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1.
Sci Rep ; 14(1): 8528, 2024 04 12.
Artículo en Inglés | MEDLINE | ID: mdl-38609446

RESUMEN

We tracked the consequences of in utero protein restriction in mice throughout their development and life course using a luciferase-based allelic reporter of imprinted Cdkn1c. Exposure to gestational low-protein diet (LPD) results in the inappropriate expression of paternally inherited Cdkn1c in the brains of embryonic and juvenile mice. These animals were characterised by a developmental delay in motor skills, and by behavioural alterations indicative of reduced anxiety. Exposure to LPD in utero resulted in significantly more tyrosine hydroxylase positive (dopaminergic) neurons in the midbrain of adult offspring as compared to age-matched, control-diet equivalents. Positron emission tomography (PET) imaging revealed an increase in striatal dopamine synthesis capacity in LPD-exposed offspring, where elevated levels of dopamine correlated with an enhanced sensitivity to cocaine. These data highlight a profound sensitivity of the developing epigenome to gestational protein restriction. Our data also suggest that loss of Cdkn1c imprinting and p57KIP2 upregulation alters the cellular composition of the developing midbrain, compromises dopamine circuitry, and thereby provokes behavioural abnormalities in early postnatal life. Molecular analyses showed that despite this phenotype, exposure to LPD solely during pregnancy did not significantly change the expression of key neuronal- or dopamine-associated marker genes in adult offspring.


Asunto(s)
Dieta con Restricción de Proteínas , Dopamina , Animales , Femenino , Ratones , Embarazo , Alelos , Inhibidor p57 de las Quinasas Dependientes de la Ciclina , Neuronas , Conducta Animal
2.
Commun Biol ; 7(1): 442, 2024 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-38600349

RESUMEN

Aryl hydrocarbon receptor (AHR) signalling integrates biological processes that sense and respond to environmental, dietary, and metabolic challenges to ensure tissue homeostasis. AHR is a transcription factor that is inactive in the cytosol but upon encounter with ligand translocates to the nucleus and drives the expression of AHR targets, including genes of the cytochrome P4501 family of enzymes such as Cyp1a1. To dynamically visualise AHR activity in vivo, we generated reporter mice in which firefly luciferase (Fluc) was non-disruptively targeted into the endogenous Cyp1a1 locus. Exposure of these animals to FICZ, 3-MC or to dietary I3C induced strong bioluminescence signal and Cyp1a1 expression in many organs including liver, lung and intestine. Longitudinal studies revealed that AHR activity was surprisingly long-lived in the lung, with sustained Cyp1a1 expression evident in discrete populations of cells including columnar epithelia around bronchioles. Our data link diet to lung physiology and also reveal the power of bespoke Cyp1a1-Fluc reporters to longitudinally monitor AHR activity in vivo.


Asunto(s)
Citocromo P-450 CYP1A1 , Receptores de Hidrocarburo de Aril , Ratones , Animales , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Luciferasas/genética , Hígado/metabolismo , Pulmón/metabolismo
3.
Sci Rep ; 13(1): 5626, 2023 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-37024615

RESUMEN

Genomic imprinting is an epigenetically mediated mechanism that regulates allelic expression of genes based upon parent-of-origin and provides a paradigm for studying epigenetic silencing and release. Here, bioluminescent reporters for the maternally-expressed imprinted gene Cdkn1c are used to examine the capacity of chromatin-modifying drugs to reverse paternal Cdkn1c silencing. Exposure of reporter mouse embryonic stem cells (mESCs) to 5-Azacytidine, HDAC inhibitors, BET inhibitors or GSK-J4 (KDM6A/B inhibitor) relieved repression of paternal Cdkn1c, either selectively or by inducing biallelic effects. Treatment of reporter fibroblasts with HDAC inhibitors or GSK-J4 resulted in similar paternal Cdkn1c activation, whereas BET inhibitor-induced loss of imprinting was specific to mESCs. Changes in allelic expression were generally not sustained in dividing cultures upon drug removal, indicating that the underlying epigenetic memory of silencing was maintained. In contrast, Cdkn1c de-repression by GSK-J4 was retained in both mESCs and fibroblasts following inhibitor removal, although this impact may be linked to cellular stress and DNA damage. Taken together, these data introduce bioluminescent reporter cells as tools for studying epigenetic silencing and disruption, and demonstrate that Cdkn1c imprinting requires distinct and cell-type specific chromatin features and modifying enzymes to enact and propagate a memory of silencing.


Asunto(s)
Metilación de ADN , Inhibidores de Histona Desacetilasas , Animales , Ratones , Impresión Genómica , Epigénesis Genética , Cromatina , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismo
4.
Commun Biol ; 6(1): 318, 2023 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-36966198

RESUMEN

Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by loss of function mutations in the dystrophin gene (Dmd), resulting in progressive muscle weakening. Here we modelled the longitudinal expression of endogenous Dmd, and its paralogue Utrn, in mice and in myoblasts by generating bespoke bioluminescent gene reporters. As utrophin can partially compensate for Dmd-deficiency, these reporters were used as tools to ask whether chromatin-modifying drugs can enhance Utrn expression in developing muscle. Myoblasts treated with different PRC2 inhibitors showed significant increases in Utrn transcripts and bioluminescent signals, and these responses were independently verified by conditional Ezh2 deletion. Inhibition of ERK1/2 signalling provoked an additional increase in Utrn expression that was also seen in Dmd-mutant cells, and maintained as myoblasts differentiate. These data reveal PRC2 and ERK1/2 to be negative regulators of Utrn expression and provide specialised molecular imaging tools to monitor utrophin expression as a therapeutic strategy for DMD.


Asunto(s)
Músculo Esquelético , Distrofia Muscular de Duchenne , Animales , Ratones , Utrofina/genética , Utrofina/metabolismo , Músculo Esquelético/metabolismo , Sistema de Señalización de MAP Quinasas , Distrofia Muscular de Duchenne/genética , Expresión Génica
5.
Cell Rep Med ; 3(11): 100810, 2022 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-36384093

RESUMEN

Glucagon analogs show promise as components of next-generation, multi-target, anti-obesity therapeutics. The biology of chronic glucagon treatment, in particular, its ability to induce energy expenditure and weight loss, remains poorly understood. Using a long-acting glucagon analog, G108, we demonstrate that glucagon-mediated body weight loss is intrinsically linked to the hypoaminoacidemia associated with its known amino acid catabolic action. Mechanistic studies reveal an energy-consuming response to low plasma amino acids in G108-treated mice, prevented by dietary amino acid supplementation and mimicked by a rationally designed low amino acid diet. Therefore, low plasma amino acids are a pre-requisite for G108-mediated energy expenditure and weight loss. However, preventing hypoaminoacidemia with additional dietary protein does not affect the ability of G108 to improve glycemia or hepatic steatosis in obese mice. These studies provide a mechanism for glucagon-mediated weight loss and confirm the hepatic glucagon receptor as an attractive molecular target for metabolic disease therapeutics.


Asunto(s)
Glucagón , Pérdida de Peso , Ratones , Animales , Glucagón/metabolismo , Metabolismo Energético/fisiología , Receptores de Glucagón/metabolismo , Ratones Obesos , Aminoácidos/farmacología
6.
Nat Commun ; 13(1): 2464, 2022 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-35513363

RESUMEN

Transmission of epigenetic information between generations occurs in nematodes, flies and plants, mediated by specialised small RNA pathways, modified histones and DNA methylation. Similar processes in mammals can also affect phenotype through intergenerational or trans-generational mechanisms. Here we generate a luciferase knock-in reporter mouse for the imprinted Dlk1 locus to visualise and track epigenetic fidelity across generations. Exposure to high-fat diet in pregnancy provokes sustained re-expression of the normally silent maternal Dlk1 in offspring (loss of imprinting) and increased DNA methylation at the somatic differentially methylated region (sDMR). In the next generation heterogeneous Dlk1 mis-expression is seen exclusively among animals born to F1-exposed females. Oocytes from these females show altered gene and microRNA expression without changes in DNA methylation, and correct imprinting is restored in subsequent generations. Our results illustrate how diet impacts the foetal epigenome, disturbing canonical and non-canonical imprinting mechanisms to modulate the properties of successive generations of offspring.


Asunto(s)
Epigénesis Genética , Impresión Genómica , Animales , Variación Biológica Poblacional , Metilación de ADN , Dieta Alta en Grasa , Femenino , Mamíferos , Ratones , Embarazo
7.
Nucleic Acids Res ; 50(6): 3379-3393, 2022 04 08.
Artículo en Inglés | MEDLINE | ID: mdl-35293570

RESUMEN

Pre-mRNA processing is an essential mechanism for the generation of mature mRNA and the regulation of gene expression in eukaryotic cells. While defects in pre-mRNA processing have been implicated in a number of diseases their involvement in metabolic pathologies is still unclear. Here, we show that both alternative splicing and alternative polyadenylation, two major steps in pre-mRNA processing, are significantly altered in non-alcoholic fatty liver disease (NAFLD). Moreover, we find that Serine and Arginine Rich Splicing Factor 10 (SRSF10) binding is enriched adjacent to consensus polyadenylation motifs and its expression is significantly decreased in NAFLD, suggesting a role mediating pre-mRNA dysregulation in this condition. Consistently, inactivation of SRSF10 in mouse and human hepatocytes in vitro, and in mouse liver in vivo, was found to dysregulate polyadenylation of key metabolic genes such as peroxisome proliferator-activated receptor alpha (PPARA) and exacerbate diet-induced metabolic dysfunction. Collectively our work implicates dysregulated pre-mRNA polyadenylation in obesity-induced liver disease and uncovers a novel role for SRSF10 in this process.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Enfermedad del Hígado Graso no Alcohólico , Poliadenilación , Proteínas Represoras/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Animales , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Ratones , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalme del ARN
8.
Nat Commun ; 12(1): 1980, 2021 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-33790300

RESUMEN

The majority of patients with systemic lupus erythematosus (SLE) have high expression of type I IFN-stimulated genes. Mitochondrial abnormalities have also been reported, but the contribution of type I IFN exposure to these changes is unknown. Here, we show downregulation of mitochondria-derived genes and mitochondria-associated metabolic pathways in IFN-High patients from transcriptomic analysis of CD4+ and CD8+ T cells. CD8+ T cells from these patients have enlarged mitochondria and lower spare respiratory capacity associated with increased cell death upon rechallenge with TCR stimulation. These mitochondrial abnormalities can be phenocopied by exposing CD8+ T cells from healthy volunteers to type I IFN and TCR stimulation. Mechanistically these 'SLE-like' conditions increase CD8+ T cell NAD+ consumption resulting in impaired mitochondrial respiration and reduced cell viability, both of which can be rectified by NAD+ supplementation. Our data suggest that type I IFN exposure contributes to SLE pathogenesis by promoting CD8+ T cell death via metabolic rewiring.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Perfilación de la Expresión Génica/métodos , Interferón Tipo I/inmunología , Lupus Eritematoso Sistémico/inmunología , Adulto , Anciano , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Femenino , Humanos , Interferón Tipo I/metabolismo , Interferón Tipo I/farmacología , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/metabolismo , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Redes y Vías Metabólicas/genética , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mitocondrias/metabolismo , Adulto Joven
10.
Cell Rep ; 21(12): 3559-3572, 2017 Dec 19.
Artículo en Inglés | MEDLINE | ID: mdl-29262334

RESUMEN

Feeding requires the integration of homeostatic drives with emotional states relevant to food procurement in potentially hostile environments. The ventromedial hypothalamus (VMH) regulates feeding and anxiety, but how these are controlled in a concerted manner remains unclear. Using pharmacogenetic, optogenetic, and calcium imaging approaches with a battery of behavioral assays, we demonstrate that VMH steroidogenic factor 1 (SF1) neurons constitute a nutritionally sensitive switch, modulating the competing motivations of feeding and avoidance of potentially dangerous environments. Acute alteration of SF1 neuronal activity alters food intake via changes in appetite and feeding-related behaviors, including locomotion, exploration, anxiety, and valence. In turn, intrinsic SF1 neuron activity is low during feeding and increases with both feeding termination and stress. Our findings identify SF1 neurons as a key part of the neurocircuitry that controls both feeding and related affective states, giving potential insights into the relationship between disordered eating and stress-associated psychological disorders in humans.


Asunto(s)
Ansiedad/fisiopatología , Emociones , Conducta Alimentaria , Hipotálamo/fisiología , Neuronas/fisiología , Animales , Ansiedad/metabolismo , Apetito , Calcio/metabolismo , Conducta Exploratoria , Femenino , Hipotálamo/citología , Hipotálamo/metabolismo , Locomoción , Masculino , Ratones , Neuronas/metabolismo , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo
11.
Cell Rep ; 18(5): 1090-1099, 2017 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-28147266

RESUMEN

Imprinted genes are regulated according to parental origin and can influence embryonic growth and metabolism and confer disease susceptibility. Here, we designed sensitive allele-specific reporters to non-invasively monitor imprinted Cdkn1c expression in mice and showed that expression was modulated by environmental factors encountered in utero. Acute exposure to chromatin-modifying drugs resulted in de-repression of paternally inherited (silent) Cdkn1c alleles in embryos that was temporary and resolved after birth. In contrast, deprivation of maternal dietary protein in utero provoked permanent de-repression of imprinted Cdkn1c expression that was sustained into adulthood and occurred through a folate-dependent mechanism of DNA methylation loss. Given the function of imprinted genes in regulating behavior and metabolic processes in adults, these results establish imprinting deregulation as a credible mechanism linking early-life adversity to later-life outcomes. Furthermore, Cdkn1c-luciferase mice offer non-invasive tools to identify factors that disrupt epigenetic processes and strategies to limit their long-term impact.


Asunto(s)
Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismo , Impresión Genómica/fisiología , Alelos , Animales , Cromatina/fisiología , Metilación de ADN/fisiología , Epigénesis Genética/fisiología , Ratones
12.
Sci Rep ; 6: 37777, 2016 11 25.
Artículo en Inglés | MEDLINE | ID: mdl-27886235

RESUMEN

Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format.


Asunto(s)
Técnicas Biosensibles , Glucosa/análisis , Microscopía Fluorescente/métodos , Esferoides Celulares , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos
13.
Sensors (Basel) ; 16(8)2016 Aug 19.
Artículo en Inglés | MEDLINE | ID: mdl-27548185

RESUMEN

We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation.


Asunto(s)
Técnicas Biosensibles/métodos , Técnicas de Cultivo de Célula , Imagen Molecular/métodos , Proteínas Quinasas/aislamiento & purificación , Quinasas de la Proteína-Quinasa Activada por el AMP , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Fluorescentes Verdes/química , Humanos , Imagen Óptica/métodos , Esferoides Celulares/citología
14.
Anal Chem ; 86(21): 10732-40, 2014 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-25303623

RESUMEN

Uracil DNA glycosylase plays a key role in DNA maintenance via base excision repair. Its role is to bind to DNA, locate unwanted uracil, and remove it using a base flipping mechanism. To date, kinetic analysis of this complex process has been achieved using stopped-flow analysis but, due to limitations in instrumental dead-times, discrimination of the "binding" and "base flipping" steps is compromised. Herein we present a novel approach for analyzing base flipping using a microfluidic mixer and two-color two-photon (2c2p) fluorescence lifetime imaging microscopy (FLIM). We demonstrate that 2c2p FLIM can simultaneously monitor binding and base flipping kinetics within the continuous flow microfluidic mixer, with results showing good agreement with computational fluid dynamics simulations.


Asunto(s)
ADN/química , Microscopía Fluorescente/métodos , Nucleótidos/química , Color , Cinética , Fotones
15.
Proc Natl Acad Sci U S A ; 111(4): 1503-8, 2014 Jan 28.
Artículo en Inglés | MEDLINE | ID: mdl-24474777

RESUMEN

Apoptotic cells are a source of autoantigens and impairment of their removal contributes to the development of autoimmunity in C1q deficiency. However, the lack of complement component 3 (C3), the predominant complement opsonin, does not predispose to autoimmunity, suggesting a modifying role of C3 in disease pathogenesis. To explore this hypothesis, here we investigated the role of C3 in the T-cell response to apoptotic cell-associated antigens. By comparing the phagosome maturation and the subsequent MHC class II presentation of a peptide derived from the internalized cargo between C3-deficient or C3-sufficient dendritic cells, we found that C3 deficiency accelerated the fusion of the apoptotic cargo with lysosomes. As a result, C3 deficiency led to impaired antigen-specific T-cell proliferation in vitro and in vivo. Notably, preopsonization of the apoptotic cells with C3 activation fragments rectified the trafficking and T-cell stimulation defects. These data indicate that activated C3 may act as a "chaperone" in the intracellular processing of an apoptotic cargo and, thus, may modulate the T-cell response to self-antigens displayed on dying cells.


Asunto(s)
Apoptosis , Autoantígenos/inmunología , Complemento C3/metabolismo , Endocitosis , Proteínas Opsoninas/metabolismo , Linfocitos T/inmunología , Animales , Ratones , Ratones Noqueados , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología
16.
PLoS One ; 7(11): e49200, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23152874

RESUMEN

We have compared the performance of two Troponin-C-based calcium FRET sensors using fluorescence lifetime read-outs. The first sensor, TN-L15, consists of a Troponin-C fragment inserted between CFP and Citrine while the second sensor, called mTFP-TnC-Cit, was realized by replacing CFP in TN-L15 with monomeric Teal Fluorescent Protein (mTFP1). Using cytosol preparations of transiently transfected mammalian cells, we have measured the fluorescence decay profiles of these sensors at controlled concentrations of calcium using time-correlated single photon counting. These data were fitted to discrete exponential decay models using global analysis to determine the FRET efficiency, fraction of donor molecules undergoing FRET and calcium affinity of these sensors. We have also studied the decay profiles of the donor fluorescent proteins alone and determined the sensitivity of the donor lifetime to temperature and emission wavelength. Live-cell fluorescence lifetime imaging (FLIM) of HEK293T cells expressing each of these sensors was also undertaken. We confirmed that donor fluorescence of mTFP-TnC-Cit fits well to a two-component decay model, while the TN-L15 lifetime data was best fitted to a constrained four-component model, which was supported by phasor analysis of the measured lifetime data. If the constrained global fitting is employed, the TN-L15 sensor can provide a larger dynamic range of lifetime readout than the mTFP-TnC-Cit sensor but the CFP donor is significantly more sensitive to changes in temperature and emission wavelength compared to mTFP and, while the mTFP-TnC-Cit solution phase data broadly agreed with measurements in live cells, this was not the case for the TN-L15 sensor. Our titration experiment also indicates that a similar precision in determination of calcium concentration can be achieved with both FRET biosensors when fitting a single exponential donor fluorescence decay model to the fluorescence decay profiles. We therefore suggest that mTFP-based probes are more suitable for FLIM experiments than CFP-based probes.


Asunto(s)
Técnicas Biosensibles , Calcio/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/metabolismo , Troponina C/metabolismo , Citosol/metabolismo , Células HEK293 , Humanos , Microscopía Fluorescente , Proteínas Mutantes/metabolismo , Conformación Proteica , Reproducibilidad de los Resultados , Temperatura , Factores de Tiempo , Volumetría
17.
Appl Opt ; 50(36): 6583-90, 2011 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-22193187

RESUMEN

We describe a new light transport model, which was applied to three-dimensional lifetime imaging of Förster resonance energy transfer in mice in vivo. The model is an approximation to the radiative transfer equation and combines light diffusion and ray optics. This approximation is well adopted to wide-field time-gated intensity-based data acquisition. Reconstructed image data are presented and compared with results obtained by using the telegraph equation approximation. The new approach provides improved recovery of absorption and scattering parameters while returning similar values for the fluorescence parameters.


Asunto(s)
Algoritmos , Transferencia Resonante de Energía de Fluorescencia/métodos , Óptica y Fotónica/métodos , Animales , Fluorescencia , Imagenología Tridimensional/métodos , Ratones , Modelos Teóricos , Dispersión de Radiación , Tomografía Óptica/métodos
18.
Biomed Opt Express ; 2(7): 1907-17, 2011 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-21750768

RESUMEN

Förster resonance energy transfer (FRET) is a powerful biological tool for reading out cell signaling processes. In vivo use of FRET is challenging because of the scattering properties of bulk tissue. By combining diffuse fluorescence tomography with fluorescence lifetime imaging (FLIM), implemented using wide-field time-gated detection of fluorescence excited by ultrashort laser pulses in a tomographic imaging system and applying inverse scattering algorithms, we can reconstruct the three dimensional spatial localization of fluorescence quantum efficiency and lifetime. We demonstrate in vivo spatial mapping of FRET between genetically expressed fluorescent proteins in live mice read out using FLIM. Following transfection by electroporation, mouse hind leg muscles were imaged in vivo and the emission of free donor (eGFP) in the presence of free acceptor (mCherry) could be clearly distinguished from the fluorescence of the donor when directly linked to the acceptor in a tandem (eGFP-mCherry) FRET construct.

19.
Chemphyschem ; 12(3): 609-26, 2011 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-21337485

RESUMEN

A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas/análisis , Línea Celular , Evaluación Preclínica de Medicamentos , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/química , Humanos , Microscopía Fluorescente , Unión Proteica , Rodaminas/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/análisis
20.
Opt Lett ; 34(18): 2772-4, 2009 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-19756100

RESUMEN

We report a three-dimensional time-resolved tomographic imaging technique for localizing protein-protein interaction and protein conformational changes in turbid media based on Förster resonant energy-transfer read out using fluorescence lifetime. This application of "tomoFRET" employs an inverse scattering algorithm utilizing the diffusion approximation to the radiative-transfer equation applied to a large tomographic data set of time-gated images. The approach is demonstrated by imaging a highly scattering cylindrical phantom within which are two thin wells containing cytosol preparations of HEK293 cells expressing TN-L15, a cytosolic genetically encoded calcium Förster resonant energy-transfer sensor. A 10 mM calcium chloride solution was added to one of the wells, inducing a protein conformation change upon binding to TN-L15, resulting in Förster resonant energy transfer and a corresponding decrease in the donor fluorescence lifetime. We successfully reconstruct spatially resolved maps of the resulting fluorescence lifetime distribution as well as of the quantum efficiency, absorption, and scattering coefficients.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Óptica y Fotónica/métodos , Cloruro de Calcio/farmacología , Línea Celular , Citosol/metabolismo , Difusión , Humanos , Fantasmas de Imagen , Conformación Proteica , Dispersión de Radiación , Siliconas/química , Factores de Tiempo
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