RESUMEN
To circumvent the limited spatial resolution of fluorescent protein imaging, we are developing genetically encoded tags for electron microscopy (EM).
Asunto(s)
Péptidos de Penetración Celular/síntesis química , Diagnóstico por Imagen/métodos , Microscopía Electrónica/métodos , Animales , Péptidos de Penetración Celular/farmacocinética , Humanos , Ingeniería de Proteínas/métodosRESUMEN
Release of cytochrome c from mitochondria is a central event in apoptotic signaling. In this study, we utilized a cytochrome c fusion that binds fluorescent biarsenical ligands (cytochrome c-4CYS (cyt. c-4CYS)) as well as cytochrome c-green fluorescent protein (cyt. c-GFP) to measure its release from mitochondria in different cell types during apoptosis. In single cells, the kinetics of cyt. c-4CYS release was indistinguishable from that of cyt. c-GFP in apoptotic cells expressing both molecules. Lowering the temperature by 7 degrees C did not affect this corelease, but further separated cytochrome c release from the subsequent decrease in mitochondrial membrane potential (DeltaPsi(m)). Cyt. c-GFP rescued respiration in cells lacking endogenous cytochrome c, and the duration of cytochrome c release was approximately 5 min in a variety of cell types induced to die by various forms of cellular stress. In addition, we could observe no evidence of caspase-dependent amplification of cytochrome c release or changes in DeltaPsi(m) preceding the release of cyt. c-GFP. We conclude that there is a general mechanism responsible for cytochrome c release that proceeds in a single step that is independent of changes in DeltaPsi(m).
Asunto(s)
Apoptosis/fisiología , Citocromos c/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Biomarcadores , Dactinomicina/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Células Jurkat , Cinética , Ligandos , Potenciales de la Membrana/efectos de los fármacos , Microscopía por Video , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Inhibidores de la Síntesis de la Proteína/farmacología , Estaurosporina/farmacología , Temperatura , Factor de Necrosis Tumoral alfa/farmacología , Rayos UltravioletaRESUMEN
The complexity and specificity of many forms of signal transduction are widely suspected to require spatial microcompartmentation of protein kinase and phosphatase activities, yet current relevant imaging methods such as phosphorylation-specific antibodies or fluorescent peptide substrates require fixation or microinjection and lack temporal or spatial resolution. We present a genetically encoded fluorescent reporter for protein kinase A (PKA) consisting of fusions of cyan fluorescent protein, a phosphoamino acid binding domain (14-3-3tau), a consensus substrate for PKA, and yellow fluorescent protein. cAMP elevations cause 25-50% changes in the ratios of yellow to cyan emissions in live cells caused by phosphorylation-induced changes in fluorescence resonance energy transfer. The reporter response was accelerated by tethering to PKA holoenzyme and slowed by localization to the nucleus. We demonstrate that deliberate redistribution of a substrate or colocalizing a substrate and PKA can modulate its susceptibility to phosphorylation by the kinase. The successful design of a fluorescent reporter of PKA activity and its application for studying compartmentalized and dynamic modulation of kinases lays a foundation for studying targeting and compartmentation of PKA and other kinases and phosphatases.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Genes Reporteros , Secuencia de Aminoácidos , Animales , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Transferencia de Energía , Fluorescencia , Humanos , Cinética , Datos de Secuencia Molecular , Fosforilación , Especificidad por SustratoRESUMEN
The complexity and specificity of many forms of signal transduction are widely believed to require spatial compartmentation of protein kinase and phosphatase activities, yet existing methods for measuring kinase activities in cells lack generality or spatial or temporal resolution. We present three genetically encoded fluorescent reporters for the tyrosine kinases Src, Abl, and epidermal growth factor (EGF) receptor. The reporters consist of fusions of cyan fluorescent protein (CFP), a phosphotyrosine binding domain, a consensus substrate for the relevant kinase, and yellow fluorescent protein (YFP). Stimulation of kinase activities in living cells with addition of growth factors causes 20-35% changes in the ratios of yellow to cyan emissions because of phosphorylation-induced changes in fluorescence resonance energy transfer (FRET). Platelet-derived growth factor (PDGF) stimulated Abl activity most strongly in actin-rich membrane ruffles, supporting the importance of this tyrosine kinase in the regulation of cell morphology. These results establish a general strategy for nondestructively imaging dynamic protein tyrosine kinase activities with high spatial and temporal resolution in single living cells.
Asunto(s)
Genes Reporteros , Proteínas Luminiscentes/genética , Proteínas Tirosina Quinasas/metabolismo , Células 3T3 , Animales , Receptores ErbB/genética , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Fosforilación , Proteínas Tirosina Quinasas/genéticaRESUMEN
We report a new and simple technique for photo-mediated temporal and spatial control of gene activation in zebrafish embryos as an alternative to the gene 'knockdown' approach using antisense, morpholino-modified oligonucleotides (morpholinos). The synthetic compound 6-bromo-4-diazomethyl-7-hydroxycoumarin (Bhc-diazo) forms a covalent bond with the phosphate moiety of the sugar-phosphate backbone of RNA, a process known as caging. The 6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc) group binds to approximately 30 sites on the phosphate moieties per 1 kb of RNA sequence. Bhc-caged mRNA undergoes photolysis (uncaging) when exposed to long-wave ultraviolet light (350 to 365 nm). We show that Bhc-caged green fluorescent protein (Gfp) mRNA has severely reduced translational activity in vitro, whereas illumination of Bhc-caged mRNA with ultraviolet light leads to partial recovery of translational activity. Bhc-caged mRNA is highly stable in zebrafish embryos. In embryos injected with Bhc-caged Gfp mRNA at the one-cell stage, GFP protein expression and fluorescence is specifically induced by ultraviolet light. We also show that, consistent with results obtained using other methods, uncaging eng2a (which encodes the transcription factor Engrailed2a) in the head region during early development causes a severe reduction in the size of the eye and enhanced development of the midbrain and the midbrain-hindbrain boundary at the expense of the forebrain.
Asunto(s)
Embrión no Mamífero/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Fotólisis , ARN Mensajero/administración & dosificación , Animales , Compuestos Azo/química , Compuestos Azo/farmacología , Cumarinas/química , Cumarinas/farmacología , ADN/administración & dosificación , ADN/química , ADN/efectos de la radiación , Embrión no Mamífero/metabolismo , Embrión no Mamífero/patología , Anomalías del Ojo/inducido químicamente , Anomalías del Ojo/embriología , Anomalías del Ojo/patología , Regulación del Desarrollo de la Expresión Génica/efectos de la radiación , Proteínas Fluorescentes Verdes , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/farmacología , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Microinyecciones , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/farmacología , Malformaciones del Sistema Nervioso/inducido químicamente , Malformaciones del Sistema Nervioso/embriología , Malformaciones del Sistema Nervioso/patología , Oryzias/genética , Prosencéfalo/anomalías , Prosencéfalo/efectos de los fármacos , Prosencéfalo/metabolismo , Biosíntesis de Proteínas/efectos de la radiación , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/química , ARN Mensajero/efectos de la radiación , Activación Transcripcional , Rayos Ultravioleta , Pez Cebra , Proteínas de Pez CebraRESUMEN
Two new fluorescent sensors for Zn(2+) that utilize fluorescein as a reporting group, Zinpyr-1 and Zinpyr-2, have been synthesized and characterized. Zinpyr-1 is prepared in one step via a Mannich reaction, and Zinpyr-2 is obtained in a multistep synthesis that utilizes 4',5'-fluorescein dicarboxaldehyde as a key intermediate. Both Zinpyr sensors have excitation and emission wavelengths in the visible range ( approximately 500 nm), dissociation constants (K(d1)) for Zn(2+) of <1 nM, quantum yields approaching unity (Phi = approximately 0.9), and cell permeability, making them well-suited for intracellular applications. A 3- to 5-fold fluorescent enhancement is observed under simulated physiological conditions corresponding to the binding of the Zn(2+) cation to the sensor, which inhibits a photoinduced electron transfer (PET) quenching pathway. The X-ray crystal structure of a 2:1 Zn(2+):Zinpyr-1 complex has also been solved, and is the first structurally characterized example of a complex of fluorescein substituted with metal binding ligands.
Asunto(s)
Fluoresceínas/síntesis química , Colorantes Fluorescentes , Zinc/análisis , Animales , Células COS/química , Cationes Bivalentes , Cristalografía por Rayos X , Fluoresceínas/química , Espectrometría de Fluorescencia , Zinc/química , Zinc/farmacologíaRESUMEN
BACKGROUND: Fluorescence resonance energy transfer (FRET) is a powerful technique for measuring molecular interactions at Angstrom distances. We present a new method for FRET that utilizes the unique spectral properties of variants of the green fluorescent protein (GFP) for large-scale analysis by flow cytometry. METHODS: The proteins of interest are fused in frame separately to the cyan fluorescent protein (CFP) or the yellow fluorescent protein (YFP). FRET between these differentially tagged fusion proteins is analyzed using a dual-laser FACSVantage cytometer. RESULTS: We show that homotypic interactions between individual receptor chains of tumor necrosis factor receptor (TNFR) family members can be detected as FRET from CFP-tagged receptor chains to YFP-tagged receptor chains. Noncovalent molecular complexation can be detected as FRET between fusions of CFP and YFP to either the intracellular or extracellular regions of the receptor chains. The specificity of the assay is demonstrated by the absence of FRET between heterologous receptor pairs that do not biochemically associate with each other. Interaction between a TNFR-like receptor (Fas/CD95/Apo-1) and a downstream cytoplasmic signaling component (FADD) can also be demonstrated by flow cytometric FRET analysis. CONCLUSIONS: The utility of spectral variants of GFP in flow cytometric FRET analysis of membrane receptors is demonstrated. This method of analyzing FRET allows probing of noncovalent molecular interactions that involve both the intracellular and extracellular regions of membrane proteins as well as proteins within the cells. Unlike biochemical methods, FRET allows the quantitative determination of noncovalent molecular associations at Angstrom level in living cells. Moreover, flow cytometry allows quantitative analyses to be carried out on a cell-by-cell basis on large number of cells. Published 2001 Wiley-Liss, Inc.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Bacterianas/análisis , Proteínas Portadoras/análisis , Citometría de Flujo/métodos , Proteínas Luminiscentes/análisis , Receptores de Superficie Celular/análisis , Receptores del Factor de Necrosis Tumoral/análisis , Transferencia de Energía , Proteína de Dominio de Muerte Asociada a Fas , Proteínas Fluorescentes Verdes , Transducción de Señal/fisiología , Espectrometría de Fluorescencia , Análisis Espectral/métodosRESUMEN
A precise pH gradient between organelles of the regulated secretory pathway is required for sorting and processing of prohormones. We studied pH regulation in live endocrine cells by targeting biotin-based pH indicators to cellular organelles expressing avidin-chimera proteins. In AtT-20 cells, we found that steady-state pH decreased from the endoplasmic reticulum (ER) (pH(ER) = 7.4 +/- 0.2, mean +/- S.D.) to Golgi (pH(G) = 6.2 +/- 0.4) to mature secretory granules (MSGs) (pH(MSG) = 5.5 +/- 0.4). Golgi and MSGs required active H(+) v-ATPases for acidification. ER, Golgi, and MSG steady-state pH values were also dependent upon the different H(+) leak rates across each membrane. However, neither steady-state pH(MSG) nor rates of passive H(+) leak were affected by Cl(-)-free solutions or valinomycin, indicating that MSG membrane potential was small and not a determinant of pH(MSG). Therefore, our data do not support earlier suggestions that organelle acidification is primarily regulated by Cl(-) conductances. Measurements of H(+) leak rates, buffer capacities, and estimates of surface areas and volumes of these organelles were applied to a mathematical model to determine the H(+) permeability (P(H+)) of each organelle membrane. We found that P(H+) decreased progressively from ER to Golgi to MSGs, and proper acidification of Golgi and MSGs required gradual decreases in P(H+) and successive increases in the active H(+) pump density.
Asunto(s)
Concentración de Iones de Hidrógeno , Membranas Intracelulares/fisiología , Orgánulos/fisiología , Hipófisis/fisiología , ATPasas de Translocación de Protón Vacuolares , Animales , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Cloruros/metabolismo , Retículo Endoplásmico/fisiología , Aparato de Golgi/fisiología , Homeostasis , Membranas Intracelulares/efectos de los fármacos , Cinética , Potenciales de la Membrana/fisiología , Ratones , Modelos Biológicos , Modelos Químicos , Orgánulos/efectos de los fármacos , Hormonas Hipofisarias/metabolismo , Precursores de Proteínas/metabolismo , ATPasas de Translocación de Protón/metabolismo , Vesículas Secretoras/fisiología , Células Tumorales Cultivadas , Valinomicina/farmacologíaRESUMEN
Yellow mutants of the green fluorescent protein (YFP) are crucial constituents of genetically encoded indicators of signal transduction and fusions to monitor protein-protein interactions. However, previous YFPs show excessive pH sensitivity, chloride interference, poor photostability, or poor expression at 37 degrees C. Protein evolution in Escherichia coli has produced a new YFP named Citrine, in which the mutation Q69M confers a much lower pK(a) (5.7) than for previous YFPs, indifference to chloride, twice the photostability of previous YFPs, and much better expression at 37 degrees C and in organelles. The halide resistance is explained by a 2.2-A x-ray crystal structure of Citrine, showing that the methionine side chain fills what was once a large halide-binding cavity adjacent to the chromophore. Insertion of calmodulin within Citrine or fusion of cyan fluorescent protein, calmodulin, a calmodulin-binding peptide and Citrine has generated improved calcium indicators. These chimeras can be targeted to multiple cellular locations and have permitted the first single-cell imaging of free [Ca(2+)] in the Golgi. Citrine is superior to all previous YFPs except when pH or halide sensitivity is desired and is particularly advantageous within genetically encoded fluorescent indicators of physiological signals.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Luminiscentes/química , Proteínas Luminiscentes/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/efectos de la radiación , Sitios de Unión , Calcio/metabolismo , Calmodulina/metabolismo , Cloruros/farmacología , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Colorantes Fluorescentes , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Proteínas Luminiscentes/efectos de la radiación , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fotólisis , Reacción en Cadena de la Polimerasa , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
To investigate the dynamics of guanosine 3',5'-cyclic monophosphate (cGMP) in single living cells, we constructed genetically encoded, fluorescent cGMP indicators by bracketing cGMP-dependent protein kinase (cGPK), minus residues 1-77, between cyan and yellow mutants of green fluorescent protein. cGMP decreased fluorescence resonance energy transfer (FRET) and increased the ratio of cyan to yellow emissions by up to 1.5-fold with apparent dissociation constants of approximately 2 microM and >100:1 selectivity for cGMP over cAMP. To eliminate constitutive kinase activity, Thr(516) of cGPK was mutated to Ala. Emission ratio imaging of the indicators transfected into rat fetal lung fibroblast (RFL)-6 showed cGMP transients resulting from activation of soluble and particulate guanylyl cyclase, respectively, by nitric oxide (NO) and C-type natriuretic peptide (CNP). Whereas all naive cells tested responded to CNP, only 68% responded to NO. Both sets of signals showed large and variable (0.5-4 min) latencies. The phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) did not elevate cGMP on its own but consistently amplified responses to NO or CNP, suggesting that basal activity of guanylate cyclase is very low and emphasizing the importance of PDEs in cGMP recycling. A fraction of RFL cells showed slowly propagating tides of cGMP spreading across the cell in response to delocalized application of NO. Biolistically transfected Purkinje neurons showed cGMP responses to parallel fiber activity and NO donors, confirming that single-cell increases in cGMP occur under conditions appropriate to cause synaptic plasticity.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Colorantes Fluorescentes , Proteínas Luminiscentes/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Proteínas Quinasas Dependientes de GMP Cíclico/química , Transferencia de Energía , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Datos de Secuencia Molecular , Ratas , SpodopteraAsunto(s)
Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Mutación , Espectrometría de Fluorescencia/métodos , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Línea Celular , Cromatografía , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Microscopía Fluorescente/métodos , Modelos Teóricos , Datos de Secuencia Molecular , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Factores de Tiempo , Zinc/farmacologíaAsunto(s)
Avidina/química , Avidina/metabolismo , Biotina/metabolismo , Microscopía Fluorescente/métodos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Animales , Biotina/síntesis química , Línea Celular , Retículo Endoplásmico/metabolismo , Fluoresceína/química , Fluoresceína/metabolismo , Colorantes Fluorescentes/farmacología , Aparato de Golgi/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Membranas Intracelulares/metabolismo , Modelos Biológicos , Transporte de Proteínas , ProtonesAsunto(s)
Bioquímica/métodos , Cisteína/química , Microscopía Fluorescente/métodos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Fluoresceínas/síntesis química , Fluoresceínas/farmacología , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/farmacología , Humanos , Datos de Secuencia Molecular , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/farmacología , TransfecciónRESUMEN
DsRed is a recently cloned 28-kDa fluorescent protein responsible for the red coloration around the oral disk of a coral of the Discosoma genus. DsRed has attracted tremendous interest as a potential expression tracer and fusion partner that would be complementary to the homologous green fluorescent protein from Aequorea, but very little is known of the biochemistry of DsRed. We now show that DsRed has a much higher extinction coefficient and quantum yield than previously reported, plus excellent resistance to pH extremes and photobleaching. In addition, its 583-nm emission maximum can be further shifted to 602 nm by mutation of Lys-83 to Met. However, DsRed has major drawbacks, such as strong oligomerization and slow maturation. Analytical ultracentrifugation proves DsRed to be an obligate tetramer in vitro, and fluorescence resonance energy transfer measurements and yeast two-hybrid assays verify oligomerization in live cells. Also, DsRed takes days to ripen fully from green to red in vitro or in vivo, and mutations such as Lys-83 to Arg prevent the color change. Many potential cell biological applications of DsRed will require suppression of the tetramerization and acceleration of the maturation.
Asunto(s)
Cnidarios/metabolismo , Proteínas Luminiscentes/metabolismo , Secuencia de Aminoácidos , Animales , Concentración de Iones de Hidrógeno , Cinética , Proteínas Luminiscentes/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Técnicas del Sistema de Dos Híbridos , Proteína Fluorescente RojaRESUMEN
DsRed, a brilliantly red fluorescent protein, was recently cloned from Discosoma coral by homology to the green fluorescent protein (GFP) from the jellyfish Aequorea. A core question in the biochemistry of DsRed is the mechanism by which the GFP-like 475-nm excitation and 500-nm emission maxima of immature DsRed are red-shifted to the 558-nm excitation and 583-nm emission maxima of mature DsRed. After digestion of mature DsRed with lysyl endopeptidase, high-resolution mass spectra of the purified chromophore-bearing peptide reveal that some of the molecules have lost 2 Da relative to the peptide analogously prepared from a mutant, K83R, that stays green. Tandem mass spectrometry indicates that the bond between the alpha-carbon and nitrogen of Gln-66 has been dehydrogenated in DsRed, extending the GFP chromophore by forming C==N==C==O at the 2-position of the imidazolidinone. This acylimine substituent quantitatively accounts for the red shift according to quantum mechanical calculations. Reversible hydration of the C==N bond in the acylimine would explain why denaturation shifts mature DsRed back to a GFP-like absorbance. The C==N bond hydrolyses upon boiling, explaining why DsRed shows two fragment bands on SDS/PAGE. This assay suggests that conversion from green to red chromophores remains incomplete even after prolonged aging.
Asunto(s)
Compuestos Cromogénicos/química , Cnidarios/química , Proteínas Luminiscentes/química , Animales , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Espectrometría de Masas , Conformación Proteica , Desnaturalización Proteica , Proteína Fluorescente RojaRESUMEN
Gene expression of intrinsically fluorescent proteins in biological systems offers new noninvasive windows into cellular function, but optimization of these probes relies on understanding their molecular spectroscopy, dynamics, and structure. Here, the photophysics of red fluorescent protein (dsRed) from discosoma (coral), providing desired longer emission/absorption wavelengths, and an improved yellow fluorescent protein mutant (Citrine) (S65G/V68L/Q69 M/S72A/T203Y) for significant comparison, are characterized by using fluorescence correlation spectroscopy and time-correlated single-photon counting. dsRed fluorescence decays as a single exponential with a 3.65 +/- 0.07-ns time constant, indicating a single emitting state/species independent of pH 4.4-9.0, in contrast with Citrine. However, laser excitation drives reversible fluorescence flicker at 10(3)-10(4) Hz between dark and bright states with a constant partition fraction f(1) = 0.42 +/- 0.06 and quantum yield of approximately 3 x 10(-3). Unlike Citrine (pKa approximately 5.7), pH-dependent proton binding is negligible (pH 3. 9-11) in dsRed. Time-resolved anisotropy of dsRed reveals rapid depolarization (211 +/- 6 ps) plus slow rotational motion (53 +/- 8 ns), in contrast with a single rotational time (16 +/- 2 ns) for Citrine. The molecular dimensions, calculated from rotational and translational diffusion, indicate that dsRed is hydrodynamically 3.8 +/- 0.4 times larger than predicted for a monomer, which suggests an oligomer (possibly a tetramer) configuration even at approximately 10(-9) M. The fast depolarization is attributed to intraoligomer energy transfer between mobile nonparallel chromophores with the initial anisotropy implying a 24 +/- 3 degrees depolarization angle. Large two-photon excitation cross sections ( approximately 100 GM at 990 nm for dsRed and approximately 50 GM at 970 nm for Citrine), advantageous for two-photon-fluorescence imaging in cells, are measured.
Asunto(s)
Proteínas Bacterianas/química , Cnidarios/química , Proteínas Luminiscentes/química , Animales , Polarización de Fluorescencia , Desnaturalización Proteica , Proteína Fluorescente RojaRESUMEN
Cytosolic calcium oscillations control signaling in animal cells, whereas in plants their importance remains largely unknown. In wild-type Arabidopsis guard cells abscisic acid, oxidative stress, cold, and external calcium elicited cytosolic calcium oscillations of differing amplitudes and frequencies and induced stomatal closure. In guard cells of the V-ATPase mutant det3, external calcium and oxidative stress elicited prolonged calcium increases, which did not oscillate, and stomatal closure was abolished. Conversely, cold and abscisic acid elicited calcium oscillations in det3, and stomatal closure occurred normally. Moreover, in det3 guard cells, experimentally imposing external calcium-induced oscillations rescued stomatal closure. These data provide genetic evidence that stimulus-specific calcium oscillations are necessary for stomatal closure.
Asunto(s)
Arabidopsis/fisiología , Señalización del Calcio , Hojas de la Planta/fisiología , ATPasas de Translocación de Protón Vacuolares , Ácido Abscísico/farmacología , Arabidopsis/citología , Arabidopsis/genética , Calcio/metabolismo , Membrana Celular/metabolismo , Frío , Retículo Endoplásmico/metabolismo , Genes de Plantas , Peróxido de Hidrógeno/farmacología , Potenciales de la Membrana , Mutación , Estrés Oxidativo , Hojas de la Planta/citología , Potasio/metabolismo , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismo , Vacuolas/metabolismoRESUMEN
Arsenite is a potent environmental toxin that causes various pathologies including cancers and skin disorders. Arsenite is believed to exert its biological effects through reaction with exposed sulfhydryl groups, especially pairs of adjacent thiols. Here, we describe the mechanism by which arsenite affects the NF-kappaB signaling pathway. Activation of transcription factor NF-kappaB depends on the integrity of the IkappaB kinase (IKK) complex. We found that arsenite potently inhibits NF-kappaB and IKK activation by binding to Cys-179 in the activation loop of the IKK catalytic subunits, IKKalpha/beta. The affinity of IKKbeta for trivalent arsenic was verified in vitro by the ability of IKKbeta to enhance the fluorescence of an arsenic-substituted fluorescein dye. The addition of 1,2-dithiol antidotes or replacement of Cys-179 with an alanine residue abolished dye binding to and arsenite inhibition of IKKbeta. Overexpression of IKKbeta (C179A) protects NF-kappaB from inhibition by arsenite, indicating that despite the involvement of a large number of distinct gene products in this activation pathway, the critical target for inhibition by arsenite is on the IKK catalytic subunits.
Asunto(s)
Arsenitos/farmacología , Cisteína/metabolismo , FN-kappa B/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Secuencia de Aminoácidos , Dominio Catalítico , Línea Celular , Cisteína/genética , Activación Enzimática/efectos de los fármacos , Fluoresceínas/metabolismo , Fluorescencia , Humanos , Quinasa I-kappa B , Datos de Secuencia Molecular , Mutación , FN-kappa B/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Subunidades de Proteína , Alineación de Secuencia , TransfecciónRESUMEN
Electrophysiology and optical indicators have been used in vertebrate systems to investigate excitable cell firing and calcium transients, but both techniques have been difficult to apply in organisms with powerful reverse genetics. To overcome this limitation, we expressed cameleon proteins, genetically encoded calcium indicators, in the pharyngeal muscle of the nematode worm Caenorhabditis elegans. In intact transgenic animals expressing cameleons, fluorescence ratio changes accompanied muscular contraction, verifying detection of calcium transients. By comparing the magnitude and duration of calcium influx in wild-type and mutant animals, we were able to determine the effects of calcium channel proteins on pharyngeal calcium transients. We also successfully used cameleons to detect electrically evoked calcium transients in individual C. elegans neurons. This technique therefore should have broad applications in analyzing the regulation of excitable cell activity in genetically tractable organisms.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/metabolismo , Calcio/metabolismo , Neuronas/metabolismo , Músculos Faríngeos/inervación , Animales , Canales de Calcio/genética , Canales de Calcio/fisiología , Regulación hacia Abajo , Electrofisiología , Proteínas del Helminto/genética , Proteínas Musculares/genética , Mutación/fisiología , Óptica y Fotónica , Isoformas de Proteínas/fisiologíaRESUMEN
Heterozygous mutations encoding abnormal forms of the death receptor Fas dominantly interfere with Fas-induced lymphocyte apoptosis in human autoimmune lymphoproliferative syndrome. This effect, rather than depending on ligand-induced receptor oligomerization, was found to stem from ligand- independent interaction of wild-type and mutant Fas receptors through a specific region in the extracellular domain. Preassociated Fas complexes were found in living cells by means of fluorescence resonance energy transfer between variants of green fluorescent protein. These results show that formation of preassociated receptor complexes is necessary for Fas signaling and dominant interference in human disease.