Diese Arbeit wird durch die intramuralen Fonds von NIAID, NIH unterstützt.

1. Vorbereitung der chemotaktischen kompetente Zellen von Dictyostelium discoideum <ol><li> Zur Erzeugung von D. discoideum-Zellen, die chemotaktisch auf die chemoattractant cAMP, Ernte wachsenden Zellen in D3-T Rich-Media aus einer Schüttelkultur bei 22 ° C sind </li><li> Waschen Sie die Zellen zweimal in non-Nährstoff-Entwicklungs-Puffer (DB-Puffer mit 5 mM Na 2 HPO 4, 5 mM KH 2 PO 4, 2 mM MgCl 2 und 0,1 mM CaCl 2). </li><li> Re-suspend-Zellen in DB-Puffer bei einer Dichte von 2x10 7 Zellen / ml. </li><li> Schütteln 10 ml Zellen in einem 250 ml-Kolben bei 100 rpm bei 22 ° C für eine Stunde. </li><li> Deliver 100 ul von 7,5 pM cAMP Lager an die 10 ml Zellen alle sechs Minuten über 6 Stunden bis zu einer Endkonzentration von 75 nM cAMP, ein Verfahren, wie cAMP pulsierende Behandlung bezeichnet erreichen. Nach 5-6 Stunden cAMP pulsierende Behandlung, D. discoideum Zellen werden chemotaktisch zuständige gegenüber cAMP-Gradienten. </li><li> Sammeln Sie die Zellen durch Zentrifugation bei 200 g für 5 min und dann die Zellen mit DB-Puffer mit 2,5 mM Koffein, und schütteln mit 200 UpM bei 22 ° C für 20 min zu Zelle zu einer chemotaktischen Situation basolate. </li></ol> 2. Imaging chemotaxing Zellen in eine sichtbare und manipulierbare Lockstoff-Gradienten <ol><li> Backfill einer Mikropipette mit einem frisch zubereiteten 30 ul Lösung von 1 nM cAMP und Alexa 594 bei 0.1μg/μl in DB-Puffer. </li><li> Bringen Sie die Femtotip zu einer Mikropipette Inhaber und verbinden Sie den Schlauch mit einer Druckversorgung Apparat, Eppendorf FemtoJet System. </li><li> Bringen Sie die Mikropipette Montage an einem Mikromanipulator (Eppendorf TransferMan NK2) motorisierten Mikromanipulator zu einem stetigen Druck bereitzustellen, um eine stabile Gradienten zu etablieren. </li><li> Montieren Sie einen Ein-und LabTek Kammer mit 6 ml DB-Puffer über einen 40x Öl Objektiv gefüllt auf einem konfokalen Mikroskop und verwenden Hellfeld-Optik, in der Mitte der Femtotip in das Blickfeld. </li><li> Schalten Sie die Druckluftversorgung und stellen Sie die Entschädigung Druck (Pc) für 70 hPa auf einen Gradienten des cAMP / Alexa 594-Gemisch herzustellen. </li><li> Visualisieren cAMP-Gradienten durch die Überwachung der Mischung der gewünschten Konzentration von cAMP und Alexa 594-Fluoreszenz unter Anregung mit 543 nm Laserlinie. </li><li> Verwenden Sie auto-Positionierung der Mikromanipulator zu Mikropipette in die gewünschte Position bringen und sie als Position 1, Position 2 und Position 3 auf den Verlauf, um die Zelle ausgesetzt sind, zu manipulieren. </li></ol> 3. Immobile nicht polarisierten Zelle System erleichtert die Bildgebung Signalwege in cAMP Gradienten Sensing beteiligt <ol><li> Nach Coffein-Behandlung, entfernen Sie ein Aliquot von Zellen und Zentrifuge bei 500g für 3 min. </li><li> Remove-Puffer und verdünnten Zellen auf 5x10 5 Zellen / ml mit frischem DB-Puffer mit 2,5 mM Koffein. </li><li> Es wird 1 ml der Zellsuspension auf eine Single-well Kammer oder 0,4 ml in jede Vertiefung eines Vier-well Kammer. </li><li> Lassen Zellen haften für 10 min, sorgfältig Pipette aus dem Puffer zu losen Zellen zu entfernen und ersetzen mit dem gleichen Volumen. </li><li> Suchen Sie die gewünschten Zellen unter dem Mikroskop und starten Bildgebung. </li><li> Für ein Experiment entworfen, um die Dynamik der Signalkomponenten in die Zellen ausgesetzt, um eine stetige Steigung Überwachung, Behandlung der Zellen mit 5,0 nM (Endkonzentration) Latrunculin B für 10 min vor den Experimenten. </li></ol> 4. Gleichzeitige Überwachung heterotrimeren G-Protein-Aktivierung und PIP 3-Produktion <ol><li> cAMP pulsierende entwickeln Zellen co-exprimierenden GαCFP und YFPGβ (G-Zellen) und Zellen, PIP 3 Indikator PH-GFP (PH-Zellen) 7. </li><li> Diese beiden Arten von Zellen, die mit 1:1-Verhältnis und die Platte sie in einem Brunnen oder 4-Well Kammern. </li><li> Erstellen und speichern Sie die Emission Fingerabdruck Referenzkurve CFP, YFP und GFP mit Lambda Stack Acquisition mode im Spektralbereich von 464 bis 624 nm mit 10 nm Breite. </li><li> Gleichzeitig Bild G-Protein-Aktivierung in G-Zellen und PIP 3-Produktion in PH-Zellen mit mit gleichem Lambda Stack Acquisition mode im Spektralbereich von 464 bis 544 nm mit einer 10 nm-Schritten. </li><li> Bewerben Linear Unmixing Funktion Zeiss 510META Software mit gespeicherten CFPand YFP-und Emissionswerte Fingerabdrücke mathematisch berechnen den Anteil der einzelnen Fluorophors in der Lambda Stack mit der GFP und YFP-Intensität in einzelnen Kanäle in G. trennen </li><li> Mit der gleichen Strategie, gelten Linear Unmixing-Funktion mit gespeichert GFP und Hintergrund-Emission Fingerabdrücke mathematisch berechnen GFP-Intensität in PH-Zellen. </li></ol> 5. Repräsentative Ergebnisse: <ol><li> Ein hervorragendes Modellsystem von D. discoideum für GPCR-vermittelte Chemotaxis. Eine soziale Amöbe, D. discoideum zeigt eine markante Chemotaxis während des Lebenszyklus. Aufgrund seiner genetischen und biochemischen Vorteile bietet D. d eine starke system zu studieren Chemotaxis. </li><li> Chemotaxis von Zellen unter einem sichtbaren und manipulierbare chemoattract Felder aus. Hier zeigen wir zunächst eine einfache Methode, um eine lineare Beziehung zwischen cAMP-Konzentration und die Intensität der einen Fluoreszenzfarbstoff Alexa 594 durch eine Verdünnungsreihe von 2 pM cAMP mit 10 pg / mL gemischt erhalten Alexa 594 (Abb. 2A). Als nächstes stellen wir eine einfache Möglichkeit, den Verlauf visualisieren darüber hinaus eine quantitative Messung der cAMP-Konzentration eines Gradienten von der Intensität der Alexa 594 (Abb. Abb.. 2B) zu etablieren. </li><li> Zellbewegung ist mit Zell-Polarisation und Richtungserkennung entkoppelt. Eine Aktin Polymerisationsinhibitor eliminiert bereits bestehenden morphologischen Polarität und verhindert auch, dass Zellbewegung unter Beibehaltung der Zellen Fähigkeit Richtungserkennung (Abb. 3A). Beschäftigung von sichtbaren und manipulierbare cAMP Stimulation garantiert die Eingabe, zum Beispiel einheitlich angewandt Anregungen oder einen Farbverlauf. Diese Methode erlaubt eine quantitative Analyse der cAMP-induzierte Umverteilung der Taste signalisiert Komponenten in der Steigung sensing Maschinen. Gemessen raumzeitliche Dynamik dieser Signalkomponenten ermöglichen es uns zu verstehen, wie die Signalisierung Netzwerk Anpassung erreicht, um eine einheitliche Anregungen bei der Erzeugung polarisierten biochemische Reaktionen auf Gradienten (Abb. 3B). </li><li> Systemische Messungen der Kinetik der chemosensing Signalisierungsnetz bei Einwirkung einer stetigen Steigung. Es ist wichtig, um die Dynamik / Kinetik der Richtungs-Sensing-Signalkomponenten zu verstehen, wie jede Komponente trägt zum Aufbau der intrazellulären Polarisation, wenn die Zellen ersten Gradienten Erfahrung zu messen. Die Anwendung des Live Cell Imaging mit einem hohen Tempo-räumlichen Auflösung zeigen wir zunächst einen biphasischen PIP3 Produktion von Zellen, die zu einem stetigen cAMP-Gradienten (Abb. 4A-C) ausgesetzt ist. Anwenden von Live Cell Imaging, haben wir systematisch Dynamik der gemessenen direktionalen-Sensing-spezifische Signalisierung Netzwerk von cAMP Stimulation PIP3 Produktion (Abb. 4D, E). </li><li> Gleichzeitige Überwachung heterotrimeren G-Protein-Aktivierung und PIP 3-Produktion auf einheitlich angewandt cAMP Stimulation. Förster resonance energy transfer (FRET abgekürzt) bietet einen effizienten Ansatz zur heterotrimeren G-Protein-Aktivierung (Dissoziation) auf cAMP Stimulation zu überwachen. Hier beschrieben wir eine bequeme einfach übernehmen für eine gleichzeitige Messung von heterotrimeren G-Protein-Aktivierung und PIP 3-Produktion durch die Überwachung der FRET ändern und Membran-Translokation von PIP 3-Sonde, PH-GFP in G und PH-Zellen, bzw. (Abb. 5 ). Ein einheitlich angewandt cAMP Stimulation löst ein hartnäckiges G-Protein-Aktivierung, während die Auslöser einer transienten PIP 3-Produktion. </li></ol><img src="/files/ftp_upload/3128/3128fig1.jpg" alt="Abbildung 1"/> Abbildung 1: Ein hervorragendes Modellsystem von D. discoideum für GPCR-vermittelte Chemotaxis. A. Schema zeigt eine kurze Signalweg des Richtungserkennung. B. cAMP Gradienten induziert eine schnelle Chemotaxis von D. discoideum Zellen. Zellen exprimieren PIP 3-Sonde, PH-GFP (Green). Gradient (Red) wird visualisiert durch Alexa 594. Balken = 50 um. <img src="/files/ftp_upload/3128/3128fig2.jpg" alt="Abbildung 2"/> Abbildung 2: Chemotaxis von Zellen unter einem sichtbaren und manipulierbare chemoattract Felder aus. A. Graph zeigt eine lineare Beziehung zwischen cAMP-Konzentration und die Intensität der einen Fluoreszenzfarbstoff Alexa 594 durch eine Verdünnungsreihe von 2 pM cAMP mit 10 pg / mL Alexa 594 gemischt. B. Quantitative Messung der cAMP-Konzentration eines Gradienten von der linearen Beziehung der cAMP-Konzentration und Intensität der fluoreszierenden Farbstoff Alexa 594 in A. <img src="/files/ftp_upload/3128/3128fig3.jpg" alt="Abbildung 3"/> Abbildung 3: Cell Motilität ist mit Zell-Polarisation und Richtungserkennung entkoppelt. A. Bild zeigt, dass immobile Zellen durch die Behandlung von Aktin Polymerisationsinhibitor Latrunculin B die Fähigkeit Richtungserkennung aufrecht zu erhalten. Zellen exprimieren PIP 3-Sonde, PH-GFP (Green). Gradient (Red) wird visualisiert durch Alexa 594. B. manipulierbare cAMP Stimulation und immobile Zelle System erlaubt es, zentrale Fragen der Richtungserkennung Adresse. Balken = 10 um. <img src="/files/ftp_upload/3128/3128fig4.jpg" alt="Abbildung 4"/> Abbildung 4: Systemische Messungen der Kinetik der chemosensing Signalisierungsnetz bei der Belichtung zu einem stetigen Gefälle. A. Montage zeigt eine biphasische PIP 3-Produktion (Grüne) der Zelle, die zu einem stetigen cAMP-Gradienten (Red) ausgesetzt ist. B. Das Bild zeigt die Regionen von Interesse (ROI) für die Messung der Kinetik der PIP 3-Produktion in C. C vorgestellt . Kinetics of PIP 3-Produktion in den Zellen, die einen stetigen Verlauf. D. Schema zeigt die Signalisierung Netzwerk von Richtungserkennung von cAMP Stimulation PIP 3-Produktion. Ihre Kinetik bei der Belichtung mit einem stetigen Gefälle ist in der gleichen Farbe durchgezogenen Linien in E vorgestellt. <img src="/files/ftp_upload/3128/3128fig5.jpg" alt="Abbildung 5"/> Abbildung 5: Gleichzeitige Überwachung multi-Ereignisse von GPCR Signalnetzwerke. A. Schema zeigt die gleichzeitige Messung von heterotrimeren G-Protein-Aktivierung und PIP 3-Produktion durch die Überwachung der FRET ändern und Membran-Translokation von PIP3 Sonde, PH-GFP in G und PH Zellen. B. Montage der Regenbogen Bilder von G-und PH-Zellen zeigt dass eine einheitliche Anwendung der cAMP Stimulation löst ein hartnäckiges G-Protein-Aktivierung in der Zelle peripheren, während die Auslöser einer transienten PIP3 Produktion. Die Zeitpunkte sind vor (0s) und nach Stimulation für 4,9 s, 10.2s und 20.4s. C. Kinetik der G-Protein-Aktivierung und PIP3 Produktion auf eine einheitliche Anwendung der cAMP Stimulation.

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Die Prozesse der chemotaktischen kompetente Stadium von Zellen Für Wildtyp D. discoideum Zellen, dauert es etwa 5 ~ 6 Stunden pulsierende Entwicklung bei Raumtemperatur, um sie in einem gut chemotaktische kompetente Stadium, in dem Zellen zeigen eine gut polarisiert zelluläre Morphologie und schnelle Zellmigration (Abb. 1) zu induzieren. Mehrere Faktoren, wie zum Beispiel cAMP-Konzentration für die Taktung, Temperatur und verschiedenen genetischen Hintergründen, kann sich auf den P...

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare </th></tr><tr><td> D3-T Growth Medien </td><td> KD Medical </td><td></td><td></td></tr><tr><td> Koffein </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 programmierbaren Timer </td><td> ChronTrol Corp </td><td></td><td></td></tr><tr><td> Miniplus 3 Schlauchpumpe </td><td> Gilbson </td><td></td><td></td></tr><tr><td> Platform Rotationsschüttler </td><td></td><td></td><td></td></tr><tr><td> FemtoJet Mikrokapillare Druckversorgung </td><td> Eppendorf </td><td></td><td></td></tr><tr><td> Einzel-und vier-und Lab-Tek II Deckglas Kammern </td><td> Nalge Nunc International </td><td></td><td></td></tr><tr><td> LSM 510 META oder gleichwertig Fluoreszenzmikroskop </td><td> Zeiss </td><td></td><td> einem 40X 1,3 NA oder 60X 1.4 NA Öl DIC Plan-Neofluar Objektiv </td></tr><tr><td> Olympus X81 oder gleichwertig </td><td> Olymp </td><td></td><td> Benötigt ein 100X 1,47 NA TIRF Objektiv </td></tr></tbody></table>

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

Viele eukaryotischen Zellen können Steigungen von chemischen Signalen in ihrer Umgebung erkennen und wandern entsprechend 1. Diese geführte Zellmigration als Chemotaxis, die wesentlich für verschiedene Zellen zur Erfüllung ihrer Funktionen wie Handel von Immunzellen und Strukturierung von neuronalen Zellen 2, 3 bezeichnet wird. Eine große Familie von G-Protein-gekoppelte Rezeptoren (GPCRs) erkennt variable kleine Peptide, wie Chemokine bekannt, die Zellmigration in vivo 4 Direktwahltasten. Das Endziel der Chemotaxis Forschung ist zu verstehen, wie ein GPCR Maschinen Sinne Chemokin-Gradienten und steuert Ereignisse, die zu Chemotaxis-Signalisierung. Zu diesem Zweck haben wir bildgebende Techniken verwenden, um zu überwachen, in Echtzeit, räumlich-zeitliche Konzentrationen von Lockstoffen, Zellbewegung in einem Gefälle von Lockstoff, vermittelt GPCR Aktivierung heterotrimerer G-Protein-und intrazellulären Signalwege in Chemotaxis von eukaryotischen Zellen 5-8 beteiligt . Die einfache eukaryotischen Organismus, Dictyostelium discoideum, zeigt chemotaxic Verhaltensweisen, die ähnlich denen der Leukozyten, und D. discoideum ist ein wichtiges Modellsystem für die Untersuchung eukaryotischer Chemotaxis. Wie frei lebenden Amöben, D. discoideum Zellen teilen in Vollmedium. Bei Hunger, in die Zellen eine Entwicklungsstörung Programm, in dem sie durch cAMP-vermittelte Chemotaxis Aggregat multicullular Strukturen zu bilden. Viele Komponenten in Chemotaxis auf cAMP beteiligt haben in D. identifiziert discoideum. Die Bindung von cAMP an einem GPCR (car1) induziert Dissoziation heterotrimerer G-Proteine ​​in Gγ und Gβγ-Untereinheiten 7, 9, 10. Gβγ-Untereinheiten aktivieren Ras, was wiederum aktiviert PI3K, Konvertieren PIP 2 in PIP 3 auf der Zellmembran 11-13. PIP 3 dienen als Bindungsstellen für Proteine ​​mit Pleckstrin Homologie (PH)-Domänen, wodurch Rekrutierung dieser Proteine ​​auf die Membran 14, 15. Die Aktivierung der Rezeptoren car1 steuert auch die Membran Verbände der PTEN, die PIP 3 dephosphoryliert PIP 2 16, 17. Die molekularen Mechanismen sind evolutionär in Chemokin GPCR-vermittelte Chemotaxis von humanen Zellen wie Neutrophile 18 erhalten. Wir präsentieren folgende Methoden zur Untersuchung von Chemotaxis von D. discoideum Zellen. 1. Vorbereitung der chemotaktische Komponente Zellen. 2. Imaging Chemotaxis von Zellen in einem cAMP-Gradienten. 3. Überwachung eines GPCR induzierte Aktivierung von heterotrimeren G-Protein in einzelnen lebenden Zellen. 4. Imaging chemoattractant ausgelösten dynamischen PIP 3 Antworten in einzelnen lebenden Zellen in Echtzeit. Unsere entwickelten bildgebenden Verfahren angewendet, um Chemotaxis von humanen Leukozyten zu studieren.

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

Hier beschreiben wir detailliert Live Cell Imaging Verfahren zur Untersuchung Chemotaxis. Wir präsentieren fluoreszenzmikroskopische Methoden zur raum-zeitlichen Dynamik der Signalwege bei der Migration von Zellen zu überwachen. Die Messung der Signal-Ereignisse ermöglicht es uns, besser zu verstehen, wie ein GPCR-Signalisierung Netzwerk Gradienten Erfassung Lockstoffe und steuert direktionale Migration von eukaryotischen Zellen erreicht.

Imaging G-Protein-gekoppelten Rezeptor (GPCR)-vermittelten Signaltransduktion, die Steuerung der Chemotaxis 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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JoVE Journal

Biology

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

17.9K Aufrufe.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. Hier beschreiben wir detailliert Live Cell Imaging Verfahren zur Untersuchung Chemotaxis. Wir präsentieren fluoreszenzmikroskopische Methoden zur raum-zeitlichen Dynamik der Signalwege bei der Migration von Zellen zu überwachen. Die Messung der Signal-Ereignisse ermöglicht es uns, besser zu verstehen, wie ein GPCR-Signalisierung Netzwerk Gradienten Erfassung Lockstoffe und steuert direktionale Migration von eukaryotischen Zellen erreicht.

Serie: 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.

In vivo Quantifizierung der G Protein-gekoppelten Rezeptor-Wechselwirkungen mit spektral aufgelöste Zwei-Photonen-Mikroskopie

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

Forschung

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.

Die Quantifizierung der Wirkung als Agonist am G-Protein-gekoppelte Rezeptoren

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-Mikroskopie zur Echtzeit-Überwachung der Signalisierung Events in lebenden Zellen mit unimolekularen Biosensors

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.

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.

Genetisch kodierte Molecular Probes, um G-Protein-gekoppelte Rezeptoren Studieren

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).

Charakterisierung von G-Protein gekoppelten Rezeptoren durch eine Fluoreszenz-basierte Calciummobilisierungsassay

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.

Visualisierung Clathrin-vermittelte Endozytose von G-Protein-gekoppelten Rezeptoren an Einzelereignis Resolution über TIRF-Mikroskopie

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.

Die Untersuchung der Funktion von Coronin A in der frühen Starvation Reaktion von<em&gt; Dictyostelium discoideum</em&gt; Durch Aggregationstests

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-gekoppelten Rezeptor-vermittelte Chemotaxis und seine Signalereignisse in Neutrophilen-like HL60-Zellen

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.

Die Untersuchung der Proteinqualitätskontrolle System der<em&gt; D. discoideum</em&gt; Verwendung von Temperaturgesteuerte 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 ...