisiología , Presión Osmótica , Fosfatidilinositol 3-Quinasas , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Tirosina Quinasas/efectos de los fármacos , Tirosina/metabolismo , Proteínas de Unión al GTP rhoRESUMEN
During cell division in the Xenopus egg (diameter 1.25 mm) new cell membrane is formed in the furrow region (rate of growth approx 4-10(4) mum2/min). Freeze-fracture electron microscopy has produced the following data. Preexisting plasma membrane faces show a reversed polarity with respect to particle distribution, i.e. more particles are attached to the E-face (density 1600-2200 particles/mum2) than to the P-face (300 particles/mum2). A frequency histogram of 2331 measured intramembranous particles does not show a continuous range of sizes. The following sizes were very obvious: 95 A (12%), 125 A (30%) and 180 A (6%). At the tips of surface protrusions both the E- and the P- face are particle-free. Nascent cell membrane fracture faces are more difficult to obtain. The particle density is low (E-face 300-500 particles/mum2). Lowering the ambient temperature to 5 degrees C for approx. 5 mins does not change the normal particle pattern, but it improves the output in nascent membrane fracture faces. The fact that in the Xenopus egg preexisting and nascent membrane regions are continuous but nevertheless maintain their highly different particle densities is noteworthy. The freeze-fracture data are discussed in relation to, among other things, the known values of the specific resistances of these membrane regions.
Asunto(s)
División Celular , Membrana Celular/fisiología , Óvulo/fisiología , Animales , División Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Citocalasinas/farmacología , Femenino , Técnica de Fractura por Congelación , Microscopía Electrónica , Óvulo/efectos de los fármacos , Óvulo/ultraestructura , XenopusRESUMEN
The fluidity of the plasma membrane is thought to affect the responsiveness of blood platelets. We measured membrane fluidity in a single cell by Fluorescence Recovery after Photobleaching (FRAP) of the lipophilic probe DiIC14. Since platelets are too small for this technique, we used the human megakaryoblastic cell-line MEG-01, which shares many properties with platelets. MEG-01 cells were cultured for 44 h with simvastatin or mevalonate to change the cholesterol content, enabling analysis of signal processing at cholesterol/phospholipid ratios (C/P) between 0.20 and 0.31. The diffusion of DiIC14 correlated inversely with the C/P ratio with lateral diffusion coefficients (D) of 3.28 x 10(-9) cm2/s at a low C/P decreasing to 2.55 x 10(-9) cm2/s at a high C/P ratio. The mobile fraction was 65% and constant at the different C/P ratios. The relation between lipid diffusion and signal processing was measured following stimulation with 10 U/ml thrombin at 22 degrees C. There were only little differences in phosphatidylinositol metabolism, Ca2+ influx or mobilization and prostaglandin I2-induced formation of cyclic AMP. At 37 degrees C, cells with a high C/P ratio showed increased phosphatidylinositol metabolism, but these differences had no major effect on the Ca2+ responses. These data demonstrate that in megakaryoblasts the lateral diffusion of lipids is inversely correlated with the C/P ratio, but within the range of 0.20-0.31 the influence on signal processing is minor.
Asunto(s)
Fluidez de la Membrana , Transducción de Señal , Adenilil Ciclasas/metabolismo , Carbocianinas , Línea Celular , Colesterol/análisis , Colorantes Fluorescentes , Humanos , Metabolismo de los Lípidos , Lovastatina/análogos & derivados , Lovastatina/farmacología , Megacariocitos/metabolismo , Ácido Mevalónico/farmacología , Fosfolípidos/análisis , Simvastatina , Fosfolipasas de Tipo C/metabolismoRESUMEN
For measuring K+ efflux from exponentially growing neuroblastoma cells (clone Neuro-2A), two methods were used, a sampling method and a washing method. Both methods indicated that K+ efflux kinetics were as from a two-compartment system, but the two compartments could only be resolved completely using the washing method. A fast compartment, containing 143 +/- 16 nmol K+/10(6) cells, was found to be associated to the cell surface, and a slow compartment, containing 151 +/- 7 nmol K+/10(6) cells, was found to represent the intracellular K+. The rate constant of the slow compartment was 0.0164 +/-0.0005 min-1, and the K+ efflux rate was 2.46 +/- 0.14 nmol K+/10(6) cells per min. Using the appropriate conditions to measure K+ influx, the kinetics of influx were equal to the kinetics of efflux, indicating steady-state conditions. In addition a comparison was made between 42K+ and 86Rb+ as radioactive tracers for K+ flux. It was found that 86Rb+ was specifically bound on both the inside and the outside of the cells, and for this reason was not a suitable tracer for studying K+ flux kinetics in neuro-2A cells. A membrane potential of -42.9 +/- 1.3 mV and intracellular K+ activity of 108.1 +/- 3.0 mM were measured using conventional and ion-selective microelectrodes. A correlation was made between the K+ flux and electrophysiological data, using the equations of electrodiffusion theory. Thus, the permeabilities of K+ and Na+ were calculated as (3.9 +/- 0.4) . 10(-8) cm/s and (0.6 and 0.1) . 10(-8) cm/s respectively, together with K+ conductance of (2.8 +/- 0.3) . 10(-6) omega-1/cm2.
Asunto(s)
Neuroblastoma/metabolismo , Potasio/metabolismo , Rubidio/metabolismo , Animales , Transporte Biológico Activo , División Celular , Línea Celular , Membrana Celular/fisiología , Cinética , Matemática , Potenciales de la Membrana , Ratones , Neoplasias Experimentales/metabolismo , TermodinámicaRESUMEN
Neuronal differentiation from totipotent precursors in vitro, is thought to require two signals: first a biophysical state (cellular aggregation) followed by a biochemical signal (retinoic acid treatment). In investigating the properties of retinoic acid-differentiated embryonic stem cell lines. However, we noted that retinoic acid treatment without prior aggregation, is sufficient to induce expression of the neuronal markers GAP-43 and NF-165. In agreement, immunohistochemistry revealed the presence of GAP-43 positive cells in these embryonic stem cell monolayers after three days of retinoic acid (RA) treatment. Furthermore an NF-165 positive subpopulation of cells was clearly observed after 4-5 days of RA treatment. The expression of these neuronal markers coincided with the appearance of electrically excitable cells, as assayed with whole cell patch clamp recording. We conclude that for neuronal differentiation of totipotent embryonic stem cells in vitro, one biochemical signal, i.e. retinoic acid treatment, is sufficient.
Asunto(s)
Neuronas/citología , Células Madre/citología , Potenciales de Acción , Animales , Agregación Celular , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proteína GAP-43 , Glicoproteínas de Membrana/análisis , Ratones , Proteínas del Tejido Nervioso/análisis , Neuronas/fisiología , Técnicas de Placa-Clamp , Células Madre/fisiología , Tretinoina/farmacologíaRESUMEN
1. Addition of 3.5 mM ATP to mouse neuroblastoma Neuro-2A cells results in a selective enhancement of the plasma membrane permeability for Na+ relative to K+, as measured by cation flux measurements and electro-physiological techniques. 2. Addition of 3.5 mM ATP to Neuro-2A cells results in a 70% stimulation of the rate of active K+ -uptake by these cells, partly because of the enhanced plasma membrane permeability for Na+. Under these conditions the pumping activity of the Neuro-2A (Na+ +K+)-ATPase is optimally stimulated with respect to its various substrate ions. 3. External ATP significantly enhances the affinity of the Neuro-2A (Na+ +K+)-ATPase for ouabain, as measured by direct [3H]ouabain-binding studies and by inhibition studies of active K+ uptake. In the presence of 3.5 mM ATP and the absence of external K+ both techniques indicate an apparent dissociation constant for ouabain of 2 X 10(-6)M. Neuro-2A cells contain (3.5 +/- 0.7) X 10(5) ouabain-binding sites per cell, giving rise to an optimal pumping activity of (1.7 +/- 0.4) X 10(-20) mol K+/min per copy of (Na+ +K+)-ATPase at room temperature.
Asunto(s)
Adenosina Trifosfato/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Línea Celular , Potenciales de la Membrana/efectos de los fármacos , Ratones , Neuroblastoma , Ouabaína/metabolismo , Unión ProteicaRESUMEN
Receptor Protein-Tyrosine Phosphatases (RPTPs) belong to the superfamily of protein-tyrosine phosphatases and have the intrinsic ability to transduce signals across the cell membrane. We are beginning to understand the role of RPTPs in development of invertebrates, due to elegant genetic studies. In contrast, relatively little is known about the role of RPTPs in vertebrate development. Signalling by RPTPs has predominantly been studied in mammalian cell systems, which has led to important insights into potential ligands, into regulation of RPTP activity and into potential RPTP substrates. Here, we will introduce the RPTPs, and discuss the function of the LAR-subfamily of RPTPs. In addition, we focus on the function and signalling of the haematopoietic RPTP, CD45. Finally, we will discuss the structure and function of RPTPalpha, the RPTP that is the subject of our studies.
Asunto(s)
Proteínas del Tejido Nervioso/fisiología , Proteínas Tirosina Fosfatasas/fisiología , Receptores de Superficie Celular/fisiología , Transducción de Señal , Animales , Caenorhabditis elegans/embriología , Drosophila/embriología , Antígenos Comunes de Leucocito/fisiología , Ratones , Tejido Nervioso/embriología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores , Receptores de Superficie Celular/metabolismoRESUMEN
Signalling between cells in the developing vertebrate embryo is essential for normal embryonic development. In the mid 1970's, signal transduction research started at the Hubrecht Laboratory with special emphasis on analysis of the signalling mechanisms that direct cell proliferation and differentiation. The introduction of in vitro model systems contributed tremendously to the success of the signal transduction research at the Hubrecht Laboratory. Initially neuroblastoma cell lines, and later embryonal carcinoma and embryonal stem cells played an important role in identification of the molecular key players in developmental signalling. For instance, embryonal carcinoma cells were used to identify and characterise polypeptide growth factors. Growth factor signalling research was extended to analysis of growth factor receptor activation. Moreover, the second messenger systems that are linked to growth factor receptors were studied, as well as the nuclear responses to growth factor receptor activation. Finally, the role of growth factor signalling in differentiation was established using embryonal carcinoma cells. Here, we will review work that was characteristic for the growth factor receptor signalling research that was done at the Hubrecht Laboratory between 1980 and the early 1990's.
Asunto(s)
Sustancias de Crecimiento/fisiología , Receptores de Factores de Crecimiento/fisiología , Transducción de Señal/fisiología , Animales , Calcio/metabolismo , Línea Celular , Embrión de Mamíferos , Embrión no Mamífero , Receptores ErbB/metabolismo , Receptores ErbB/fisiología , Sustancias de Crecimiento/metabolismo , Fosfatos de Inositol/metabolismo , Sistemas de Mensajero Secundario/fisiología , Factores de Crecimiento Transformadores/metabolismoRESUMEN
An improved method is presented for a quantitative analysis of freeze-fractured biological membranes. The analysis provides the numerical distribution of intramembrane particles (IMP) as a function of their diameter and a measure for the lateral IMP-distribution as a function of the IMP-diameter in terms that allow a statistical comparison between different replicas. In analyzing the lateral IMP-distribution a distinction between random, aggregative and dispersive distributions can be made for different IMP-diameter classes. Such an analysis makes the interpretation of the ultrastructural appearance of biological membranes possible in terms of the dynamic behavior of the represented membrane components. The method is demonstrated by comparing the structure of the plasma membrane of synchronized neuroblastoma cells after glutaraldehyde and formaldehyde fixation at pH 6.0.
Asunto(s)
Membrana Celular/ultraestructura , Técnicas Citológicas , Animales , Línea Celular , Fijadores , Formaldehído , Técnica de Fractura por Congelación , Glutaral , Ratones , Microscopía Electrónica , NeuroblastomaRESUMEN
In murine C1300 neuroblastoma cells, clone Neuro 2A, the major fraction of the necessary increase in cell surface area during the cell cycle occurs within a short period around mitosis. During this period cell cycle-related modulations in a number of structural, dynamic and transport properties are most prominent. In this study we have examined the mechanism of rapid plasma membrane growth during mitosis, and the resulting changes in the ultrastructural features of the plasma membrane, by scanning and freeze-fracture electron microscopy as well as by electron microscopy of ultrathin sections. Our observations show that plasma membrane growth occurs by the fusion with and the incorporation into the plasma membrane of cytoplasmic multilamellar, lipidic membrane vesicles. Such vesicles are not observed at other times in the cell cycle. As a consequence, IMP-free domains appear transiently in the mitotic and early post-mitotic plasma membrane. Comparison of replicas prepared from glutaraldehyde-fixed cells and unfixed, ultrarapidly frozen cells showed that aldehyde fixation artefactually induces a bleb-like appearance of these domains. The IMP-free domains disappear in the G1-phase as a result of the mobilization and lateral redistribution of membrane components. It is argued that mitotic membrane growth by preferential incorporation of membrane lipids not only serves to accomodate for the necessary increase in cell surface area, but also provides a mechanism for plasma membrane-mediated regulation of the cell cycle.
Asunto(s)
Membrana Celular/ultraestructura , Neuroblastoma/ultraestructura , Animales , Células Clonales/ultraestructura , Técnica de Fractura por Congelación , Metabolismo de los Lípidos , Ratones , Microscopía Electrónica de Rastreo , MitosisRESUMEN
BACKGROUND: Dimerization is an important regulatory mechanism of single membrane-spanning receptors. For instance, activation of receptor protein-tyrosine kinases (RPTKs) involves dimerization. Structural, functional and biochemical studies suggested that the enzymatic counterparts of RPTKs, the receptor protein-tyrosine phosphatases (RPTPs), are inhibited by dimerization, but whether RPTPs actually dimerize in living cells remained to be determined. RESULTS: In order to assess RPTP dimerization, we have assayed Fluorescence Resonance Energy Transfer (FRET) between chimeric proteins of cyan- and yellow-emitting derivatives of green fluorescent protein, fused to RPTPalpha, using three different techniques: dual wavelength excitation, spectral imaging and fluorescence lifetime imaging. All three techniques suggested that FRET occurred between RPTPalpha -CFP and -YFP fusion proteins, and thus that RPTPalpha dimerized in living cells. RPTPalpha dimerization was constitutive, extensive and specific. RPTPalpha dimerization was consistent with cross-linking experiments, using a non-cell-permeable chemical cross-linker. Using a panel of deletion mutants, we found that the transmembrane domain was required and sufficient for dimerization. CONCLUSIONS: We demonstrate here that RPTPalpha dimerized constitutively in living cells, which may be mediated by the transmembrane domain, providing strong support for the model that dimerization is involved in regulation of RPTPs.
Asunto(s)
Proteínas Tirosina Fosfatasas/metabolismo , Receptores de Superficie Celular , Reactivos de Enlaces Cruzados/química , Dimerización , Transferencia de Energía , Fluorescencia , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Microscopía Fluorescente , Estructura Terciaria de Proteína , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores , Proteínas Recombinantes de Fusión/análisis , Espectrometría de Fluorescencia , Células Tumorales CultivadasRESUMEN
The regulation of phospholipase C has been investigated in both intact and streptolysin-O permeabilized human HeLa carcinoma cells. Stimulation of phospholipase C by histamine and guanosine-5'-O-thiotriphosphate (GTP[S]) requires the presence of at least 10 nM free Ca2+, but is not significantly further increased by raising [Ca2+]i to greater than 10(-6) M. The pH optimum of the inositol phosphate response is at pH 6.8, while small changes in intracellular pH, as occur during hormonal stimulation (0.2-0.4 unit) attenuate the histamine/GTP[S]-induced stimulation of phospholipase C. Increasing cellular cAMP levels, either through addition of cell permeable cAMP analogues to intact cells or by stimulation with isoproterenol, does not affect histamine responsiveness, arguing against cross-talk between both signalling pathways. In contrast, we found that the response to histamine and/or GTP[S] is largely inhibited after brief pretreatment of the cells with phorbol esters or synthetic diacylglycerol prior to permeabilization, suggesting that protein kinase C exerts feedback inhibition at the level of, or downstream from, the putative GTP-binding protein.
Asunto(s)
Calcio/farmacología , Guanosina Trifosfato/análogos & derivados , Histamina/farmacología , Fosfatidilinositoles/metabolismo , Proteína Quinasa C/metabolismo , Tionucleótidos/farmacología , Ácido Egtácico/farmacología , Guanosina Trifosfato/farmacología , Células HeLa/citología , Células HeLa/efectos de los fármacos , Células HeLa/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Fosfatos de Inositol/metabolismo , Cinética , Fosfolipasas de Tipo C/metabolismoRESUMEN
Addition of bradykinin to mouse N1E-115 neuroblastoma cells evokes a rapid but transient rise in cytoplasmic free Ca2+ concentration ([Ca2+]i). The [Ca2+]i rise is accompanied by a transient membrane hyperpolarization, due to a several-fold increase in K+ conductance, followed by a prolonged depolarizing phase. Pretreatment of the cells with a Ca2+-ionophore abolishes the hormone-induced hyperpolarization but leaves the depolarizing phase intact. The transient hyperpolarization can be mimicked by iontophoretic injection of IP3(1,4,5) or Ca2+, but not by injection of IP3(1,3,4), IP4(1,3,4,5) or Mg2+ into the cells. Instead, IP3(1,3,4) evokes a small but significant membrane depolarization in about 50% of the cells tested. Microinjected IP4(1,3,4,5) has no detectable effect, nor has treatment of the cells with phorbol esters. These results suggest that, while IP3(1,4,5) triggers the release of stored Ca2+ to hyperpolarize the membrane, IP3(1,3,4) may initiate a membrane depolarization.