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1.
FASEB J ; 28(3): 1375-85, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24334549

RESUMEN

The ß-strands of GFP form a rigid barrel that protects the chromophore from external influence. Herein, we identified specific mutations in ß-strand 7 that render the chromophore sensitive to interactions of GFP with another protein domain. In the process of converting the FRET-based protein kinase A (PKA) sensor AKAR2 into a single-wavelength PKA sensor containing a GFP and a quencher, we discovered that the quencher was not required and that the sensor response relied on changes in GFP intrinsic fluorescence. The identified mutations in ß-strand 7 render GFP fluorescence intensity and lifetime sensitive to conformational changes of the PKA-sensing domain. In addition, sensors engineered from the GCaMP2 calcium indicator to incorporate a conformation-sensitive GFP (csGFP) exhibited calcium-dependent fluorescence changes. We further demonstrate that single GFP sensors report PKA dynamics in dendritic spines of neurons from brain slices on 2-photon imaging with a high signal-to-baseline ratio and minimal photobleaching. The susceptibility of GFP variants to dynamic interactions with other protein domains provides a new approach to generate single wavelength biosensors for high-resolution imaging.


Asunto(s)
Técnicas Biosensibles , Proteínas Fluorescentes Verdes/genética , Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fluorescencia , Proteínas Fluorescentes Verdes/química
2.
Anal Bioanal Chem ; 407(14): 4183-93, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25814274

RESUMEN

It is generally acknowledged that the popular cyan and yellow fluorescent proteins carried by genetically encoded reporters suffer from strong pH sensitivities close to the physiological pH range. We studied the consequences of these pH responses on the intracellular signals of model Förster resonant energy transfer (FRET) tandems and FRET-based reporters of cAMP-dependent protein kinase activity (AKAR) expressed in the cytosol of living BHK cells, while changing the intracellular pH by means of the nigericin ionophore. Although the simultaneous pH sensitivities of the donor and the acceptor may mask each other in some cases, the magnitude of the perturbations can be very significant, as compared to the functional response of the AKAR biosensor. Replacing the CFP donor by the spectrally identical, but pH-insensitive Aquamarine variant (pK1/2 = 3.3) drastically modifies the biosensor pH response and gives access to the acid transition of the yellow acceptor. We developed a simple model of pH-dependent FRET and used it to describe the expected pH-induced changes in fluorescence lifetime and ratiometric signals. This model qualitatively accounts for most of the observations, but reveals a complex behavior of the cytosolic AKAR biosensor at acid pHs, associated to additional FRET contributions. This study underlines the major and complex impact of pH changes on the signal of FRET reporters in the living cell.


Asunto(s)
Proteínas Bacterianas/química , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Fluorescentes Verdes/química , Proteínas Luminiscentes/química , Animales , Línea Celular , Cricetinae , Concentración de Iones de Hidrógeno , Ionóforos/farmacología , Nigericina/farmacología
3.
J Physiol ; 591(13): 3197-214, 2013 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-23551948

RESUMEN

The cAMP/protein kinase A (PKA) signalling cascade is ubiquitous, and each step in this cascade involves enzymes that are expressed in multiple isoforms. We investigated the effects of this diversity on the integration of the pathway in the target cell by comparing prefrontal cortical neurones with striatal neurones which express a very specific set of signalling proteins. The prefrontal cortex and striatum both receive dopaminergic inputs and we analysed the dynamics of the cAMP/PKA signal triggered by dopamine D1 receptors in these two brain structures. Biosensor imaging in mouse brain slice preparations showed profound differences in the D1 response between pyramidal cortical neurones and striatal medium spiny neurones: the cAMP/PKA response was much stronger, faster and longer lasting in striatal neurones than in pyramidal cortical neurones. We identified three molecular determinants underlying these differences: different activities of phosphodiesterases, particularly those of type 4, which strongly damp the cAMP signal in the cortex but not in the striatum; stronger adenylyl cyclase activity in the striatum, generating responses with a faster onset than in the cortex; and DARPP-32, a phosphatase inhibitor which prolongs PKA action in the striatum. Striatal neurones were also highly responsive in terms of gene expression since a single sub-second dopamine stimulation is sufficient to trigger c-Fos expression in the striatum, but not in the cortex. Our data show how specific molecular elements of the cAMP/PKA signalling cascade selectively enable the principal striatal neurones to respond to brief dopamine stimuli, a critical process in incentive learning.


Asunto(s)
Cuerpo Estriado/fisiología , Dopamina/fisiología , Neuronas/fisiología , 1-Metil-3-Isobutilxantina/farmacología , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/farmacología , Animales , Técnicas Biosensibles , Colforsina/farmacología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Agonistas de Dopamina/farmacología , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Inhibidores de Fosfodiesterasa/farmacología , Corteza Prefrontal/fisiología , Proteínas Proto-Oncogénicas c-fos/metabolismo
4.
J Neurosci ; 30(17): 6143-51, 2010 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-20427672

RESUMEN

We investigated the role of phosphodiesterases (PDEs) in the integration of cAMP signals and protein kinase A (PKA) activity following beta-adrenergic stimulation, by carrying out real-time imaging of male mouse pyramidal cortical neurons expressing biosensors to monitor cAMP levels (Epac1-camps and Epac2-camps300) or PKA activity (AKAR2). In the soma, isoproterenol (ISO) increased the PKA signal to approximately half the maximal response obtained with forskolin, with a characteristic beta(1) pharmacology and an EC(50) of 4.5 nm. This response was related to free cAMP levels in the submicromolar range. The specific type 4 PDE (PDE4) inhibitor rolipram had a very small effect alone, but strongly potentiated the PKA response to ISO. Blockers of other PDEs had no effect. PDE4 thus acts as a brake in the propagation of the beta(1)-adrenergic signal from the membrane to the bulk somatic cytosol. The results for a submembrane domain were markedly different, whether recorded with a PKA-sensitive potassium current related to the slow AHP or by two-photon imaging of small distal dendrites. The responses to ISO were stronger than in the bulk cytosol. This is consistent with the cAMP/PKA signal being strong at the membrane, as shown by electrophysiology, and favored in cellular domains with a high surface area to volume ratio, in which this signal was detected by imaging. Rolipram alone also produced a strong cAMP/PKA signal, revealing tonic cAMP production. PDE4 thus appears as a crucial integrator with different physiological implications in different subcellular domains.


Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Lóbulo Parietal/enzimología , Células Piramidales/enzimología , Adenilil Ciclasas/metabolismo , Agonistas de Receptores Adrenérgicos beta 1 , Agonistas Adrenérgicos beta/farmacología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Membrana Celular/metabolismo , Fármacos del Sistema Nervioso Central/farmacología , Colforsina/farmacología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dendritas/efectos de los fármacos , Dendritas/enzimología , Dendritas/metabolismo , Técnicas In Vitro , Isoproterenol/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Lóbulo Parietal/efectos de los fármacos , Lóbulo Parietal/metabolismo , Inhibidores de Fosfodiesterasa 4 , Inhibidores de Fosfodiesterasa/farmacología , Potasio/metabolismo , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Rolipram/farmacología
5.
J Biol Chem ; 285(10): 6922-36, 2010 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-20048388

RESUMEN

The RecQ family helicases catalyze the DNA unwinding reaction in an ATP hydrolysis-dependent manner. We investigated the mechanism of DNA unwinding by the Escherichia coli RecQ helicase using a new sensitive helicase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. The FCCS-based assay can be used to measure the unwinding activity under both single and multiple turnover conditions with no limitation related to the size of the DNA strands constituting the DNA substrate. We found that the monomeric helicase was sufficient to perform the unwinding of short DNA substrates. However, a significant increase in the activity was observed using longer DNA substrates, under single turnover conditions, originating from the simultaneous binding of multiple helicase monomers to the same DNA molecule. This functional cooperativity was strongly dependent on several factors, including DNA substrate length, the number and size of single-stranded 3'-tails, and the temperature. Regarding the latter parameter, a strong cooperativity was observed at 37 degrees C, whereas only modest or no cooperativity was observed at 25 degrees C regardless of the nature of the DNA substrate. Consistently, the functional cooperativity was found to be tightly associated with a cooperative DNA binding mode. We also showed that the cooperative binding of helicase to the DNA substrate indirectly accounts for the sigmoidal dependence of unwinding activity on ATP concentration, which also occurs only at 37 degrees C but not at 25 degrees C. Finally, we further examined the influences of spontaneous DNA rehybridization (after helicase translocation) and the single-stranded DNA binding property of helicase on the unwinding activity as detected in the FCCS assay.


Asunto(s)
ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , ADN/química , ADN/metabolismo , Escherichia coli/enzimología , RecQ Helicasas/metabolismo , Espectrometría de Fluorescencia/métodos , ADN/genética , ADN de Cadena Simple/genética , Escherichia coli/genética , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , RecQ Helicasas/genética , Espectrometría de Fluorescencia/instrumentación
6.
J Neurosci Res ; 88(4): 695-711, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19798746

RESUMEN

Responses of three bioluminescent Ca(2+) sensors were studied in vitro and in neurons from brain slices. These sensors consisted of tandem fusions of green fluorescent protein (GFP) with the photoproteins aequorin, obelin, or a mutant aequorin with high Ca(2+) sensitivity. Kinetics of GFP-obelin responses to a saturating Ca(2+) concentration were faster than those of GFP-aequorin at all Mg(2+) concentrations tested, whereas GFP-mutant aequorin responses were the slowest. GFP-photoproteins were efficiently expressed in pyramidal neurons following overnight incubation of acute neocortical slices with recombinant Sindbis viruses. Expression of GFP-photoproteins did not result in conspicuous modification of morphological or electrophysiological properties of layer V pyramidal cells. The three sensors allowed the detection of Ca(2+) transients associated with action potential discharge in single layer V pyramidal neurons. In these neurons, depolarizing steps of increasing amplitude elicited action potential discharge of increasing frequency. Bioluminescent responses of the three sensors were similar in several respects: detection thresholds, an exponential increase with stimulus intensity, photoprotein consumptions, and kinetic properties. These responses, which were markedly slower than kinetics measured in vitro, increased linearly during the action potential discharge and decayed exponentially at the end of the discharge. Onset slopes increased with stimulus intensity, whereas decay kinetics remained constant. Dendritic light emission contributed to whole-field responses, but the spatial resolution of bioluminescence imaging was limited to the soma and proximal apical dendrite. Nonetheless, the high signal-to-background ratio of GFP-photoproteins allowed the detection of Ca(2+) transients associated with 5 action potentials in single neurons upon whole-field bioluminescence recordings.


Asunto(s)
Calcio/metabolismo , Corteza Cerebral/citología , Proteínas Luminiscentes/metabolismo , Neuronas/metabolismo , Potenciales de Acción/fisiología , Animales , Animales Recién Nacidos , Señalización del Calcio/fisiología , Línea Celular Transformada , Cricetinae , Proteínas Fluorescentes Verdes/genética , Técnicas In Vitro , Líquido Intracelular/metabolismo , Cinética , Luz , Proteínas Luminiscentes/genética , Técnicas de Placa-Clamp/métodos , Ratas , Ratas Wistar
7.
Nat Commun ; 11(1): 6249, 2020 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-33288743

RESUMEN

The bioactive vitamin D3, 1α,25(OH)2D3, plays a central role in calcium homeostasis by controlling the activity of the vitamin D receptor (VDR) in various tissues. Hypercalcemia secondary to high circulating levels of vitamin D3 leads to hypercalciuria, nephrocalcinosis and renal dysfunctions. Current therapeutic strategies aim at limiting calcium intake, absorption and resorption, or 1α,25(OH)2D3 synthesis, but are poorly efficient. In this study, we identify WBP4 as a new VDR interactant, and demonstrate that it controls VDR subcellular localization. Moreover, we show that the vitamin D analogue ZK168281 enhances the interaction between VDR and WBP4 in the cytosol, and normalizes the expression of VDR target genes and serum calcium levels in 1α,25(OH)2D3-intoxicated mice. As ZK168281 also blunts 1α,25(OH)2D3-induced VDR signaling in fibroblasts of a patient with impaired vitamin D degradation, this VDR antagonist represents a promising therapeutic option for 1α,25(OH)2D3-induced hypercalcemia.


Asunto(s)
Calcio/metabolismo , Hipercalcemia/metabolismo , Receptores de Calcitriol/metabolismo , Vitamina D/farmacología , Animales , Calcitriol/análogos & derivados , Calcitriol/farmacología , Línea Celular , Línea Celular Tumoral , Citosol/metabolismo , Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Hipercalcemia/genética , Hipercalcemia/prevención & control , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ratas , Receptores de Calcitriol/genética , Vitamina D/análogos & derivados
8.
Cell Metab ; 31(3): 503-517.e8, 2020 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-32130882

RESUMEN

Alteration of brain aerobic glycolysis is often observed early in the course of Alzheimer's disease (AD). Whether and how such metabolic dysregulation contributes to both synaptic plasticity and behavioral deficits in AD is not known. Here, we show that the astrocytic l-serine biosynthesis pathway, which branches from glycolysis, is impaired in young AD mice and in AD patients. l-serine is the precursor of d-serine, a co-agonist of synaptic NMDA receptors (NMDARs) required for synaptic plasticity. Accordingly, AD mice display a lower occupancy of the NMDAR co-agonist site as well as synaptic and behavioral deficits. Similar deficits are observed following inactivation of the l-serine synthetic pathway in hippocampal astrocytes, supporting the key role of astrocytic l-serine. Supplementation with l-serine in the diet prevents both synaptic and behavioral deficits in AD mice. Our findings reveal that astrocytic glycolysis controls cognitive functions and suggest oral l-serine as a ready-to-use therapy for AD.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Astrocitos/metabolismo , Disfunción Cognitiva/metabolismo , Glucólisis , Serina/biosíntesis , Administración Oral , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/fisiopatología , Animales , Astrocitos/efectos de los fármacos , Sitios de Unión , Encéfalo/patología , Encéfalo/fisiopatología , Disfunción Cognitiva/patología , Disfunción Cognitiva/fisiopatología , Metabolismo Energético/efectos de los fármacos , Femenino , Glucosa/metabolismo , Glucólisis/efectos de los fármacos , Humanos , Masculino , Ratones Transgénicos , Persona de Mediana Edad , Plasticidad Neuronal/efectos de los fármacos , Fosfoglicerato-Deshidrogenasa/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/administración & dosificación , Serina/farmacología , Serina/uso terapéutico , Memoria Espacial/efectos de los fármacos
9.
Front Cell Neurosci ; 12: 216, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30072874

RESUMEN

The impairment of cerebral glucose utilization is an early and predictive biomarker of Alzheimer's disease (AD) that is likely to contribute to memory and cognition disorders during the progression of the pathology. Yet, the cellular and molecular mechanisms underlying these metabolic alterations remain poorly understood. Here we studied the glucose metabolism of supragranular pyramidal cells at an early presymptomatic developmental stage in non-transgenic (non-Tg) and 3xTg-AD mice, a mouse model of AD replicating numerous hallmarks of the disease. We performed both intracellular glucose imaging with a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose biosensor and transcriptomic profiling of key molecular elements of glucose metabolism with single-cell multiplex RT-PCR (scRT-mPCR). We found that juvenile pyramidal cells exhibit active glycolysis and pentose phosphate pathway at rest that are respectively enhanced and impaired in 3xTg-AD mice without alteration of neuronal glucose uptake or transcriptional modification. Given the importance of glucose metabolism for neuronal survival, these early alterations could initiate or at least contribute to the later neuronal dysfunction of pyramidal cells in AD.

10.
eNeuro ; 2(4)2015.
Artículo en Inglés | MEDLINE | ID: mdl-26465004

RESUMEN

Type 10A phosphodiesterase (PDE10A) is highly expressed in the striatum, in striatonigral and striatopallidal medium-sized spiny neurons (MSNs), which express D1 and D2 dopamine receptors, respectively. PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear. We analyzed the effects of PDE10A inhibition in vivo by immunohistochemistry, and imaged cAMP, cAMP-dependent protein kinase A (PKA), and cGMP signals with biosensors in mouse brain slices. PDE10A inhibition in mouse striatal slices produced a steady-state increase in intracellular cAMP concentration in D1 and D2 MSNs, demonstrating that PDE10A regulates basal cAMP levels. Surprisingly, the PKA-dependent AKAR3 phosphorylation signal was strong in D2 MSNs, whereas D1 MSNs remained unresponsive. This effect was also observed in adult mice in vivo since PDE10A inhibition increased phospho-histone H3 immunoreactivity selectively in D2 MSNs in the dorsomedial striatum. The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for protein phosphatase-1 inhibition. These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs. This study shows that PDE10A inhibitors share with antipsychotic medications the property of activating preferentially PKA-dependent signaling in D2 MSNs.

11.
Methods Mol Biol ; 1071: 175-94, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24052389

RESUMEN

Cyclic-AMP dependent protein kinase (PKA) is present in most branches of the animal kingdom, and is an example in the nervous system where a kinase effector integrates the cellular effects of various neuromodulators. The recent development of FRET-based biosensors, such as AKAR, now allows the direct measurement of PKA activation in living cells by simply measuring the ratio between the fluorescence emission at the CFP and YFP wavelengths upon CFP excitation. This novel approach provides data with a temporal resolution of a few seconds at the cellular and even subcellular level, opening a new avenue of understanding the integration processes in space and time. Our protocol has been optimized to study morphologically intact mature neurons and we describe how simple and cheap wide-field imaging, as well as more elaborate two-photon imaging, allows real-time monitoring of PKA activation in pyramidal cortical neurons in neonate rodent brain slices. In addition, many practical details presented here also pertain to image analysis in other cellular preparations, such as cultured cells. Finally, this protocol can also be applied to the various other CFP-YFP-based FRET biosensors that are available for other kinases or other intracellular signals. It is likely that this kind of approach will be generally applicable to a broad range of assays in the near future.


Asunto(s)
Técnicas Biosensibles/métodos , Encéfalo/citología , Imagen Molecular/métodos , Encéfalo/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Espacio Intracelular/metabolismo , Fosforilación , Fotones , Transducción de Señal
12.
Biotechnol J ; 9(2): 192-202, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24478276

RESUMEN

Cyclic adenosine monophosphate (cAMP) and the cyclic-AMP-dependent protein kinase (PKA) regulate a plethora of cellular functions in virtually all eukaryotic cells. In neurons, the cAMP/PKA signaling cascade controls a number of biological properties such as axonal growth, pathfinding, efficacy of synaptic transmission, regulation of excitability, or long term changes. Genetically encoded optical biosensors for cAMP or PKA are considerably improving our understanding of these processes by providing a real-time measurement in living neurons. In this review, we describe the recent progress made in the creation of biosensors for cAMP or PKA activity. These biosensors revealed profound differences in the amplitude of the cAMP signal evoked by neuromodulators between various neuronal preparations. These responses can be resolved at the level of individual neurons, also revealing differences related to the neuronal type. At the sub-cellular level, biosensors reported different signal dynamics in domains like dendrites, cell body, nucleus, and axon. Combining this imaging approach with pharmacology or genetic models points at phosphodiesterases and phosphatases as critical regulatory proteins. Biosensor imaging will certainly emerge as a forefront tool to decipher the subtle mechanics of intracellular signaling. This will certainly help us to understand the mechanism of action of current drugs and foster the development of novel molecules for neuropsychiatric diseases.


Asunto(s)
Técnicas Biosensibles , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Transducción de Señal , Animales , Encéfalo/citología , Encéfalo/metabolismo , Química Encefálica , Ratones
13.
J Biol Chem ; 283(41): 27838-27849, 2008 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-18697740

RESUMEN

Integration catalyzed by integrase (IN) is a key process in the retrovirus life cycle. Many biochemical or structural human immunodeficiency virus, type 1 (HIV-1) IN studies have been severely impeded by its propensity to aggregate. We characterized a retroviral IN (primate foamy virus (PFV-1)) that displays a solubility profile different from that of HIV-1 IN. Using various techniques, including fluorescence correlation spectroscopy, time-resolved fluorescence anisotropy, and size exclusion chromatography, we identified a monomer-dimer equilibrium for the protein alone, with a half-transition concentration of 20-30 mum. We performed specific enzymatic labeling of PFV-1 IN and measured the fluorescence resonance energy transfer between carboxytetramethylrhodamine-labeled IN and fluorescein-labeled DNA substrates. FRET and fluorescence anisotropy highlight the preferential binding of PFV-1 IN to the 3'-end processing site. Sequence-specific DNA binding was not observed with HIV-1 IN, suggesting that the intrinsic ability of retroviral INs to bind preferentially to the processing site is highly underestimated in the presence of aggregates. IN is in a dimeric state for 3'-processing on short DNA substrates, whereas IN polymerization, mediated by nonspecific contacts at internal DNA positions, occurs on longer DNAs. Additionally, aggregation, mediated by nonspecific IN-IN interactions, occurs preferentially with short DNAs at high IN/DNA ratios. The presence of either higher order complex is detrimental for specific activity. Ionic strength favors catalytically competent over higher order complexes by selectively disrupting nonspecific IN-IN interactions. This counteracting effect was not observed with polymerization. The synergic effect on the selection of specific/competent complexes, obtained by using short DNA substrates under high salt conditions, may have important implications for further structural studies in IN.DNA complexes.


Asunto(s)
ADN/química , Integrasas/química , Modelos Químicos , Virus Espumoso de los Simios/enzimología , Proteínas Virales/química , Animales , Catálisis , Transferencia Resonante de Energía de Fluorescencia , Cobayas , Concentración Osmolar
14.
Plant J ; 50(2): 338-46, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17376167

RESUMEN

The hypersensitive response has been mostly studied by molecular and biochemical methods after sample destruction. The development of imaging techniques allows the monitoring of physiological changes before any signs of cell death. Here, we follow the early steps of a hypersensitive-like response induced by the bacterial elicitor harpin in Nicotiana sp. We describe cytological modifications after inoculation of the harpin protein, using confocal fluorescence microscopy (CFM) and optical coherence tomography (OCT), an interferometric-based microscopy. The changes detected by CFM occurred 5 h after harpin infiltration and corresponded to a redistribution of the chloroplasts from the upper to the inner regions of the palisade mesophyll cells which could be related to a perturbation in the microtubule network. Using OCT, we were able to detect a decrease in chloroplast backscattered signal as early as 30 min after harpin infiltration. A simple physical model, which accounted for the structure and distribution of thylakoid membranes, suggested that this loss of scattering could be associated with a modification in the refractive index of the thylakoid membranes. Our OCT observations were correlated with a decrease in photosynthesis, emphasizing changes in chloroplast structure as one of the earliest hallmarks of plant hypersensitive cell death.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/farmacología , Cloroplastos/metabolismo , Tomografía de Coherencia Óptica/métodos , Apoptosis/efectos de los fármacos , Cloroplastos/efectos de los fármacos , Microscopía Confocal , Microscopía Fluorescente , Fotosíntesis/efectos de los fármacos , Nicotiana/citología , Nicotiana/efectos de los fármacos , Nicotiana/fisiología , Tomografía de Coherencia Óptica/instrumentación
15.
J Virol ; 81(15): 7924-32, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17507472

RESUMEN

The heterodimer Ku70/80 Ku is the DNA-binding component of the DNA-PK complex required for the nonhomologous end-joining pathway. It participates in numerous nuclear processes, including telomere and chromatin structure maintenance, replication, and transcription. Ku interacts with retroviral preintegration complexes and is thought to interfere with the retroviral replication cycle, in particular the formation of 2-long terminal repeat (LTR) viral DNA circles, viral DNA integration, and transcription. We describe here the effect of Ku80 on both provirus integration and the resulting transgene expression in cells transduced with retroviral vectors. We found that transgene expression was systematically higher in Ku80-deficient xrs6 cells than in Ku80-expressing CHO cells. This higher expression was observed irrespective of the presence of the viral LTR and was also not related to the nature of the promoter. Real-time PCR monitoring of the early viral replicative steps demonstrated that the absence of Ku80 does not affect the efficiency of transduction. We analyzed the transgene distributions localization in nucleus by applying a three-dimensional reconstruction model to two-dimensional fluorescence in situ hybridization images. This indicated that the presence of Ku80 resulted in a bias toward the transgenes being located at the periphery of the nucleus associated with their being repressed; in the absence of this factor the transgenes tend to be randomly distributed and actively expressed. Therefore, although not strictly required for retroviral integration, Ku may be involved in targeting retroviral elements to chromatin domains prone to gene silencing.


Asunto(s)
Antígenos Nucleares/metabolismo , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Retroviridae/genética , Transgenes , Animales , Antígenos Nucleares/genética , Células CHO , Cricetinae , Cricetulus , Proteínas de Unión al ADN/genética , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Hibridación Fluorescente in Situ , Autoantígeno Ku , Regiones Promotoras Genéticas , Integración Viral
16.
J Biol Chem ; 281(32): 22707-19, 2006 Aug 11.
Artículo en Inglés | MEDLINE | ID: mdl-16774912

RESUMEN

The 3'-processing of the extremities of viral DNA is the first of two reactions catalyzed by HIV-1 integrase (IN). High order IN multimers (tetramers) are required for complete integration, but it remains unclear which oligomer is responsible for the 3'-processing reaction. Moreover, IN tends to aggregate, and it is unknown whether the polymerization or aggregation of this enzyme on DNA is detrimental or beneficial for activity. We have developed a fluorescence assay based on anisotropy for monitoring release of the terminal dinucleotide product in real-time. Because the initial anisotropy value obtained after DNA binding and before catalysis depends on the fractional saturation of DNA sites and the size of IN.DNA complexes, this approach can be used to study the relationship between activity and binding/multimerization parameters in the same assay. By increasing the IN:DNA ratio, we found that the anisotropy increased but the 3'-processing activity displayed a characteristic bell-shaped behavior. The anisotropy values obtained in the first phase were predictive of subsequent activity and accounted for the number of complexes. Interestingly, activity peaked and then decreased in the second phase, whereas anisotropy continued to increase. Time-resolved fluorescence anisotropy studies showed that the most competent form for catalysis corresponds to a dimer bound to one viral DNA end, whereas higher order complexes such as aggregates predominate during the second phase when activity drops off. We conclude that a single IN dimer at each extremity of viral DNA molecules is required for 3'-processing, with a dimer of dimers responsible for the subsequent full integration.


Asunto(s)
ADN/química , Integrasa de VIH/química , VIH-1/enzimología , Anisotropía , Catálisis , ADN Viral/química , Dimerización , Cinética , Modelos Químicos , Modelos Estadísticos , Nucleótidos/química , Estructura Terciaria de Proteína , Temperatura , Factores de Tiempo
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