Questo lavoro è supportato dal fondo intramurale dal NIAID, NIH.

1. Preparazione delle cellule competenti chemiotattici di Dictyostelium discoideum <ol><li> Per generare cellule di D. discoideum che sono chemiotattica al campo chemoattractant, le cellule crescono in vendemmia rich media D3-T da una cultura agitazione a 22 ° C. </li><li> Lavare le cellule due volte in non-nutrienti sviluppo tampone (buffer DB contenente 5 mM Na 2 HPO 4, 5 mM KH 2 PO 4, 2 mM MgCl 2, e 0,1 mM CaCl 2). </li><li> Risospendere le cellule nel buffer DB ad una densità di 2x10 7 cellule / ml. </li><li> Agitare 10 cellule ml in un pallone da 250 ml a 100 rpm a 22 ° C per un&#39;ora. </li><li> Consegnare 100 l di 7,5 mM magazzino cAMP al 10 celle ml ogni sei minuti più di 6 ore per raggiungere una concentrazione finale di 75 nM cAMP, un processo designato come trattamento cAMP pulsante. Dopo 5-6 ore di trattamento cAMP pulsante, D. cellule discoideum diventare chemiotattica competente verso gradiente cAMP. </li><li> Raccogliere le cellule per centrifugazione a 200 g per 5 minuti e poi risospendere le cellule con un tampone DB contenente 2,5 mM di caffeina, e si agita a 200 giri al minuto a 22 ° C per 20 min a basolate cella a una situazione chemiotattica. </li></ol> 2. Imaging cellule chemotaxing in un gradiente chemiotattico visibile e manipolabile <ol><li> Riporto una micropipetta con appena preparato 30 ml di soluzione 1 mM cAMP e Alexa 594 a 0.1μg/μl nel buffer DB. </li><li> Collegare il Femtotip al titolare micropipetta e collegare il tubo di un apparato pressione di alimentazione, Eppendorf sistema FemtoJet. </li><li> Fissare il gruppo micropipetta ad un micromanipolatore (Eppendorf TransferMan NK2) micromanipolatore motorizzato per assicurare una pressione costante al fine di stabilire un gradiente stabile. </li><li> Montare una sola camera LabTek ben riempito con 6 ml di tampone DB su una lente olio 40X su un microscopio confocale e l&#39;utilizzo luminoso-campo dell&#39;ottica, il centro Femtotip nel campo di vista. </li><li> Accendere l&#39;alimentazione di pressione e di impostare la pressione di compensazione (Pc) per 70 hPa di stabilire un gradiente del campo / Alexa 594 miscela. </li><li> Visualizza gradiente cAMP attraverso il monitoraggio della miscela di concentrazione desiderata di cAMP e Alexa 594 fluorescenza con eccitazione con una linea di 543 nm laser. </li><li> Utilizzare la funzione di auto-posizionamento del micromanipolatore per mettere micropipetta nelle posizioni desiderate e impostarle come posizione 1, posizione 2 e posizione 3 di manipolare il gradiente a cui cellule sono esposti. </li></ol> 3. Immobile sistema a celle nonpolarized facilita eventi di imaging segnale coinvolte nel cAMP gradiente di rilevamento <ol><li> Dopo il trattamento la caffeina, rimuovere una aliquota di cellule e centrifugare a 500g per 3 min. </li><li> Eliminare la soluzione tampone e diluire le cellule a 5x10 5 cellule / ml con tampone DB fresco contenente 2,5 mM caffeina. </li><li> Applicare 1 ml di sospensione cellulare in un singolo bene da camera o 0,4 ml di ciascun pozzetto di una ben quattro camera. </li><li> Consentono alle cellule di aderire per 10 minuti, con attenzione pipetta fuori il buffer per rimuovere le cellule distaccati e sostituirlo con lo stesso volume. </li><li> Individuare le cellule al microscopio desiderato e iniziare imaging. </li><li> Per un esperimento progettato per monitorare le dinamiche di componenti di segnalamento nelle cellule esporre ad una pendenza, trattare le cellule con 5,0 mM (concentrazione finale) Latrunculin B per 10 minuti prima degli esperimenti. </li></ol> 4. L&#39;attivazione simultanea di monitoraggio eterotrimeriche G proteine ​​e PIP 3 Produzione <ol><li> cAMP pulsare sviluppare cellule co-esprimono GαCFP e YFPGβ (cellule G) e cellule che esprimono PIP 3 Indicatore PH-GFP (cellule PH) 7. </li><li> Mescolare i due tipi di cellule con rapporto 1:1 e piastra in camere di un bene o 4-bene. </li><li> Creare e salvare l&#39;emissione delle impronte digitali di riferimento della curva CFP, YFP e GFP utilizzando la modalità Stack Lambda acquisizione entro l&#39;intervallo spettrale 464-624 nm con una larghezza di 10 nm. </li><li> Contemporaneamente l&#39;attivazione delle proteine ​​G immagine nelle cellule G e PIP 3 Produzione in cellule PH utilizzando con la stessa modalità di acquisizione Stack Lambda all&#39;interno della gamma spettrale 464-544 nm con incrementi di 10 nm. </li><li> Applicare la funzione lineare Unmixing del software Zeiss 510META utilizzando salvato CFPand YFP e le impronte digitali di emissioni di calcolare matematicamente il contributo di ciascun fluoroforo nel Stack Lambda per separare il PCP e l&#39;intensità YFP nei singoli canali in G. </li><li> Con la stessa strategia, applicare la funzione lineare Unmixing usando GFP salvate e le impronte digitali emissione sfondo per calcolare matematicamente intensità GFP in cellule PH. </li></ol> 5. Rappresentante dei risultati: <ol><li> Un sistema eccellente modello di D. discoideum per chemiotassi mediata GPCR. un&#39;ameba sociale, D. discoideum esibisce una chemiotassi colpisce durante il ciclo di vita. Grazie ai vantaggi genetici e biochimici, D. D offre una potente system studiare chemiotassi. </li><li> Chemiotassi di cellule al campo visibile e manipolabile chemoattract. Qui, prima mostra una metodologia semplice per ottenere una relazione lineare tra la concentrazione di cAMP e l&#39;intensità di un colorante fluorescente Alexa 594 da una serie di diluizioni di 2 cAMP mM mescolati con 10 mg / ml Alexa 594 (Fig. 2A). Successivamente, forniamo un modo semplice per visualizzare la pendenza, inoltre, a stabilire una misura quantitativa delle concentrazioni di cAMP di un gradiente per l&#39;intensità di Alexa 594 (Fig. fig. 2B). </li><li> Motilità cellulare è staccata con polarizzazione delle cellule e di rilevamento direzionale. Un inibitore di polimerizzazione di actina elimina preesistenti polarità morfologica e impedisce anche il movimento delle cellule, pur mantenendo la capacità delle cellule &#39;di rilevamento direzionale (Fig. 3A). Occupazione di stimolazione cAMP visibile e manipolabile garantisce l&#39;ingresso, per esempio stimolazioni applicate uniformemente o un gradiente. Questo metodo permette un&#39;analisi quantitativa di cAMP indotta redistribuzione dei principali componenti di segnalazione nel macchinario gradiente di rilevamento. Misurata la dinamica spazio-temporale di queste componenti di segnalazione ci permettono di capire come la rete di segnalazione raggiunge adattamento alle stimolazioni uniforme, mentre la generazione polarizzata risposte biochimiche a gradienti (Fig. 3B). </li><li> Misure sistemiche di cinetica di chemosensing rete di segnalazione in caso di esposizione ad una pendenza. E &#39;fondamentale per misurare la dinamica / cinetica di direzionale-sensing componenti di segnalamento per capire come ogni componente contribuisce alla creazione di polarizzazione intracellulare quando le cellule esperienza primo gradiente. Applicazione di imaging cellulare dal vivo con un ritmo alto-risoluzione spaziale, per prima cosa mostrano un bifasico PIP3 produzione di cellule che è esposto ad un gradiente di cAMP costante (Fig. 4A-C). L&#39;applicazione di immagini dal vivo di cellule, abbiamo misurato sistematicamente le dinamiche del direzionale-sensing specifica rete di segnalazione da stimolazione cAMP a PIP3 produzione (Fig. 4D, E). </li><li> Monitoraggio simultaneo eterotrimeriche attivazione della proteina G e PIP 3 Produzione su stimolazione cAMP applicata uniformemente. Förster risonanza trasferimento di energia (abbreviato FRET) fornisce un approccio efficace per il monitoraggio eterotrimeriche attivazione della proteina G (dissociazione) al momento della stimolazione cAMP. Qui, abbiamo descritto un conveniente facile adottare sistemi per la misurazione simultanea di eterotrimeriche attivazione della proteina G e PIP 3 di produzione attraverso il monitoraggio della FRET cambiamento e traslocazione della membrana del PIP 3 sonda, PH-GFP in G e cellule PH, rispettivamente (Fig. 5 ). Una stimolazione cAMP applicate uniformemente innesca una persistente attivazione della proteina G, mentre che innesca un transitorio PIP 3 di produzione. </li></ol><img src="/files/ftp_upload/3128/3128fig1.jpg" alt="Figura 1"/> Figura 1: Un sistema eccellente modello di D. discoideum per chemiotassi mediata GPCR. A. Schema mostra un percorso breve di segnalazione del rilevamento direzionale. B. gradiente cAMP induce la chemiotassi rapida D. discoideum cellule. Cellule esprimono PIP 3 sonda, PH-GFP (Green). Gradiente (Red) è visualizzato da Alexa 594. Barra di scala = 50 micron. <img src="/files/ftp_upload/3128/3128fig2.jpg" alt="Figura 2"/> Figura 2: chemiotassi di cellule al campo visibile e manipolabile chemoattract. A. Il grafico mostra una relazione lineare tra la concentrazione di cAMP e l&#39;intensità di un colorante fluorescente Alexa 594 da una serie di diluizioni di 2 cAMP mM mescolati con 10 mg / ml Alexa 594. B. misura quantitativa della concentrazione di cAMP di un gradiente dalla relazione lineare della concentrazione di cAMP e l&#39;intensità del colorante fluorescente Alexa 594 in A. <img src="/files/ftp_upload/3128/3128fig3.jpg" alt="Figura 3"/> Figura 3: motilità cellulare è staccata con polarizzazione delle cellule e di rilevamento direzionale. Immagine A. dimostra che le cellule immobile dal trattamento degli inibitori della polimerizzazione Latrunculin B mantenere la capacità di rilevamento direzionale. Cellule esprimono PIP 3 sonda, PH-GFP (Green). Gradiente (Red) è visualizzato da Alexa 594. B. cAMP manipolabile stimolazione e immobili sistema a celle permette di affrontare le questioni chiave di rilevamento direzionale. Barra di scala = 10μm. <img src="/files/ftp_upload/3128/3128fig4.jpg" alt="Figura 4"/> Figura 4: misure sistemiche di cinetica di chemosensing rete di segnalazione in caso di esposizione ad una pendenza. A. Montage mostra un bifasico PIP 3 Produzione (verde) della cellula, che è esposto ad un gradiente di cAMP costante (rosso). B. immagine mostra le regioni di interesse (ROI) per la misura della cinetica di PIP 3 Produzione presentato in C. C . Cinetica oPIP f 3 della produzione nelle cellule esposte a una pendenza costante. D. schema mostra la rete di segnalazione del rilevamento direzionale dalla stimolazione cAMP al PIP 3 di produzione. La cinetica in caso di esposizione ad una pendenza è presentato nelle linee dello stesso colore solido in E. <img src="/files/ftp_upload/3128/3128fig5.jpg" alt="Figura 5"/> Figura 5: monitoraggio simultaneo a più eventi di reti GPCR segnalazione. Schema A. mostra misurazione simultanea di eterotrimeriche attivazione della proteina G e PIP 3 di produzione attraverso il monitoraggio della FRET cambiamento e traslocazione della membrana di PIP3 sonda, PH-GFP in G e cellule PH, rispettivamente. B. Montaggio di immagini arcobaleno di G e cellule PH mostra che una stimolazione cAMP applicate uniformemente innesca una persistente attivazione della proteina G al cellulare periferico, mentre che innesca un transitorio PIP3 produzione. I punti ora vengono prima (0s) e dopo stimolazione per 4.9s, e 10.2s 20.4s. C. Cinetica di attivazione delle proteine ​​G e PIP3 produzione su una stimolazione cAMP applicata uniformemente.

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I processi di raggiungere chemiotattici stadio competente delle cellule Per il tipo selvaggio D. cellule discoideum, ci vogliono circa 5 ~ 6 ore pulsare lo sviluppo a temperatura ambiente per indurli in un pozzo-chemiotattica fase competenti durante il quale le cellule visualizzare una morfologia ben polarizzata cellulare e la migrazione cellulare rapida (Fig. 1). Diversi fattori, quali la concentrazione di cAMP per pulsare, la temperatura e diversi background genetico, posson...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> D3-T crescita media </td><td> KD medici </td><td></td><td></td></tr><tr><td> Caffeina </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> cAMP </td><td> Sigma </td><td></td><td></td></tr><tr><td> ChronTrol XT timer programmabile </td><td> ChronTrol Corp </td><td></td><td></td></tr><tr><td> Miniplus 3 pompa peristaltica </td><td> Gilbson </td><td></td><td></td></tr><tr><td> Piattaforma rotativo </td><td></td><td></td><td></td></tr><tr><td> FemtoJet pressione di alimentazione microcapillare </td><td> Eppendorf </td><td></td><td></td></tr><tr><td> Singolo e quattro ben Lab-Tek II coprioggetti camere </td><td> Nalge Nunc internazionale </td><td></td><td></td></tr><tr><td> LSM 510 META o equivalente microscopio a fluorescenza </td><td> Zeiss </td><td></td><td> uno 1,3 NA 40X o 60X 1,4 NA olio DIC Plan-Neofluar obiettivo lente </td></tr><tr><td> Olympus X81 o equivalente </td><td> Olimpo </td><td></td><td> Richiede un 100X 1,47 NA TIRF lente dell&#39;obiettivo </td></tr></tbody></table>

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

Molte cellule eucariotiche in grado di rilevare gradienti di segnali chimici nei loro ambienti e di conseguenza migrano 1. Questa migrazione delle cellule guidata è denominato come chemiotassi, che è essenziale per varie cellule di svolgere le loro funzioni come il traffico delle cellule immunitarie ed il modello di cellule neuronali 2, 3. Una grande famiglia di proteine ​​G recettori accoppiati (GPCR) rileva variabile piccoli peptidi, noto come chemochine, per dirigere la migrazione delle cellule in vivo 4. L&#39;obiettivo finale della chemiotassi ricerca è quello di capire come un macchinario sensi GPCR chemochine gradienti ei controlli di segnalazione eventi che portano alla chemiotassi. A tal fine, usiamo tecniche di imaging per monitorare, in tempo reale, le concentrazioni di fattori chemiotattici spazio-temporale, il movimento delle cellule in un gradiente di chemiotattico, GPCR mediata eterotrimerica attivazione di proteine ​​G, e gli eventi di segnalazione intracellulare coinvolte nella chemiotassi di cellule eucariotiche 5-8 . L&#39;organismo semplice eucariotiche, Dictyostelium discoideum, display chemotaxic comportamenti che sono simili a quelli dei leucociti, e D. discoideum è un modello di sistema chiave per lo studio chemiotassi degli eucarioti. Come amebe a vita libera, D. discoideum cellule si dividono in media ricchi. Al momento la fame, le cellule entrare in un programma di sviluppo in cui si aggregano attraverso cAMP-mediata chemiotassi per formare strutture multicullular. Molti componenti coinvolte nella chemiotassi di cAMP sono stati identificati in D. discoideum. Il legame di cAMP ad un GPCR (car1) induce la dissociazione di eterotrimeriche G-proteine ​​in Gγ e subunità Gβγ 7, 9, 10. Subunità Gβγ attivare Ras, che a sua volta attiva PI3K, la conversione PIP 2 nel PIP 3 sulla membrana cellulare 11-13. PIP 3 fungono da siti di legame per le proteine ​​con omologia Pleckstrin (PH) domini, in modo da reclutare queste proteine ​​alla membrana 14, 15. Attivazione dei recettori car1 controlla anche le associazioni membrana di PTEN, che defosforila PIP PIP 3 a 2 16, 17. I meccanismi molecolari sono evolutivamente conservati in chemochine GPCR-mediata chemiotassi di cellule umane come i neutrofili 18. Vi presentiamo i seguenti metodi per lo studio della chemiotassi D. discoideum cellule. 1. Preparazione delle cellule componente chemiotattici. 2. Imaging chemiotassi di cellule in un gradiente di cAMP. 3. Monitoraggio di una attivazione indotta GPCR di eterotrimeriche G-proteina in cellule vive sola. 4. Imaging chemoattractant-triggered PIP dinamico 3 risposte a singole cellule vive in tempo reale. I nostri metodi di imaging sviluppato può essere applicato per studiare chemiotassi dei leucociti umani.

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

Qui, descriviamo dettagliati vivere metodi di imaging per lo studio delle cellule chemiotassi. Vi presentiamo fluorescenza metodi microscopici di monitorare le dinamiche spazio-temporali di segnalazione eventi nella migrazione delle cellule. Misurazione di eventi di segnalazione ci permette di capire meglio come un GPCR di segnalazione rete raggiunge gradiente di rilevamento di fattori chemiotattici e controlli migratori direzionale delle cellule eucariotiche.

Receptor Imaging proteina G accoppiata (GPCR)-mediata eventi di segnalazione che chemiotassi di controllo di 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 Views.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. Qui, descriviamo dettagliati vivere metodi di imaging per lo studio delle cellule chemiotassi. Vi presentiamo fluorescenza metodi microscopici di monitorare le dinamiche spazio-temporali di segnalazione eventi nella migrazione delle cellule. Misurazione di eventi di segnalazione ci permette di capire meglio come un GPCR di segnalazione rete raggiunge gradiente di rilevamento di fattori chemiotattici e controlli migratori direzionale delle cellule eucariotiche.

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.

Quantificazione in vivo di G interazioni proteina recettore accoppiato con spettralmente risolte microscopia a due fotoni

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

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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.

Quantificare attività agonista a G recettori accoppiati alla proteina

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 per Monitoraggio in tempo reale di segnalazione di eventi in cellule vive Uso Biosensori unimolecolare

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.

Imaging cellulare dal vivo dei primi autofagia Eventi: Omegasomes e oltre

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 geneticamente codificati a studiare G recettori accoppiati alle proteine

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).

Caratterizzazione di G recettori accoppiati alla proteina da un calcio di Mobilitazione Assay Basato sulla fluorescenza

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.

Visualizzazione Clathrin endocitosi mediata da G recettori accoppiati a proteine ​​a risoluzione singolo evento tramite 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.

Indagare la funzione di Coronin A nel precoce fame di risposta<em&gt; Discoideum Dictyostelium</em&gt; Per aggregazione Dosaggi

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 recettore-mediata chemiotassi ed i suoi eventi di segnalazione nelle cellule HL60 neutrofili-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.

Studiando la proteina del sistema di controllo di qualità di<em&gt; D. discoideum</em&gt; Uso Cellulare Imaging a temperatura controllata

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