Este trabalho é apoiado pelo fundo intramural do NIAID, NIH.

1. Preparação de células competentes quimiotática de Dictyostelium discoideum <ol><li> Para gerar células discoideum D. que são quimiotáticos para o acampamento chemoattractant, células colheita crescendo em D3-T rich media de uma cultura agitação a 22 ° C. </li><li> Lave as células duas vezes em não-nutrientes tampão de desenvolvimento (buffer DB contendo 5 mM Na 2 HPO 4, 5 mM KH 2 PO 4, 2 mM MgCl 2, e 0,1 mM CaCl 2). </li><li> Re-suspender as células em tampão DB a uma densidade de 2x10 7 células / ml. </li><li> Agite 10 células ml em um frasco de 250 ml a 100 rpm a 22 ° C durante uma hora. </li><li> Entregar 100 l de 7,5 mM de ações cAMP aos 10 células ml cada seis minutos mais de 6 horas para obter uma concentração final de 75 nM cAMP, um processo designado como tratamento cAMP pulsante. Após 5-6 horas de tratamento cAMP pulsante, D. células discoideum se tornar competente na direção do gradiente quimiotático cAMP. </li><li> Coletar células por centrifugação a 200 g por 5 min e depois ressuspender as células com tampão contendo 2,5 DB cafeína mM, e agitar a 200 rpm a 22 ° C por 20 min para basolate célula para uma situação quimiotáticos. </li></ol> 2. Células imagem chemotaxing em um gradiente chemoattractant visível e manipulável <ol><li> Backfill micropipeta um com um recém-preparados 30 mL de solução de 1 mM cAMP e Alexa 594 em 0.1μg/μl em tampão DB. </li><li> Anexar o Femtotip a um detentor micropipeta e conectar o tubo a um aparelho de alimentação de pressão, Eppendorf sistema FemtoJet. </li><li> Prender o conjunto de micropipeta para um micromanipulador (Eppendorf TransferMan NK2) motorizada micromanipulador para fornecer uma pressão constante, a fim de estabelecer um gradiente estável. </li><li> Montar uma bem-câmara LabTek preenchido com 6 ml de tampão DB sobre uma lente de 40X de óleo em um microscópio confocal e uso brilhante de campo óptica, o centro Femtotip para o campo de visão. </li><li> Ligue a fonte de pressão e ajustar a pressão de compensação (Pc) para 70 hPa para estabelecer um gradiente de campo / Alexa 594 mistura. </li><li> Visualize gradiente cAMP através do monitoramento da mistura de concentração desejada de cAMP e fluorescência Alexa 594 usando uma linha de excitação com laser de 543 nm. </li><li> Use auto-posicionamento função do micromanipulador para colocar micropipeta para as posições desejadas e defini-las como posição 1, posição 2, 3 e posição para manipular o gradiente a que estão expostos a célula. </li></ol> 3. Sistema de célula de imóveis nonpolarized facilita eventos de imagens de sinalização envolvidos na cAMP gradiente de sensoriamento <ol><li> Após o tratamento a cafeína, retire uma alíquota de células e centrifugar a 500 g por 3 min. </li><li> Remover tampão e diluir as células a 5x10 5 células / ml com tampão contendo 2,5 DB fresco cafeína mM. </li><li> Aplicar 1 ml de suspensão de células de uma única câmara-assim ou 0,4 ml a cada poço de uma câmara de quatro bem. </li><li> Permitir que as células aderem por 10 min, com cuidado pipeta fora do tampão para remover as células solto e substituir com o mesmo volume. </li><li> Localizar as células desejadas sob o microscópio e começar de imagem. </li><li> Para um experimento projetado para monitorar a dinâmica de sinalização nas células componentes expondo a um gradiente estabilizado, tratar as células com 5,0 mM (concentração final) Latrunculin B por 10 min antes dos experimentos. </li></ol> 4. Simultânea de monitoramento heterotrimeric ativação da proteína G e PIP 3 produção <ol><li> cAMP pulsando desenvolver células co-expressando GαCFP e YFPGβ (células G) e células que expressam PIP 3 Indicador de PH GFP (células PH) 7. </li><li> Misture estes dois tipos de células com relação de 1:1 e placa-los em câmaras de um bem ou quatro poços. </li><li> Criar e salvar a emissão de impressões digitais de referência da curva PCP, YFP e GFP utilizando Lambda modo de Aquisição de pilha dentro da faixa espectral 464-624 nm com uma largura de 10 nm. </li><li> Simultaneamente ativação da proteína G de imagem em células G e produção PIP 3 em células PH usando com o mesmo modo de Aquisição Stack Lambda dentro da faixa espectral 464-544 nm com incrementos de 10 nm. </li><li> Aplicar função desmistura Linear Zeiss de software usando 510META salvo CFPand YFP e impressões digitais de emissão para calcular matematicamente a contribuição de cada fluoróforo na Stack Lambda para separar o PCP ea intensidade YFP em canais individuais em G. </li><li> Com a mesma estratégia, aplicar função desmistura Linear usando GFP salvos e impressões digitais de emissão de fundo para calcular matematicamente intensidade GFP em células PH. </li></ol> 5. Resultados representativos: <ol><li> Um sistema excelente modelo de D. discoideum para GPCR quimiotaxia mediada. A ameba social, D. discoideum exibe uma impressionante quimiotaxia durante o ciclo de vida. Devido às suas vantagens genéticas e bioquímicas, D. d fornece uma poderosa sistema para estudar a quimiotaxia. </li><li> Quimiotaxia de células em um campo visível e manipulável chemoattract. Aqui, mostramos primeiro uma metodologia simples para obter uma relação linear entre a concentração de cAMP e da intensidade de um corante fluorescente Alexa 594 por uma série de diluição de 2 cAMP M misturado com 10 mg / mL Alexa 594 (Fig. 2A). Em seguida, fornecer uma maneira fácil de visualizar o gradiente, além disso, estabelecer uma medida quantitativa da concentração de cAMP de um gradiente pela intensidade do Alexa 594 (Fig. Fig.. 2B). </li><li> Motilidade celular é desacoplado com polarização celular e detecção direcional. Um inibidor de polimerização de actina elimina pré-existentes polaridade morfológicas e também impede a movimentação das células, mantendo a capacidade das células de detecção direcional (Fig. 3A). Emprego de estimulação cAMP visível e manipulável garante a entrada, por exemplo estímulos uniformemente aplicada ou um gradiente. Este método permite uma análise quantitativa de AMPc induzida redistribuição dos principais componentes de sinalização nas máquinas gradiente de detecção. Medida dinâmica espaço-temporal destes componentes de sinalização nos permitem compreender como a rede de sinalização alcança a adaptação aos estímulos uniforme ao gerar polarizada respostas bioquímicas a gradientes (Fig. 3B). </li><li> Medições sistêmica da cinética de chemosensing rede de sinalização após a exposição a um gradiente constante. É fundamental para medir a dinâmica / cinética da direcionais de detecção de sinalização componentes para entender como cada componente contribui para o estabelecimento de polarização intracelular quando as células experiência primeiro gradiente. Aplicação de imagens de células vivas com um ritmo de alta resolução espacial, primeiro mostrar uma produção PIP3 bifásica de celular que é exposto a um gradiente de cAMP constante (Fig. 4A-C). Aplicação de imagens de células vivas, temos sistematicamente medida dinâmica da direcionais de detecção de rede de sinalização específicas de estímulo para cAMP PIP3 de produção (Fig. 4D, E). </li><li> Simultânea de monitoramento ativação da proteína G e heterotrimeric PIP 3 a estimulação da produção de AMPc uniformemente aplicadas. Förster ressonância a transferência de energia (abreviado FRET) constitui uma estratégia eficiente para monitorar a ativação da proteína G heterotrimeric (dissociação) com a estimulação do AMPc. Aqui, descrevemos um sistema de fácil adotar conveniente para uma medição simultânea de ativação da proteína G heterotrimeric e produção PIP 3 monitorando o FRET mudança e translocação de membrana de PIP 3 sonda, PH GFP-in G e células PH, respectivamente (Fig. 5 ). A estimulação cAMP uniformemente aplicadas desencadeia uma ativação da proteína G persistente, enquanto que desencadeia um transiente PIP 3 produção. </li></ol><img src="/files/ftp_upload/3128/3128fig1.jpg" alt="Figura 1"/> Figura 1: Um sistema excelente modelo de D. discoideum para quimiotaxia mediada GPCR. A. Esquema mostra um caminho breve sinalização de detecção direcional. B. gradiente AMPc induz quimiotaxia rápida de D. células discoideum. Células expressam sonda PIP 3, PH-GFP (Green). Gradiente (Red) é visualizado por Alexa 594. Barra de escala = 50 ìm. <img src="/files/ftp_upload/3128/3128fig2.jpg" alt="Figura 2"/> Figura 2: Quimiotaxia de células sob uma campos visíveis e manipuláveis ​​chemoattract. A. Gráfico mostra uma relação linear entre a concentração de cAMP e da intensidade de um corante fluorescente Alexa 594 por uma série de diluição de 2 cAMP M misturado com 10 mg / mL Alexa 594. B. Determinação quantitativa da concentração de cAMP de um gradiente pela relação linear da concentração de cAMP e intensidade do corante fluorescente Alexa em 594 A. <img src="/files/ftp_upload/3128/3128fig3.jpg" alt="Figura 3"/> Figura 3: motilidade celular é desacoplado com polarização celular e detecção direcional. Imagem A. mostra que as células imóvel pelo tratamento de actina inibidor de polimerização Latrunculin B manter a capacidade de detecção direcional. Células expressam sonda PIP 3, PH-GFP (Green). Gradiente (Red) é visualizado por Alexa 594. B. cAMP manipulável sistema de célula de estimulação e imóvel permite abordar questões-chave de detecção direcional. Barra de escala = 10μm. <img src="/files/ftp_upload/3128/3128fig4.jpg" alt="Figura 4"/> Figura 4: medidas sistêmicas da cinética de chemosensing rede de sinalização após a exposição a um gradiente constante. A. Montagem mostra um bifásico PIP 3 produção (Verde) do celular que é exposto a um gradiente de cAMP estacionário (vermelho). B. A imagem mostra as regiões de interesse (ROIs) para a medição da cinética de produção PIP 3 apresentados em C. C . Kinetics of PIP 3 de produção nas células expostas a um gradiente constante. D. Esquema mostra a rede de sinalização de detecção direcional de estímulo à produção de cAMP PIP 3. Sua cinética após a exposição a um gradiente de equilíbrio é apresentado nas linhas de mesma cor sólida em E. <img src="/files/ftp_upload/3128/3128fig5.jpg" alt="Figura 5"/> Figura 5: monitoramento simultâneo de vários eventos de redes GPCR sinalização. Esquema de A. mostra a medição simultânea de ativação da proteína G heterotrimeric e produção PIP 3 monitorando o FRET mudança e translocação de membrana de PIP3 sonda, PH-GFP em células G e PH, respectivamente. B. Montagem de imagens arco-íris de G e células PH mostra que um estímulo cAMP uniformemente aplicadas desencadeia uma ativação da proteína G persistente na célula periféricos, enquanto que desencadeia uma produção PIP3 transitória. Os momentos são antes (0s) e após a estimulação para 4.9s, 10.2s e 20.4s. C. Cinética de ativação da proteína G e PIP3 produção em cima de um estímulo cAMP uniformemente aplicadas.

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Os processos de atingir estágio competente quimiotáticos de células Para o tipo selvagem D. células discoideum, leva cerca de 5 ~ 6 horas pulsando desenvolvimento à temperatura ambiente para induzi-los em um estágio bem quimiotáticos competente durante o qual células apresentam uma morfologia bem polarizada celular e migração celular rápida (Fig. 1). Vários fatores, tais como a concentração de cAMP pulsação, temperatura e diferentes origens genéticas, podem af...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> D3-T meios de crescimento </td><td> KD Medical </td><td></td><td></td></tr><tr><td> Cafeína </td><td> Sigma </td><td></td><td></td></tr><tr><td> Latrunculin B </td><td> Sondas moleculares </td><td></td><td></td></tr><tr><td> Alexa 594 </td><td> Sondas moleculares </td><td></td><td></td></tr><tr><td> cAMP </td><td> Sigma </td><td></td><td></td></tr><tr><td> ChronTrol temporizador programável XT </td><td> ChronTrol Corp </td><td></td><td></td></tr><tr><td> Miniplus 3 bomba peristáltica </td><td> Gilbson </td><td></td><td></td></tr><tr><td> Plataforma agitador rotativo </td><td></td><td></td><td></td></tr><tr><td> FemtoJet alimentação de pressão microcapilar </td><td> Eppendorf </td><td></td><td></td></tr><tr><td> Único e quatro bem-Lab-Tek II lamela câmaras </td><td> Nalge Nunc Internacional </td><td></td><td></td></tr><tr><td> LSM META 510 ou microscópio de fluorescência equivalente </td><td> Zeiss </td><td></td><td> um 40X 60X 1,3 ou 1,4 NA NA óleo lente objetiva DIC Plano Neofluar- </td></tr><tr><td> Olympus X81 ou equivalente </td><td> Olimpo </td><td></td><td> Requer um 100X 1,47 NA TIRF lente objetiva </td></tr></tbody></table>

imaging g-protein coupled receptor (gpcr)-mediated signaling events that control chemotaxis of dictyostelium discoideum

Muitas células eucarióticas pode detectar gradientes de sinais químicos em seus ambientes e migrar de acordo 1. Esta migração celular guiada é referido como quimiotaxia, que é essencial para várias células para realizar suas funções, tais como tráfico de células do sistema imunológico e padronização de células neuronais 2, 3. A grande família de G receptores acoplados à proteína (GPCRs) detecta variável pequenos peptídeos, conhecido como quimiocinas, para dirigir a migração celular in vivo 4. O objetivo final da quimiotaxia pesquisa é compreender como um sentidos máquinas GPCR quimiocina gradientes e controles de sinalização eventos que levam a quimiotaxia. Para este fim, usamos técnicas de imagem para monitorar, em tempo real, as concentrações de espaço-temporal de quimioatrativos movimento celular, em um gradiente de chemoattractant, GPCR mediada ativação da proteína G-heterotrimeric, e eventos de sinalização intracelular envolvidas na quimiotaxia de células eucarióticas 08/05 . O organismo simples eucarióticas, Dictyostelium discoideum, exibe comportamentos quimiotático que são semelhantes aos de leucócitos, e D. discoideum é um sistema modelo para estudar a quimiotaxia chave eucarióticas. Como amebas de vida livre, D. discoideum células dividem-se em meio rico. Após a fome, as células entrar em um programa de desenvolvimento em que agregada através AMPc mediada por quimiotaxia para formar estruturas multicullular. Muitos componentes envolvidos na quimiotaxia ao AMPc foram identificados em D. discoideum. A ligação de AMPc a um GPCR (car1) induz a dissociação da heterotrimeric G-proteínas em Gγ e subunidades Gβγ 7, 9, 10. Subunidades Gβγ ativar Ras, que por sua vez ativa PI3K, convertendo PIP PIP 2 em 3 na membrana celular 11-13. PIP 3 servem como sítios de ligação para proteínas com pleckstrin Homologia (PH) domínios, assim, o recrutamento dessas proteínas para a membrana 14, 15. Ativação de receptores car1 também controla as associações de membrana de PTEN, que desfosforila PIP PIP 3 a 2 16, 17. Os mecanismos moleculares são evolutivamente conservada em quimiotaxia quimiocina GPCR mediada das células humanas, tais como neutrófilos 18. Nós apresentamos métodos a seguir para estudar a quimiotaxia de D. células discoideum. 1. Preparação de células componentes quimiotáticos. 2. Quimiotaxia de imagem de células em um gradiente de cAMP. 3. Monitoramento de uma ativação induzida de GPCR heterotrimeric proteína G-in único de células vivas. 4. Imagem PIP chemoattractant dinâmica desencadeada três respostas em um único células vivas em tempo real. Nossos métodos de imagem desenvolvida pode ser aplicada para estudar a quimiotaxia de leucócitos humanos.

Watch this Scientific Journal Video about Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum at JoVE.com

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Aqui, descrevemos os métodos detalhados de células vivas de imagem para investigar quimiotaxia. Apresentamos fluorescência métodos microscópicos para monitorar a dinâmica espaço-temporal dos eventos de sinalização em células migratórias. Medição dos eventos de sinalização nos permite entender melhor como uma rede de sinalização GPCR-alcança gradiente de detecção de quimioatrativos e controles de migração direcional das células eucarióticas.

Receptor de imagem proteína G-Coupled (GPCR) mediada por eventos de sinalização que Quimiotaxia de Controle de Dictyostelium discoideum</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

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Biology

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

17.9K visualizações.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. Aqui, descrevemos os métodos detalhados de células vivas de imagem para investigar quimiotaxia. Apresentamos fluorescência métodos microscópicos para monitorar a dinâmica espaço-temporal dos eventos de sinalização em células migratórias. Medição dos eventos de sinalização nos permite entender melhor como uma rede de sinalização GPCR-alcança gradiente de detecção de quimioatrativos e controles de migração direcional das células eucarióticas.

Vídeo: Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

Em Quantificação in vivo de proteínas G Interações Receptor Acoplado usando espectral Resolvido Microscopia de dois fótons

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Biologia

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

Quantificar atividade agonista no G receptores acoplados à proteína

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET Microscopia para Monitoramento em Tempo Real de eventos de sinalização em células vivas utilizando biossensores unimolecular

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Imagens de células vivas dos primeiros autofagia Eventos: Omegasomes and Beyond

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

Geneticamente codificados Molecular Probes para estudar G receptores acoplados à proteína

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

Caracterização dos receptores acoplados à proteína por uma mobilização de cálcio Ensaio baseado em fluorescência

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

Visualizando Endocitose clatrina mediada por receptores acoplados à proteína a resolução de evento único através de microscopia TIRF

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular development program, during which single cells chemotax towards aggregation centers1-4. This process is dependent on the release of cyclic adenosine monophosphate (cAMP)5. cAMP is produced in waves through the concerted action of adenylate cyclase and phosphodiesterases, and binds to G protein-coupled cAMP receptors6,7. A widely used assay to analyze the mechanisms involved in the developmental cycle of the lower eukaryote Dictyostelium discoideum is based on the observation of cell aggregation in submerged conditions8,9. This protocol describes the analysis of the role of coronin A in the developmental cycle by starvation in tissue-culture plates submerged in balanced salt solution (BSS)10. Coronin A is a member of the widely conserved protein family of coronins that have been implicated in a wide variety of activities11,12. Dictyostelium cells lacking coronin A are unable to form multicellular aggregates, and this defect can be rescued by supplying pulses of cAMP, suggesting that coronin A acts upstream of the cAMP cascade10. The techniques described in these studies provide robust tools to investigate functions of proteins during the initial stages of the developmental cycle of Dictyostelium discoideum upstream of the cAMP cascade. Therefore, utilizing this aggregation assay may allow the further study of coronin A function and advance our understanding of coronin biology.

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

Investigando a função de Coronin A no Precoce fome resposta de<em&gt; Dictyostelium discoideum</em&gt; Por agregação Ensaios

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Imagiologia G protein-coupled receptor mediada por quimiotaxia e seus eventos de sinalização em células de HL60 de Neutrófilos-like

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

Estudando a Proteína de Qualidade Sistema de Controle de<em&gt; D. discoideum</em&gt; Usando temperatura controlada Imagens ao vivo celular

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...