Este trabajo es apoyado por el fondo de intramuros del NIAID, NIH.

1. Preparación de células competentes quimiotáctica de Dictyostelium discoideum <ol><li> Para generar células D. discoideum que son quimiotácticas al campo quimiotáctica, las células de la cosecha que crece en D3-T de medios enriquecidos a partir de una cultura de agitación a 22 ° C. </li><li> Lavar las células dos veces en buffer no nutrientes en el desarrollo (DB buffer conteniendo 5 mM Na 2 HPO 4, 5 mM KH 2 PO 4, 2 mM de MgCl 2 y 0,1 mM CaCl 2). </li><li> Vuelva a suspender las células en un tampón de DB a una densidad de 2x10 7 células / ml. </li><li> Agitar 10 ml de células en un frasco de 250 ml a 100 rpm a 22 ° C durante una hora. </li><li> Entregar 100 l de 7,5 M de valores de AMPc a las 10 células ml cada seis minutos más de 6 horas para conseguir una concentración final de 75 nM de cAMP, un proceso designado como el tratamiento de cAMP pulso. Después de 5-6 horas de tratamiento AMPc pulso, D. células discoideum se quimiotáctica competentes hacia gradiente de cAMP. </li><li> Recoger las células por centrifugación a 200 g por 5 min y luego suspender las células con tampón DB contiene 2,5 mM de cafeína, y agitar a 200 rpm a 22 ° C durante 20 minutos para basolate celular a una situación quimiotácticos. </li></ol> 2. Imágenes chemotaxing células en un gradiente quimiotáctico visible y manipulable <ol><li> Relleno de una micropipeta con un recién preparada la solución de 30 l de AMPc M 1 y Alexa 594 en 0.1μg/μl en tampón DB. </li><li> Conecte el Femtotip al titular de la micropipeta y conectar el tubo a un aparato de presión de alimentación, sistema de Eppendorf FemtoJet. </li><li> Fije el conjunto de micropipeta a un micromanipulador (Eppendorf TransferMan NK2) motorizados micromanipulador para proporcionar una presión constante a fin de establecer un gradiente estable. </li><li> Montar una cámara de un bien Labtek lleno con 6 ml de tampón de DB en una lente de 40x de aceite en un microscopio confocal y el uso brillante campo de la óptica, el centro de la Femtotip en el campo de visión. </li><li> Abra el suministro de presión y establecer la compensación de presión (Pc) por 70 hPa para establecer un gradiente de la cAMP / Alexa 594 mezcla. </li><li> Visualizar gradiente de AMPc por el control de la mezcla de concentración deseada de AMPc y Alexa 594 fluorescencia utilizando la excitación con una línea de 543 nm láser. </li><li> El uso de auto-posicionamiento en función de la micropipeta micromanipulador para poner a las posiciones deseadas y los puso como la posición 1, posición 2, y 3 posiciones para manipular el gradiente de la celda que están expuestos. </li></ol> 3. Sistema inmóvil de células no polarizadas facilita eventos de imagen de señalización implicadas en la detección de gradiente de AMPc <ol><li> Después de tratamiento con cafeína, eliminar una parte alícuota de las células y se centrifuga a 500 g por 3 min. </li><li> Quitar buffer y diluir las células a 5x10 5 células / ml con tampón DB fresco que contiene 2,5 mM de cafeína. </li><li> Aplicar 1 ml de suspensión celular a una cámara de un solo bien o 0,4 ml a cada pocillo de una cámara de cuatro pozos. </li><li> Permiten que las células se adhieran durante 10 minutos, con cuidado de la pipeta tampón para eliminar las células sueltas y reemplazar con el mismo volumen. </li><li> Localizar las células deseadas bajo el microscopio y empezar de imágenes. </li><li> Para un experimento diseñado para controlar la dinámica de componentes de señalización en las células de la exposición a un gradiente estable, el tratamiento de las células con 5,0 M (concentración final) latrunculin B durante 10 minutos antes de los experimentos. </li></ol> 4. Heterotrimeric simultánea de vigilancia y activación de la proteína G PIP 3 producción <ol><li> AMPc pulso del desarrollo de células co-expresando GαCFP y YFPGβ (células G) y las células que expresan PIP 3 indicador de PH-GFP (células PH) 7. </li><li> Mezclar estos dos tipos de células con una relación 1:1 y la placa de ellos en cámaras de un solo bien o 4 bien. </li><li> Crear y guardar las emisiones de huellas dactilares curva de referencia de la PPC, YFP y las buenas prácticas agrarias con la pila Lambda modo de adquisición en el rango espectral desde 464 hasta 624 nm con una anchura de 10 nm. </li><li> Al mismo tiempo la imagen G activación de la proteína en las células G y PIP 3 en células PH se utiliza con el modo de adquisición de Lambda misma pila en el rango espectral desde 464 hasta 544 nm con incrementos de 10 nm. </li><li> Aplicar la función desmezcla lineal de Zeiss con software 510META salvado CFPand YFP y las huellas digitales de emisión para calcular matemáticamente la contribución de cada fluoróforo en la pila de Lambda para separar la política pesquera común y la intensidad YFP en canales individuales de G. </li><li> Con la misma estrategia, aplicar la función desmezcla lineal utilizando GFP salvado y las huellas digitales de fondo de emisión para calcular matemáticamente la intensidad de GFP en las células PH. </li></ol> 5. Los resultados representativos: <ol><li> Un excelente sistema modelo de D. discoideum de quimiotaxis mediada por GPCR. Una ameba social, D. discoideum exhibe una quimiotaxis en huelga durante el ciclo de vida. Debido a sus ventajas genéticas y bioquímicas, D. D proporciona un potente sistema para estudiar la quimiotaxis. </li><li> Quimiotaxis de las células bajo un campo visible y manipulable chemoattract. Aquí, lo primero que muestran una metodología sencilla para obtener una relación lineal entre los campos de concentración y la intensidad de un tinte fluorescente Alexa 594 por una serie de dilución de AMPc M 2 con 10 mg / ml Alexa 594 (Fig. 2A). A continuación, ofrecemos una forma sencilla de visualizar la pendiente, por otra parte, para establecer una medida cuantitativa de la concentración de cAMP de un gradiente de la intensidad de Alexa 594 (Fig. fig. 2B). </li><li> Motilidad celular no se acopla con la polarización celular y la detección de dirección. Un inhibidor de la polimerización de la actina elimina preexistentes polaridad morfológica y también evita el movimiento de células, manteniendo la capacidad de las células de detección de dirección (Fig. 3A). El empleo de la estimulación AMPc visible y manipulable garantiza la entrada, por ejemplo, los estímulos aplicados de manera uniforme o un degradado. Este método permite un análisis cuantitativo de AMPc inducida por la redistribución de los componentes clave de señalización en el mecanismo de detección de gradiente. Mide la dinámica espacio-temporal de los componentes de señalización nos permite entender cómo la red de señalización logra la adaptación a los estímulos uniforme mientras que la generación polarizada respuestas bioquímicas de los gradientes (Fig. 3B). </li><li> Medidas sistémicas de la cinética de chemosensing red de señalización con la exposición a un gradiente estable. Es muy importante para medir la dinámica / cinética de la dirección de detección de componentes de señalización para entender cómo cada componente contribuye a la creación de la polarización intracelular cuando las células de la experiencia primer gradiente. Aplicación de imágenes de células vivas, con un tempo-espacial de alta resolución, lo primero que muestran una bifásica PIP3 producción de células que se expone a un gradiente de AMPc constante (Fig. 4A-C). La aplicación de imágenes de células vivas, hemos medido sistemáticamente la dinámica de la dirección de detección de la red de señalización específica de la estimulación de la producción de cAMP a PIP3 (Fig. 4D, E). </li><li> El monitoreo simultáneo heterotrimeric activación de la proteína G y PIP 3 producción a la estimulación AMPc aplicación uniforme. Förster resonancia de transferencia de energía (FRET abreviado) proporciona un método eficiente para controlar la activación de la proteína G heterotrimeric (disociación) a la estimulación del AMPc. En este sentido, se describe una práctica fácil de adoptar el sistema para la medición simultánea de la activación de proteínas G heterotrimeric y la producción de PIP 3 por el control de la FRET cambio y el desplazamiento de la membrana de la sonda PIP 3, PH-GFP en las células G y PH, respectivamente (Fig. 5 ). Una estimulación AMPc aplicación uniforme provoca una persistente activación de la proteína G, mientras que desencadena una PIP transitoria 3 de producción. </li></ol><img src="/files/ftp_upload/3128/3128fig1.jpg" alt="Figura 1"/> Figura 1: Un sistema excelente modelo de D. discoideum de quimiotaxis mediada por GPCR. A. Esquema muestra una vía de señalización breve de detección de dirección. B. gradiente de AMPc induce la quimiotaxis rápida de D. células discoideum. Células expresan PIP 3 de la sonda, PH-GFP (Green). Gradiente (rojo) se visualiza por Alexa 594. Barra de escala = 50 micras. <img src="/files/ftp_upload/3128/3128fig2.jpg" alt="Figura 2"/> Figura 2: La quimiotaxis de las células bajo un campo visible y manipulable chemoattract. A. El gráfico muestra una relación lineal entre los campos de concentración y la intensidad de un tinte fluorescente Alexa 594 por una serie de dilución de AMPc M 2 con 10 mg / mL Alexa 594. Medición B. cuantitativa de los campos de concentración de un gradiente de la relación lineal de los campos de concentración y la intensidad de fluorescencia tinte Alexa 594 en A. <img src="/files/ftp_upload/3128/3128fig3.jpg" alt="Figura 3"/> Figura 3: la motilidad celular no se acopla con la polarización celular y la detección de dirección. A. Imagen muestra que las células inmóviles por el tratamiento de inhibidor de la polimerización de la actina B latrunculin mantener la capacidad de detección de dirección. Células expresan PIP 3 de la sonda, PH-GFP (Green). Gradiente (rojo) se visualiza por Alexa 594. B. AMPc manipulables estimulación e inmóvil sistema celular permite hacer frente a cuestiones clave de la detección de dirección. Barra de escala = 10μm. <img src="/files/ftp_upload/3128/3128fig4.jpg" alt="Figura 4"/> Figura 4: Medidas sistémicas de la cinética de chemosensing red de señalización con la exposición a un gradiente estable. A. Montaje muestra un PIP bifásica producción de 3 (verde) de la célula que está expuesto a un gradiente de AMPc constante (rojo). B. La imagen muestra las regiones de interés (ROI) para la medición de la cinética de la producción de PIP 3 se presentan en C. C . cinética oPIP f 3 en la producción de las células expuestas a un gradiente estable. D. esquema muestra la red de señalización de los sensores de dirección de la estimulación del AMPc a la producción de PIP 3. Su cinética de la exposición a un gradiente constante se presenta en las líneas del mismo color sólido en E. <img src="/files/ftp_upload/3128/3128fig5.jpg" alt="Figura 5"/> Figura 5: Simultánea de monitoreo multi-eventos de las redes de señalización de GPCR. A. Esquema muestra la medición simultánea de la activación de proteínas G heterotrimeric y la producción de PIP 3 por el control de la FRET cambio y el desplazamiento de la membrana de la sonda PIP3, PH-GFP en las células G y PH, respectivamente. B. Montaje de imágenes de arco iris de G y las células PH muestra que la estimulación AMPc aplicación uniforme provoca una persistente activación de la proteína G en el periférico de células, mientras que provoca un transitorio PIP3 producción. Los dos momentos son antes (0s) y después de la estimulación de 4.9s, y 10.2s 20.4s. C. Cinética de la activación de la proteína G y PIP3 de producción en una estimulación AMPc aplicación uniforme.

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No hay conflictos de interés declarado.

El proceso de llegar a la etapa quimiotáctica de las células competentes De tipo salvaje D. células discoideum, se tarda unos 5 a 6 horas pulsando el desarrollo a temperatura ambiente para inducir a un estado competente bien quimiotácticas durante el cual las células presentan una morfología celular y la migración celular polarizada y rápida (Fig. 1). Varios factores, tales como los campos de concentración de pulso, temperatura, y diferentes fondos genéticos, pueden ...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> D3-T medios de cultivo </td><td> KD Médico </td><td></td><td></td></tr><tr><td> Cafeína </td><td> Sigma </td><td></td><td></td></tr><tr><td> Latrunculin B </td><td> Las sondas moleculares </td><td></td><td></td></tr><tr><td> Alexa 594 </td><td> Las sondas moleculares </td><td></td><td></td></tr><tr><td> AMPc </td><td> Sigma </td><td></td><td></td></tr><tr><td> ChronTrol XT temporizador programable </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 rotatorio </td><td></td><td></td><td></td></tr><tr><td> FemtoJet fuente de presión capilar se </td><td> Eppendorf </td><td></td><td></td></tr><tr><td> De uno y cuatro bien Lab-Tek II cubreobjetos cámaras </td><td> Nalge Nunc Internacional </td><td></td><td></td></tr><tr><td> LSM 510 META microscopio de fluorescencia o equivalente </td><td> Zeiss </td><td></td><td> un 40X 60X o 1.3 NA 1.4 NA petróleo DIC Plan-Neofluar lente objetivo </td></tr><tr><td> Olympus X81 o equivalente </td><td> Olimpo </td><td></td><td> Requiere un 100X 1,47 NA TIRF lente del objetivo </td></tr></tbody></table>

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

Muchas células eucariotas pueden detectar gradientes de señales químicas en sus entornos y en consecuencia la migración 1. Esta migración de las células guiadas se conoce como quimiotaxis, que es esencial para varias células para llevar a cabo sus funciones, como el tráfico de las células inmunes y los patrones de las células neuronales 2, 3. Una gran familia de receptores de la proteína G-acoplada (GPCR) detecta variable de péptidos pequeños, conocidos como quimiocinas, para dirigir la migración celular in vivo 4. El objetivo final de la quimiotaxis de investigación es entender cómo un GPCR sentidos maquinaria gradientes de quimioquinas y los controles de los eventos de señalización que conducen a la quimiotaxis. Para ello, utilizamos técnicas de imagen para controlar, en tiempo real, las concentraciones de espacio-temporal del movimiento quimiotácticos de células, en un gradiente de quimioatrayente, mediada por la activación de GPCR heterotrimeric G-proteína, y los eventos intracelulares de señalización implicadas en la quimiotaxis de las células eucariotas 5.8 . El organismo simple eucariotas, Dictyostelium discoideum, muestra comportamientos quimiotaxis que son similares a los de los leucocitos, y D. discoideum es un modelo de sistema clave para el estudio de la quimiotaxis de eucariotas. Como amebas de vida libre, D. las células se dividen discoideum en medio rico. Sobre el hambre, las células entran en un programa de desarrollo en el que agregada a través de AMPc mediada la quimiotaxis para formar estructuras multicullular. Muchos de los componentes involucrados en la quimiotaxis de los campamentos han sido identificados en el D. discoideum. La unión del AMPc a un GPCR (CAR1) induce la disociación de heterotrimeric proteínas G en Gγ y 7 subunidades Gβγ, 9, 10. Subunidades Gβγ activar Ras, que a su vez activa la PI3K, convirtiendo PIP PIP 2 en 3 en la membrana celular 11-13. PIP 3 sirven como sitios de unión para las proteínas con homología pleckstrin (PH) dominios, con lo que reclutar a estas proteínas de la membrana 14, 15. La activación de receptores CAR1 también controla las asociaciones de la membrana de PTEN, que desfosforila PIP PIP 3 a 2 16, 17. Los mecanismos moleculares que son conservadas evolutivamente en las quimiocinas GPCR mediada por quimiotaxis de las células humanas, como los neutrófilos 18. Se presentan los siguientes métodos para el estudio de la quimiotaxis de D. células discoideum. 1. Preparación de las células componentes quimiotácticos. 2. Imágenes de la quimiotaxis de las células en un gradiente de cAMP. 3. Vigilancia de la activación inducida por GPCR de heterotrimeric G-proteína en las células individuales en vivo. 4. Imágenes quimioatrayente activada por PIP dinámica 3 respuestas en una sola las células vivas en tiempo real. Nuestra desarrollado métodos de imagen se pueden aplicar para estudiar la quimiotaxis de los leucocitos 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

A continuación, describimos los métodos detallados en vivo de imágenes de células para la investigación de la quimiotaxis. Se presenta fluorescencia microscópica métodos para controlar la dinámica espacio-temporal de los eventos de señalización en las células que migran. La medición de la señalización de eventos nos permite entender mejor cómo una red de señalización de GPCR logra gradiente de detección de factores quimiotácticos y los controles de la migración direccional de las células eucariotas.

Receptor de imagen acoplados a proteína G (GPCR) mediada por eventos de señalización que la quimiotaxis de control 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 ...

imaging-g-protein-coupled-receptor-gpcr-mediated-signaling-events

Research

JoVE Journal

Biology

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

17.9K vistas.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. A continuación, describimos los métodos detallados en vivo de imágenes de células para la investigación de la quimiotaxis. Se presenta fluorescencia microscópica métodos para controlar la dinámica espacio-temporal de los eventos de señalización en las células que migran. La medición de la señalización de eventos nos permite entender mejor cómo una red de señalización de GPCR logra gradiente de detección de factores quimiotácticos y los controles de la m...

Video: 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.

En la cuantificación in vivo de interacciones de proteínas G del receptor acoplado con espectralmente resuelve de dos fotones de Microscopía

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

Investigación

Biología

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.

La cuantificación de la actividad agonista en el G receptores acoplados a proteínas

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 Microscopía de Monitoreo en tiempo real de eventos de señalización en las células vivas Uso de biosensores unimoleculares

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.

Imágenes de células vivas de las primeras autofagia Eventos: Omegasomes y más allá

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.

Codificados genéticamente-sondas moleculares para Estudiar G receptores acoplados a proteínas

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).

Caracterización de G receptores acoplados a proteína por un ensayo de movilización de calcio basado en fluorescencia

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.

Visualización de endocitosis mediada por clatrina de G receptores acoplados a proteínas en la Resolución Individual de eventos a través de TIRF de Microscopía

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.

La investigación de la función de Coronin A en la temprana respuesta del hambre de<em&gt; Dictyostelium discoideum</em&gt; Los ensayos de agregación por

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.

Imaging G acoplados a proteínas mediada por el receptor de quimiotaxis y sus eventos de señalización en células HL60 neutrófilos como

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.

Estudiar el Sistema de Control de Calidad de la Proteína<em&gt; D. discoideum</em&gt; Uso de imágenes de células vivas con temperatura controlada

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