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1.
Angew Chem Int Ed Engl ; 57(41): 13686-13690, 2018 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-30084526

RESUMO

Fluorescence barcoding based on nanoparticles provides many advantages for multiparameter imaging. However, creating different concentration-independent codes without mixing various nanoparticles and by using single-wavelength excitation and emission for multiplexed cellular imaging is extremely challenging. Herein, we report the development of quantum dots (QDs) with two different SiO2 shell thicknesses (6 and 12 nm) that are coated with two different lanthanide complexes (Tb and Eu). FRET from the Tb or Eu donors to the QD acceptors resulted in four distinct photoluminescence (PL) decays, which were encoded by simple time-gated (TG) PL intensity detection in three individual temporal detection windows. The well-defined single-nanoparticle codes were used for live cell imaging and a one-measurement distinction of four different cells in a single field of view. This single-color barcoding strategy opens new opportunities for multiplexed labeling and tracking of cells.


Assuntos
Európio/química , Transferência Ressonante de Energia de Fluorescência/métodos , Nanopartículas , Pontos Quânticos , Térbio/química
2.
Angew Chem Int Ed Engl ; 53(40): 10718-22, 2014 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-25115848

RESUMO

Luminescent europium complexes are used in a broad range of applications as a result of their particular emissive properties. The synthesis and application of bright, highly water-soluble, and negatively charged sulfonic- or carboxylic acid derivatives of para-substituted aryl-alkynyl triazacyclononane complexes are described. Introduction of the charged solubilizing moieties suppresses cellular uptake or adsorption to living cells making them applicable for labeling and performing assays on membrane receptors. These europium complexes are applied to monitor fluorescent ligand binding on cell-surface proteins with time-resolved Förster resonance energy transfer (TR-FRET) assays in plate-based format and using TR-FRET microscopy.


Assuntos
Compostos Aza/análise , Complexos de Coordenação/análise , Európio/análise , Transferência Ressonante de Energia de Fluorescência/métodos , Substâncias Luminescentes/análise , Microscopia/métodos , Piperidinas/análise , Receptores Acoplados a Proteínas-G/metabolismo , Compostos Aza/metabolismo , Complexos de Coordenação/metabolismo , Európio/metabolismo , Células HEK293 , Humanos , Ligantes , Substâncias Luminescentes/metabolismo , Piperidinas/metabolismo , Ligação Proteica , Receptores Acoplados a Proteínas-G/análise , Solubilidade , Água/química
3.
Chemistry ; 20(28): 8636-46, 2014 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-24938657

RESUMO

A series of europium and terbium complexes based on a functionalized triazacyclononane carboxylate or phosphinate macrocyclic ligand is described. The influence of the anionic group, that is, carboxylate, methylphosphinate, or phenylphosphinate, on the photophysical properties was studied and rationalized on the basis of DFT calculated structures. The nature, number, and position of electron-donating or electron-withdrawing aryl substituents were varied systematically within the same phenylethynyl scaffold in order to optimize the brightness of the corresponding europium complexes and investigate their two-photon absorption properties. Finally, the europium complexes were examined in cell-imaging applications, and selected terbium complexes were studied as potential oxygen sensors.


Assuntos
Alquinos/química , Compostos Aza/química , Európio/química , Compostos Organometálicos/química , Piperidinas/química , Térbio/química , Ligantes , Estrutura Molecular
4.
Nat Protoc ; 8(7): 1307-20, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23764938

RESUMO

G protein-coupled receptors (GPCRs) and their ligands are traditionally characterized by radioligand-binding experiments. These experiments yield excellent quantitative data, but have low temporal and spatial resolution. In addition, the use of radioligands presents safety concerns. Here we provide a general procedure for an alternative approach with high temporal and spatial resolution, based on Tb(+)-labeled fluorescent receptor ligands and time-resolved fluorescence resonance energy transfer (TR-FRET). This protocol and its design are detailed here for the parathyroid hormone receptor, a class B GPCR, and its fluorescently labeled 34-amino acid peptide ligand, but it can be easily modified for other receptors and their appropriately labeled ligands. We discuss three protocol options that use Tb(+)-labeled fluorescent ligands: a time-resolved fluorescence separation option that works on native receptors but requires separation of bound and unbound ligand; a TR-FRET option using SNAP-tag-labeled receptors for high-throughput screening; and a TR-FRET option that uses fluorescently labeled antibodies directed against an epitope engineered into the Flag-labeled receptors' N terminus. These protocol options can be used as standard procedures with very high signal-to-background ratios in order to characterize ligands and their receptors in living cells and in cell membranes via straightforward plate-reader measurements.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Ligantes , Receptores Acoplados a Proteínas-G/metabolismo , Corantes Fluorescentes/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Receptores Acoplados a Proteínas-G/análise , Térbio/química , Teriparatida/análogos & derivados , Teriparatida/química , Teriparatida/metabolismo , Fatores de Tempo
5.
Proc Natl Acad Sci U S A ; 110(4): 1512-7, 2013 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-23297228

RESUMO

To maintain homeostasis, hypothalamic neurons in the arcuate nucleus must dynamically sense and integrate a multitude of peripheral signals. Blood-borne molecules must therefore be able to circumvent the tightly sealed vasculature of the blood-brain barrier to rapidly access their target neurons. However, how information encoded by circulating appetite-modifying hormones is conveyed to central hypothalamic neurons remains largely unexplored. Using in vivo multiphoton microscopy together with fluorescently labeled ligands, we demonstrate that circulating ghrelin, a versatile regulator of energy expenditure and feeding behavior, rapidly binds neurons in the vicinity of fenestrated capillaries, and that the number of labeled cell bodies varies with feeding status. Thus, by virtue of its vascular connections, the hypothalamus is able to directly sense peripheral signals, modifying energy status accordingly.


Assuntos
Regulação do Apetite/fisiologia , Grelina/sangue , Hipotálamo/fisiologia , Animais , Barreira Hematoencefálica/fisiologia , Permeabilidade Capilar , Ingestão de Alimentos/fisiologia , Jejum/fisiologia , Hipotálamo/irrigação sanguínea , Hipotálamo/citologia , Masculino , Eminência Mediana/irrigação sanguínea , Eminência Mediana/citologia , Eminência Mediana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica , Modelos Neurológicos , Neurônios/fisiologia
6.
Anal Biochem ; 408(2): 253-62, 2011 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-20937574

RESUMO

The growth hormone secretagogue receptor type 1a (GHS-R1a) belongs to class A G-protein-coupled receptors (GPCR). This receptor mediates pleiotropic effects of ghrelin and represents a promising target for dysfunctions of growth hormone secretion and energy homeostasis including obesity. Identification of new compounds which bind GHS-R1a is traditionally achieved using radioactive binding assays. Here we propose a new fluorescence-based assay, called Tag-lite binding assay, based on a fluorescence resonance energy transfer (FRET) process between a terbium cryptate covalently attached to a SNAP-tag fused GHS-R1a (SNAP-GHS-R1a) and a high-affinity red fluorescent ghrelin ligand. The long fluorescence lifetime of the terbium cryptate allows a time-resolved detection of the FRET signal. The assay was made compatible with high-throughput screening by using prelabeled cells in suspension under a 384-well plate format. K(i) values for a panel of 14 compounds displaying agonist, antagonist, or inverse agonist properties were determined using both the radioactive and the Tag-lite binding assays performed on the same batches of GHS-R1a-expressing cells. Compound potencies obtained in the two assays were nicely correlated. This study is the first description of a sensitive and reliable nonradioactive binding assay for GHS-R1a in a format amenable to high-throughput screening.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Ligantes , Receptores de Grelina/antagonistas & inibidores , Ligação Competitiva , Complexos de Coordenação/química , Éteres de Coroa/química , Agonismo Inverso de Drogas , Células HEK293 , Humanos , Cinética , Receptores de Grelina/agonistas , Receptores de Grelina/metabolismo , Térbio/química
7.
EMBO J ; 30(1): 32-42, 2011 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-21063387

RESUMO

Seven-transmembrane domain (7TM) receptors have important functions in cell-cell communication and can assemble into dimers or oligomers. Such complexes may allow specific functional cross-talk through trans-activation of interacting 7TMs, but this hypothesis requires further validation. Herein, we used the GABAB receptor, which is composed of two distinct subunits, GABAB1, which binds the agonist, and GABAB2, which activates G proteins, as a model system. By using a novel orthogonal-labelling approach compatible with time-resolved FRET and based on ACP- and SNAP-tag technologies to verify the heterodimerization of wild-type and mutated GABAB subunits, we demonstrate the existence of a direct allosteric coupling between the 7TMs of GABAB heterodimers. Indeed, a GABAB receptor, in which the GABAB2 extracellular domain was deleted, was still capable of activating G proteins. Furthermore, synthetic ligands for the GABAB2 7TM could increase agonist affinity at the GABAB1 subunit in this mutated receptor. In addition to bringing new information on GABAB receptor activation, these data clearly demonstrate the existence of direct trans-activation between the 7TM of two interacting proteins.


Assuntos
Receptores de GABA-B/química , Receptores de GABA-B/metabolismo , Regulação Alostérica , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Humanos , Mutação , Multimerização Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Receptores de GABA-B/genética , Transfecção
8.
J Biomol Screen ; 15(10): 1248-59, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20974902

RESUMO

G-protein-coupled receptors (GPCRs) are crucial cell surface receptors that transmit signals from a wide range of extracellular ligands. Indeed, 40% to 50% of all marketed drugs are thought to modulate GPCR activity, making them the major class of targets in the drug discovery process. Binding assays are widely used to identify high-affinity, selective, and potent GPCR drugs. In this field, the use of radiolabeled ligands has remained so far the gold-standard method. Here the authors report a less hazardous alternative for high-throughput screening (HTS) applications by the setup of a nonradioactive fluorescence-based technology named Tag-lite(®). Selective binding of various fluorescent ligands, either peptidic or not, covering a large panel of GPCRs from different classes is illustrated, particularly for chemokine (CXCR4), opioid (δ, µ, and κ), and cholecystokinin (CCK1 and CCK2) receptors. Affinity constants of well-known pharmacological agents of numerous GPCRs are in line with values published in the literature. The authors clearly demonstrate that the Tag-lite binding assay format can be successfully and reproducibly applied by using different cellular materials such as transient or stable recombinant cells lines expressing SNAP-tagged GPCR. Such fluorescent-based binding assays can be performed with adherent cells or cells in suspension, in 96- or 384-well plates. Altogether, this new technology offers great advantages in terms of flexibility, rapidity, and user-friendliness; allows easy miniaturization; and makes it completely suitable for HTS applications.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Cricetinae , Avaliação Pré-Clínica de Medicamentos/métodos , Fluorescência , Células HEK293 , Humanos , Ligantes , Receptor de Colecistocinina A/metabolismo , Receptor de Colecistocinina B/metabolismo , Receptores CXCR4/metabolismo , Receptores Opioides/metabolismo
9.
Nat Chem Biol ; 6(8): 587-94, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20622858

RESUMO

G protein-coupled receptor (GPCR) oligomers have been proposed to play critical roles in cell signaling, but confirmation of their existence in a native context remains elusive, as no direct interactions between receptors have been reported. To demonstrate their presence in native tissues, we developed a time-resolved FRET strategy that is based on receptor labeling with selective fluorescent ligands. Specific FRET signals were observed with four different receptors expressed in cell lines, consistent with their dimeric or oligomeric nature in these transfected cells. More notably, the comparison between FRET signals measured with sets of fluorescent agonists and antagonists was consistent with an asymmetric relationship of the two protomers in an activated GPCR dimer. Finally, we applied the strategy to native tissues and succeeded in demonstrating the presence of oxytocin receptor dimers and/or oligomers in mammary gland.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Oligopeptídeos/química , Receptores Acoplados a Proteínas-G/metabolismo , Algoritmos , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos , Células COS , Linhagem Celular , Chlorocebus aethiops , Dimerização , Antagonistas dos Receptores de Dopamina D2 , Feminino , Corantes Fluorescentes , Ligantes , Glândulas Mamárias Animais/metabolismo , Modelos Moleculares , Oligopeptídeos/metabolismo , Ensaio Radioligante , Ratos , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/metabolismo , Receptores Acoplados a Proteínas-G/agonistas , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores de Ocitocina/agonistas , Receptores de Ocitocina/antagonistas & inibidores , Receptores de Ocitocina/metabolismo , Receptores de Vasopressinas/agonistas , Receptores de Vasopressinas/metabolismo
10.
Nat Methods ; 5(6): 561-7, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18488035

RESUMO

Cell-surface proteins are important in cell-cell communication. They assemble into heterocomplexes that include different receptors and effectors. Elucidation and manipulation of such protein complexes offers new therapeutic possibilities. We describe a methodology combining time-resolved fluorescence resonance energy transfer (FRET) with snap-tag technology to quantitatively analyze protein-protein interactions at the surface of living cells, in a high throughput-compatible format. Using this approach, we examined whether G protein-coupled receptors (GPCRs) are monomers or assemble into dimers or larger oligomers--a matter of intense debate. We obtained evidence for the oligomeric state of both class A and class C GPCRs. We also observed different quaternary structure of GPCRs for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA): whereas metabotropic glutamate receptors assembled into strict dimers, the GABA(B) receptors spontaneously formed dimers of heterodimers, offering a way to modulate G-protein coupling efficacy. This approach will be useful in systematic analysis of cell-surface protein interaction in living cells.


Assuntos
Biofísica/métodos , Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Mapeamento de Interação de Proteínas/métodos , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Células COS , Chlorocebus aethiops , Dimerização , Humanos , Modelos Biológicos , Estrutura Quaternária de Proteína , Receptores de GABA-B/química , Propriedades de Superfície , Ácido gama-Aminobutírico
11.
Anal Biochem ; 358(1): 126-35, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16965760

RESUMO

Phospholipase C beta (PLC-beta)-coupled G protein-coupled receptor (GPCR) activities traditionally are assessed by measuring Ca2+ triggered by D-myo-inositol 1,4,5-trisphosphate (IP3), a PLC-beta hydrolysis product, or by measuring the production of inositol phosphate using cumbersome radioactive assays. A specific detection of IP3 production was also established using IP3 binding proteins. The short lifetime of IP3 makes this detection very challenging in measuring GPCR responses. Indeed, this IP3 rapidly enters the metabolic inositol phosphate cascade. It has been known for decades that lithium chloride (LiCl) leads to D-myo-inositol 1-phosphate accumulation on GPCR activation by inhibiting inositol monophosphatase, the final enzyme of the IP3 metabolic cascade. We show here that IP1 can be used as a surrogate of IP3 to monitor GPCR activation. We developed a novel homogeneous time-resolved fluorescence (HTRF) assay that correlates perfectly with existing methods and is easily amenable to high-throughput screening. The IP-One assay was validated on various GPCR models. It has the advantage over the traditional Ca2+ assay of allowing the measurement of inverse agonist activity as well as the analysis of PLC-beta activity in any nontransfected primary cultures. Finally, the high assay specificity for D-myo-inositol 1 monophosphate (IP1(1)) opens new possibilities in developing selective assays to study the functional roles of the various isoforms of inositol phosphates.


Assuntos
Inositol 1,4,5-Trifosfato , Fosfatos de Inositol , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Células CHO , Cricetinae , Humanos , Inositol 1,4,5-Trifosfato/química , Fosfatos de Inositol/química , Fosfolipases Tipo C/metabolismo
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