Ce travail est soutenu par le fonds intra-muros du NIAID, NIH.

1. Préparation du chimiotactisme des cellules compétentes de Dictyostelium discoideum <ol><li> Pour générer des cellules D. discoideum qui sont chimiotactiques au camp chimiotactique, la récolte des cellules de plus en plus D3-t rich media à partir d&#39;une culture en agitant à 22 ° C. </li><li> Laver les cellules deux fois en mémoire tampon non nutritifs développement (buffer DB contenant 5 mM Na 2 HPO 4, 5 mM KH 2 PO 4, MgCl2 2 mM, 0,1 mM CaCl et 2). </li><li> Re-suspendre les cellules dans un tampon de DB à une densité de 2x10 7 cellules / ml. </li><li> Agiter 10 ml de cellules dans un flacon de 250 ml à 100 rpm à 22 ° C pendant une heure. </li><li> Livrer 100 ul de 7,5 uM AMPc stocks aux 10 cellules par ml toutes les six minutes de plus de 6 heures pour obtenir une concentration finale de 75 nM d&#39;AMPc, un processus désigné comme le traitement de l&#39;AMPc pulsation. Après 5-6 heures de traitement pulsation AMPc, D. cellules discoideum deviennent compétents chimiotactiques envers gradient d&#39;AMPc. </li><li> Recueillir les cellules par centrifugation à 200 g pendant 5 min puis remettre les cellules avec le tampon DB contenant 2,5 mM de caféine, et secouer à 200 rpm à 22 ° C pendant 20 min à basolate cellule à une situation chimiotactiques. </li></ol> 2. Imagerie cellules chemotaxing dans un gradient chimiotactique visible et manipulable <ol><li> Remblai d&#39;une micropipette avec un fraîchement préparés solution à 30 pi de 1 uM AMPc et Alexa 594 au 0.1μg/μl dans le tampon de la DB. </li><li> Fixez le Femtotip à un titulaire de micropipette et raccorder le tuyau à un appareil d&#39;alimentation de pression, Eppendorf FemtoJet système. </li><li> Fixez l&#39;ensemble micropipette pour un micromanipulateur (Eppendorf TransferMan NK2) motorisé micromanipulateur pour fournir une pression constante en vue d&#39;établir un gradient stable. </li><li> Monter une chambre d&#39;un bien LabTek rempli de 6 ml de tampon DB sur une lentille d&#39;huile 40X sur un microscope confocal et l&#39;utilisation brillante optique de champ, le centre Femtotip dans le champ de vision. </li><li> Allumez l&#39;alimentation de pression et régler la pression de compensation (PC) pour 70 hPa pour établir un gradient de l&#39;AMPc / Alexa 594 mélange. </li><li> Visualisez gradient d&#39;AMPc par la surveillance du mélange de concentration souhaitée de l&#39;AMPc et Alexa 594 fluorescence à l&#39;aide d&#39;excitation avec une ligne de 543 nm du laser. </li><li> Utiliser l&#39;auto-positionnement en fonction de la micromanipulateur pour mettre micropipette pour les positions souhaitées et de les définir comme la position 1, position 2, 3 et position de manipuler le gradient à laquelle la cellule sont exposés. </li></ol> 3. Immobile système cellulaire non polarisé facilite des événements de signalisation impliquées dans l&#39;imagerie de l&#39;AMPc gradient de détection <ol><li> Après le traitement de caféine, enlever une partie aliquote de cellules et centrifuger à 500 g pendant 3 min. </li><li> Retirer du tampon et de diluer les cellules à 5x10 5 cellules / ml avec le tampon DB frais contenant 2,5 mM de caféine. </li><li> Appliquer 1 ml de suspension cellulaire à une chambre simple bien ou 0,4 ml dans chaque puits d&#39;une chambre de quatre puits. </li><li> Laisser les cellules adhèrent pendant 10 min, soigneusement la pipette hors du tampon pour enlever les cellules seules et remplacer par le même volume. </li><li> Repérez les cellules sous le microscope désirée et commencer l&#39;imagerie. </li><li> Pour une expérience conçue pour surveiller la dynamique de la signalisation dans les cellules composantes exposer à un gradient constant, de traiter les cellules avec 5,0 uM (concentration finale) Latrunculin B pendant 10 min avant les expériences. </li></ol> 4. Simultanée hétérotrimériques surveillance protéine G activation et la production PIP 3 <ol><li> AMPc pulsation développer des cellules co-exprimant GαCFP et YFPGβ (cellules G) et des cellules exprimant le PIP 3 Indicateur PH-GFP (cellules PH) 7. </li><li> Mélanger ces deux types de cellules avec un rapport 1:1 et plaque eux dans les chambres d&#39;un bien ou 4-même. </li><li> Créez et enregistrez des émissions de référence d&#39;empreintes digitales courbe de CFP, YFP et la GFP en utilisant le mode d&#39;acquisition Lambda Stack dans la gamme spectrale de 464 à 624 nm avec une largeur de 10 nm. </li><li> Simultanément activation de la protéine G d&#39;image dans les cellules G et 3 PIP production dans des cellules en utilisant PH avec mode Lambda Acquisition même pile dans la gamme spectrale de 464 à 544 nm avec un incrément de 10 nm. </li><li> Appliquer la fonction linéaire de Déconvolution Zeiss 510META logiciels utilisant sauvé CFPand YFP et les empreintes digitales d&#39;émission pour calculer mathématiquement la contribution de chaque fluorophore dans la pile lambda de se séparer de la PCP et de l&#39;intensité YFP dans les canaux individuels dans G. </li><li> Avec la même stratégie, appliquer la fonction linéaire Déconvolution utilisant la GFP et les empreintes digitales enregistrées des émissions de fond pour calculer mathématiquement l&#39;intensité de GFP dans les cellules PH. </li></ol> 5. Les résultats représentatifs: <ol><li> Un excellent modèle de D. discoideum pour chimiotactisme GPCR médiation. Une amibe sociale, D. discoideum présente un chimiotactisme frappante durant le cycle de vie. En raison de ses avantages génétiques et biochimiques, D. D fournit une puissante système d&#39;étudier le chimiotactisme. </li><li> Chimiotactisme des cellules sous un champs visible et manipulable chemoattract. Ici, nous montrons d&#39;abord une méthode simple pour obtenir une relation linéaire entre la concentration d&#39;AMPc et l&#39;intensité d&#39;un colorant fluorescent Alexa 594 par une série de dilution de 2 uM AMPc mélangé avec 10 ug / ml Alexa 594 (figure 2A). Ensuite, nous offrent un moyen facile de visualiser la pente, en outre, d&#39;établir une mesure quantitative de la concentration d&#39;AMPc d&#39;un gradient de l&#39;intensité des Alexa 594 (fig. Fig. 2B). </li><li> Motilité cellulaire est découplée de la polarisation cellulaire et la détection directionnelle. Un inhibiteur polymérisation de l&#39;actine élimine pré-existantes de polarité morphologique et empêche également le mouvement des cellules tout en préservant la capacité des cellules de détection directionnel (figure 3A). Emploi de la stimulation de l&#39;AMPc visible et manipulable garantit l&#39;entrée, par exemple des stimulations appliquées uniformément ou avec un dégradé. Cette méthode permet une analyse quantitative de l&#39;AMPc induite par la redistribution des principaux composants de signalisation dans les machines de détection de gradient. Mesuré dynamique spatio-temporelle de ces composants de signalisation nous permettent de comprendre comment le réseau de signalisation réalise l&#39;adaptation aux stimulations uniforme tout en générant des réponses biochimiques polarisée aux gradients (figure 3B). </li><li> Mesures systémiques de la cinétique des chemosensing réseau de signalisation à l&#39;exposition à un gradient constant. Il est essentiel de mesurer la dynamique / cinétique de la signalisation directionnelle de détection de composantes de comprendre comment chaque élément contribue à l&#39;établissement de la polarisation intracellulaire lorsque les cellules d&#39;expérience du premier gradient. Application de l&#39;imagerie de cellules vivantes avec un tempo-spatiale haute résolution, nous montrons d&#39;abord une biphasique PIP3 production de cellule qui est exposée à un gradient d&#39;AMPc stable (Fig. 4A-C). Application imagerie des cellules vivantes, nous avons systématiquement mesuré dynamique de direction à détection de réseau de signalisation spécifiques de la stimulation de la production d&#39;AMPc d&#39;PIP3 (Fig. 4D, E). </li><li> Contrôle de l&#39;activation simultanée des protéines G hétérotrimériques et PIP 3 lors de la stimulation de production d&#39;AMPc appliquées uniformément. Förster énergie de résonance de transfert (en abrégé FRET) fournit une approche efficace pour contrôler l&#39;activation des protéines G hétérotrimériques (dissociation) lors de la stimulation de l&#39;AMPc. Ici, nous avons décrit une pratique et facile d&#39;adopter le système pour une mesure simultanée de l&#39;activation des protéines G hétérotrimériques et PIP 3 Production en surveillant le FRET changement et translocation membranaire de PIP 3 sonde, PH-GFP dans les cellules G et PH, respectivement (Fig. 5 ). Une stimulation de l&#39;AMPc appliquée uniformément déclenche une activation de la protéine G persistante tout ce qui déclenche un PIP transitoires de production 3. </li></ol><img src="/files/ftp_upload/3128/3128fig1.jpg" alt="Figure 1"/> Figure 1: un excellent modèle de D. discoideum de la chimiotaxie médiée GPCR. A. Schéma montre une voie de signalisation de détection brève directionnelle. B. AMPc induit le chimiotactisme gradient rapide de D. cellules discoideum. Cellules expriment PIP 3 sonde, PH-GFP (Green). Dégradé (rouge) est visualisée par Alexa 594. Barre d&#39;échelle = 50 microns. <img src="/files/ftp_upload/3128/3128fig2.jpg" alt="Figure 2"/> Figure 2: chimiotactisme des cellules sous un champs visible et manipulable chemoattract. A. Le graphique montre une relation linéaire entre la concentration d&#39;AMPc et l&#39;intensité d&#39;un colorant fluorescent Alexa 594 par une série de dilution de 2 uM AMPc mélangé avec 10 ug / ml Alexa 594. B. La mesure quantitative de la concentration d&#39;AMPc d&#39;un gradient par la relation linéaire de la concentration de l&#39;AMPc et l&#39;intensité des colorant fluorescent Alexa 594 dans A. <img src="/files/ftp_upload/3128/3128fig3.jpg" alt="Figure 3"/> Figure 3: la motilité cellulaire est découplée de la polarisation cellulaire et la détection directionnelle. A. Image montre que les cellules immobile par le traitement de l&#39;inhibiteur de la polymérisation de l&#39;actine Latrunculin B maintenir la capacité de détection directionnel. Cellules expriment PIP 3 sonde, PH-GFP (Green). Dégradé (rouge) est visualisée par Alexa 594. B. AMPc cellulaire manipulable système de stimulation et immobile permet de répondre aux questions clés de la détection directionnelle. Barre d&#39;échelle = 10 microns. <img src="/files/ftp_upload/3128/3128fig4.jpg" alt="Figure 4"/> Figure 4: mesures systémiques de la cinétique des chemosensing réseau de signalisation à l&#39;exposition à un gradient constant. A. Montage montre un PIP biphasique de production 3 (verte) de la cellule qui est exposée à un gradient d&#39;AMPc constante (rouge). B. image montre les régions d&#39;intérêt (ROI) pour la mesure de la cinétique de PIP 3 de production présentées dans C. C . Kinetics oPIP F 3 la production dans les cellules exposées à un gradient constant. D. schéma montre le réseau de signalisation de détection directionnelle de la stimulation de l&#39;AMPc à la PIP 3 de la production. Leur cinétique après exposition à un gradient d&#39;équilibre est présenté dans les lignes de même couleur solide dans E. <img src="/files/ftp_upload/3128/3128fig5.jpg" alt="Figure 5"/> Figure 5: simultanée de surveillance multi-événements des réseaux de signalisation des RCPG. Schéma A. montre la mesure simultanée de l&#39;activation des protéines G hétérotrimériques et PIP 3 Production en surveillant le FRET changement et la translocation membranaire des PIP3 sonde, PH-GFP dans les cellules G et PH, respectivement. B. Montage d&#39;images d&#39;arc-en-G et les cellules PH montre qu&#39;une stimulation de l&#39;AMPc appliquée uniformément déclenche une activation de la protéine G persistante à la cellule périphérique, tout ce qui déclenche une passagère PIP3 production. Les points de temps sont avant (0s) et après stimulation par 4.9s, 10.2s et 20.4s. C. Cinétique de l&#39;activation des protéines G et PIP3 production sur une stimulation de l&#39;AMPc appliquées uniformément.

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Aucun conflit d&#39;intérêt déclaré.

Le processus d&#39;atteindre l&#39;étape chimiotactiques compétentes des cellules Pour type sauvage D. cellules discoideum, il faut environ 5 ~ 6 heures pulsation de développement à la température ambiante pour les inciter à une étape bien chimiotactiques compétents au cours de laquelle les cellules présentent une morphologie bien polarisée cellulaire et la migration cellulaire rapide (Fig. 1). Plusieurs facteurs, tels que la concentration d&#39;AMPc d&#39;impulsions...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires </th></tr><tr><td> Médias croissance D3-T </td><td> KD médicale </td><td></td><td></td></tr><tr><td> La caféine </td><td> Sigma </td><td></td><td></td></tr><tr><td> Latrunculin B </td><td> Molecular Probes </td><td></td><td></td></tr><tr><td> Alexa 594 </td><td> Molecular Probes </td><td></td><td></td></tr><tr><td> AMPc </td><td> Sigma </td><td></td><td></td></tr><tr><td> ChronTrol XT minuterie programmable </td><td> ChronTrol Corp </td><td></td><td></td></tr><tr><td> Miniplus 3 pompe péristaltique </td><td> Gilbson </td><td></td><td></td></tr><tr><td> Platform agitateur rotatif </td><td></td><td></td><td></td></tr><tr><td> Approvisionnement FemtoJet pression microcapillaire </td><td> Eppendorf </td><td></td><td></td></tr><tr><td> Simple et quatre ainsi Lab-Tek II lamelle chambres </td><td> Nalge Nunc International </td><td></td><td></td></tr><tr><td> LSM 510 META ou équivalent microscope à fluorescence </td><td> Zeiss </td><td></td><td> une 40X 60X 1,3 NA ou NA 1.4 du pétrole lentilles DIC objectif du Plan-Neofluar </td></tr><tr><td> Olympus X81 ou équivalent </td><td> Olympus </td><td></td><td> Nécessite un 100X 1,47 NA TIRF lentille d&#39;objectif </td></tr></tbody></table>

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

Beaucoup de cellules eucaryotes peuvent détecter des gradients de signaux chimiques dans leur environnement et de migrer en conséquence 1. Cette migration cellulaire guidée est mentionné que le chimiotactisme, qui est essentiel pour différentes cellules pour mener à bien leurs fonctions telles que le trafic des cellules immunitaires et la structuration des cellules neuronales 2, 3. Une grande famille de G-récepteurs couplés aux protéines (RCPG) détecte la variable de petits peptides, connu sous le nom de chimiokines, de diriger la migration cellulaire in vivo 4. L&#39;objectif final de chimiotactisme de recherche est de comprendre comment un gradient de chimiokine GPCR machines et des commandes de signalisation sens des événements menant à la chimiotaxie. À cette fin, nous utilisons des techniques d&#39;imagerie pour suivre, en temps réel, les concentrations spatio-temporelle des mouvements chimiotactiques cellulaire, dans un gradient de chimiotactique, GPCR activation médiée par des protéines G hétérotrimériques, et les signaux intracellulaires impliqués dans le chimiotactisme des cellules eucaryotes 5-8 . L&#39;organisme simple eucaryotes, Dictyostelium discoideum, affiche des comportements chemotaxic qui sont similaires à celles des leucocytes, et D. discoideum est un système modèle pour l&#39;étude des principaux chimiotactisme eucaryotes. Comme amibes libres, D. discoideum cellules se divisent en milieu riche. Après la famine, les cellules entrer dans un programme de développement global dans lequel elles par le biais de l&#39;AMPc à médiation chimiotactisme pour former des structures multicullular. De nombreux composants impliqués dans le chimiotactisme au camp ont été identifiés dans D. discoideum. La liaison de l&#39;AMPc à un GPCR (CAR1) induit la dissociation des protéines G hétérotrimériques dans Gγ et 7 sous-unités Gßy, 9, 10. Sous-unités Gßy activer Ras, qui active à son tour la PI3K, la conversion PIP PIP 2 en 3 sur la membrane cellulaire de 11 à 13. PIP 3 servent de sites de liaison pour des protéines ayant une homologie avec pleckstrine (PH) domaines, recrutant ainsi ces protéines à la membrane 14, 15. L&#39;activation des récepteurs CAR1 contrôle également les associations de la membrane de PTEN, qui déphosphoryle PIP PIP 3 à 2 16, 17. Les mécanismes moléculaires sont évolutivement conservées dans des chimiokines GPCR médiée par le chimiotactisme des cellules humaines telles que les neutrophiles 18. Nous présentons des méthodes suivantes pour étudier le chimiotactisme des D. cellules discoideum. 1. Préparation des cellules composant chimiotactiques. 2. Imagerie chimiotactisme des cellules dans un gradient d&#39;AMPc. 3. Surveillance d&#39;une activation induite des RCPG G hétérotrimériques protéines dans des cellules vivantes simples. 4. Imagerie chimioattractive déclenché PIP dynamique 3 réponses simples cellules vivantes en temps réel. Nos méthodes d&#39;imagerie développée peut être appliquée à l&#39;étude du chimiotactisme des leucocytes humains.

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

Ici, nous décrivons les méthodes détaillées direct imagerie cellulaire pour enquêter sur le chimiotactisme. Nous présentons la fluorescence des méthodes microscopiques pour surveiller la dynamique spatio-temporelle des événements de signalisation dans les cellules en migration. Mesure des événements de signalisation nous permet de mieux comprendre comment un réseau de signalisation GPCR réalise gradient de détection et de contrôle des chimiotactiques migration directionnelle des cellules eucaryotes.

Récepteur d&#39;imagerie couplés aux protéines G (RCPG) à médiation événements de signalisation qui contrôle chimiotactisme des 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 vues.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. Ici, nous décrivons les méthodes détaillées direct imagerie cellulaire pour enquêter sur le chimiotactisme. Nous présentons la fluorescence des méthodes microscopiques pour surveiller la dynamique spatio-temporelle des événements de signalisation dans les cellules en migration. Mesure des événements de signalisation nous permet de mieux comprendre comment un réseau de signalisation GPCR réalise gradient de détection et de contrôle des chimiotactiques migrat...

Vidéo: 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.

Dans la quantification in vivo des interactions des protéines G récepteurs couplés utilisant résolution spectrale microscopie à deux photons

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

Recherche

Biologie

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.

Quantifier l&#39;activité agoniste à G récepteurs couplés aux protéines

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 microscopie pour Surveillance en temps réel des événements de signalisation dans les cellules vivantes en utilisant Biocapteurs unimoléculaires

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.

Vivre imagerie cellulaire de l&#39;autophagie premiers événements: Omegasomes et au-delà

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.

Molecular Probes génétiquement codés pour étude g récepteurs couplés à la protéine

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).

Caractérisation des récepteurs de protéines G couplées par une mobilisation de calcium dosage à base de fluorescence

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.

Visualisation endocytose clathrine de G récepteurs couplés aux protéines à un seul événement résolution par microscopie 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.

Étude de la fonction de Coronin A dans la réponse Starvation précoce de<em&gt; Dictyostelium discoideum</em&gt; Par agrégation Assays

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 Protein-Coupled Receptor-médiée chimiotactisme et ses événements de signalisation dans les cellules HL60 neutrophiles comme

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.

L&#39;étude de la protéine système de contrôle de la qualité des<em&gt; D. discoideum</em&gt; Utilisation de cellules vivantes d&#39;imagerie à température contrôlée

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