Wir danken für die großzügige Unterstützung mehrerer Stiftungen und wohltätigen Spendern (siehe SakmarLab.org).

1. Standortspezifische Genetische Einbau von unnatürlichen Aminosäuren in GPCRs <ol><li> Aufrechterhaltung HEK293T-Zellen in DMEM (4,5 g / l Glucose, 2 mM Glutamin), das mit 10% fötalem Rinderserum (FBS) ergänzt war, bei 37 ° C in einem 5% CO 2-Atmosphäre. </li><li> Transfektion die Zellen 60-80% Konfluenz in einem 10-cm-Platte mit Lipofectamine Plus-Reagenz gewachsen. <ol><li> 750 ul DMEM, mit 10 ul Plus-Reagens, 3,5 ug GPCR-cDNA (pMT4. Rho oder pcDNA 3.1. CCR5), der das Amber-Stopcodon an einer gewünschten Position, 3,5 &amp;mgr; g des cDNA-Suppressor-tRNA (pSVB.Yam) und 0,35 ug der mutierten Aminoacyl-tRNA-Synthetase-cDNA für p-Azido-L-phenylalanin (pcDNA.RS). Inkubieren bei Raumtemperatur für 15 min. Auch eine ähnliche Transfektion mit der Gew. GPCR-cDNA (ein Amber-Stopp-Codon enthalten), um als Kontrolle dienen. Fügen Sie diese Mischung auf 750 ul DMEM mit 17 ul Lipofectamine. Nach Gleichgewichtseinstellung 15 min bei Raumtemperatur bringen dieGesamtvolumen von 4 ml. </li><li> Saugen Medien auf 10-cm-Platte gelten Transfektion Mischung, um Zellen und werden wieder zu 37 ° C in 5% CO 2-Atmosphäre. Nach 4-6 Stunden, ergänzen die Zellen mit 4 ml DMEM mit 20% FBS und 1 mM AZF. </li></ol></li><li> Am nächsten Tag ersetzt das Wachstumsmedium mit DMEM mit 10% FBS und 0,5 mM AZF. </li><li> Ernten Sie die Zellen 48 Stunden nach der Transfektion die Expression zu analysieren, oder fahren Sie in den folgenden Abschnitten beschrieben Photo oder Markierungsverfahren. <ol><li> Expression des GPCR-Amber-Mutante bestätigt in Gegenwart von LF in den Wachstumsmedien. Wir tun dies durch Western-Immunoblot-Detektion. Lysieren das Zellpellet in 1% (w / v) Dodecyl-β-D-maltosid (DDM) in 20 mM Tris-HCl, pH 7,5, 100 mM NaCl, 1 h bei 4 ° C Zentrifugieren Sie das Lysat bei 16.000 xg und zur Lösung der Überstand unter reduzierenden Bedingungen durch SDS-PAGE. Übertragung auf eine PVDF-Membran und führen Immunoblotanalyse in voller Länge Rezeptor-Expression erkennen, mit the mAb 1D4 (National Cell Culture Center), die eine gentechnisch Epitop am C-Terminus des Rezeptors erkennt. 31 </li></ol></li></ol> 2. Zuordnen eines Ligandenbindungsstelle durch gezielte Photovernetzung <ol><li> 48 Stunden nach der Transfektion von Zellen, Medien absaugen und ersetzen mit 4 ml 1x Phosphate Buffered Saline (PBS). Rückkehr zu 37 º C für 15 min. </li><li> Resuspendieren der Zellen mit 1x PBS und Transfer in ein Falcon-Röhrchen. Zentrifugieren bei 1.500 rpm für 2 min auf die Zellen zu pelletieren, und saugen Sie den Überstand. </li><li> Zellpellet in 1 × Hanks gepufferte Salzlösung mit 20 mM HEPES, pH 7,5, 0,2% BSA und tritiierten Liganden bei Sättigungsbindungskonzentration für die erforderliche Zeitdauer und Temperatur, um Rezeptor-Liganden-Komplexbildung zu gewährleisten. </li><li> Übertragen Sie die Zellsuspension auf eine 24-Well-oder 96-Well-Platte für Photoaktivierung von AZF bei 365 nm (z. B. mit einem UV-A-Lampe) für 15 min bei 4 ° C Maintain die Platte auf Eis, um die Probenerwärmung und Zell-Lyse zu vermeiden. </li><li> Übertragen Sie die Zellen, um ein Eppendorf-Röhrchen, Zentrifugen, um die Zellen zu pelletieren, den Überstand und zur Analyse. Die Pellets können bei -20 ° C zur weiteren Verwendung gelagert werden. </li><li> Resuspendieren des Zellpellets in Lyse-Puffer, der 1% (w / v) DDM, 20 mM Tris-HCl, pH 7,5 und Protease-Inhibitoren. Nach 1-stündiger Inkubation bei 4 ° C zentrifugieren Sie das Zelllysat für 10 min bei 16.000 × g, und den Überstand in ein neues Röhrchen. </li><li> Hinzufügen mAb 1D4-Sepharose-2B-Harz zu dem Überstand und Inkubation über Nacht bei 4 ° C, um die GPCR immunopurify mit dem C-Terminus konstruiert mAb 1D4-Epitop. Am nächsten Tag Waschen der Kügelchen mehrmals mit Lyse-Puffer. Eluat-Proben mit 1% SDS bei 37 ° C für 1 Stunde unter Schütteln. </li><li> Übertragen eines Teils des Eluenten in ein 15 ml Fläschchen mit Szintillationsflüssigkeit und zählen auf einem Beta-Szintillationszähler, die Menge der spezifischen Bindung des tritiierten Liganden zu quantifizieren thE-Rezeptor. </li><li> Beheben Sie den restlichen mAb 1D4 Laufmittel gereinigt durch Standard-Gel-Elektrophorese. Wir verwenden ein 4-12% SDS-PAGE-Gel und dann halbtrockenen Transfer auf eine PVDF-Membran für Immunoblotting. Wir sind auch eine Spur mit Standard-Protein-Leiter, um visuelle Annäherung der Molekulargewicht zu führen. <ol><li> Sperrung der PVDF-Membran mit 5% Milch in TBS-Puffer, enthaltend 0,05% Tween-20. Sonde die Membran in voller Länge Rezeptor-Expression mit mAb 1D4 gefolgt von HRP-konjugierte Anti-Maus-IgG Sekundäranti bestätigen. </li><li> Behandeln mit verstärkter Chemilumineszenz (ECL)-Reagens und setzen Membran Autoradiographie Film Banden sichtbar. </li></ol></li><li> Schneiden der Membran mit einer Rasierklinge, um jede Probenspur zu trennen, gefolgt von Schneiden zu bestimmten Molekulargewichtsmarkern. Übertragen Membransegmente Fläschchen mit Szintillationsflüssigkeit. Quantifizierung der Menge an Tritium in jeder Membransegment durch Zählen auf einem Beta-Szintillationszähler. </li><li> Identifizieren Sie die positivephotovernetzen mit der Detektion von Tritium in der scheinbaren molekularen Masse gleich der Summe derjenigen der GPCR und dem Liganden. </li></ol> 3. Gezielte Epitop-Tagging und Zelloberflächen Enzyme-linked Immunosorbent Assay (ELISA) <ol><li> 24 Stunden nach der Transfektion, waschen Sie die Zellen mit 1 ml 1x PBS und trypsinize mit 1 ml 0,25% Trypsin für 3 min. Ergänzt mit 9 ml DMEM, enthaltend 10% FBS und 0,5 mM AZF. Die Zellen und zählen die Zelldichte. Wir verwenden eine Standard-Zählkammer. </li><li> Pre-Behandlung eine hohe Bindungs, klare Boden 96-Well-Platte mit 100 ml 0,01 mg / ml Poly-D-Lysin pro Vertiefung. Inkubation für 30 Minuten bei Raumtemperatur, immer wieder waschen mit 1x PBS und an der Luft trocknen. </li><li> Platte 200 ul der transfizierten Zellen in einer Dichte von 6 × 10 4 - 8 Stück 10 4 Zellen / Well auf 96-Well-Platte und zurück auf 37 ° C in 5% CO 2-Atmosphäre. </li><li> Am nächsten Tag vorbereiten Zellen für die Kennzeichnung. Behutsam dreimal mit 100 ul / 1x PBS to entfernen Sie AZF enthalten Wachstumsmedien. Sicherzustellen Zellen haften bleiben, zum Beispiel durch Verwendung von PBS, das Ca 2 + und Mg 2 +. </li><li> Bereiten 50-200 uM FLAG-Triarylphosphins Markierungsreagenzes in 1x HBSS / PBS aus einer Stamm 5-20 mM in PBS erhalten. Bewerben 60 ml in jede Vertiefung (in dreifacher Ausfertigung für jede Amber-Mutante) und zurück auf 37 ° C für 30 min bis 4 Stunden. Achten Sie auf eine Reihe von Brunnen ohne Label-Behandlung in 1x HBSS / PBS. Auch eine Gewichtskontrolle GPCR mit AZF-GPCR-Varianten vergleichen. </li><li> Waschen der Zellen 3-mal mit 100 &amp;mgr; l / Vertiefung Blockierungspuffer, BB (1x PBS, 0,5% BSA), um nicht umgesetztes Etikett vollständig zu entfernen. </li><li> Bewerben 100 ul / 4% Paraformaldehyd (aus einer 16% Lager in 1x PBS) zu den Zellen. Inkubation für 20 min bei Raumtemperatur. Dreimal mit BB waschen. </li><li> Führen Sie Standard-Zelloberfläche ELISA-Protokoll, um markierten Rezeptor und Rezeptor-Expression zu erkennen. Inkubation mit primärem Antikörper für 1,5 h auf Eis, gefolgt von Sekundär eintibody Inkubation bei Raumtemperatur für 1 Stunde. <ol><li> 100 l Anti-FLAG-M2-Antikörper (z. B. 1:2000 Verdünnung des Antikörpers in BB) an FLAG-Peptid-Epitop des FLAG-Triarylphosphins Markierungsreagenzes zu erkennen. Sonde auch ohne Etikett behandelten Vertiefungen als Negativkontrolle. Hinzufügen anti-CCR5-2D7 (wir verwenden eine 1:500 Verdünnung)-Antikörper mit einem getrennten Satz von Vertiefungen, um zu bestimmen wt oder AZF-GPCR-Zelloberflächenexpression. </li><li> Mit BB waschen Zellen dreimal, und Inkubation mit 100 &amp;mgr; l HRP-konjugiertes Anti-Maus-IgG-Sekundärantikörper (1:2000 Verdünnung). </li><li> Sorgfältig waschen Sie die Zellen fünfmal mit BB. In 50 ul Detektionspuffer: Amplex Rot, 20 mM H 2 O 2, 1x PBS (1:10:90 Verhältnis) und Inkubation 15 Minuten bei Raumtemperatur. Sammeln spektralen Daten auf einer Fluoreszenz Multi-Well-Plattenlesegerät bei λ ex = 530 und λ em = 590. </li></ol></li></ol> 4. Bioorthogonale Fluoreszenzmarkierung eines GPCR <ol><li> Lyse10 7 Zellen, geerntet oder AZF Gew.-Rhodopsin-Varianten in 1 ml Lösungspuffer, enthaltend 1% (w / v) DM, 50 mM HEPES oder Tris-HCl, pH 6,8, 100 mM NaCl, 1 mM CaCl 2 und Protease-Inhibitoren für 1 h bei 4 ° C. Zentrifugieren Sie die Zelllysat bei 15.000 g für 10 min und sammeln die Überstandsfraktion. </li><li> 100 l-mAb 1D4 Sepharose 2B Rezeptor Harz erfassen mit Hilfe der C-Terminus entwickelt 1D4 Epitop. Inkubation für 12 Stunden bei 4 ° C. Dreimal mit 0,1% Das Harz (w / v) DDM in 0,1 M Natriumphosphatpuffer, pH 7,3. </li><li> Mit 0,1 mM Fluorescein-Phosphin in einem Gesamtvolumen von 0,3 - 0,5 mL an Rezeptor auf der Immun-Affinitätsmatrix (mAb 1D4-Sepharose 2B) immobilisiert beschriften. Inkubation für 12 Stunden bei Raumtemperatur. Zentrifuge und entfernen Stand. Das Harz, um nicht umgesetztes Etikett zu entfernen. </li><li> Eluieren des markierten Rezeptors mit 100 ml Elutionspuffer, enthaltend 0,1% (w / v) DDM und 0,33 mg / ml Nonapeptid (C9-Peptid gegen die Epitop 1D4)in 2 mM Phosphat-Puffer, pH 6,0 durch Inkubation auf Eis für 1 Stunde. </li><li> Beheben markierten Proben durch SDS-PAGE unter reduzierenden Bedingungen. Kurz waschen Gele in PBS und dann visualisieren Kennzeichnung von AZF-GPCR durch in-Gel-Fluoreszenz. Verwenden Sie beispielsweise einen konfokalen Fluoreszenz-Scanners mit einem 488-nm-Laserwellenlänge an den Rezeptor mit Fluorescein modifizierte erkennen. </li></ol>

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S., MacKenzie, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+to+rhodopsin:+characterization,+cross-reactivity,+and+application+as+structural+probes.">Monoclonal antibodies to rhodopsin: characterization, cross-reactivity, and application as structural probes.</a> Biochemistry. 22, 653-660 (1983).</li><li>Gether, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agonists+induce+conformational+changes+in+transmembrane+domains+III+and+VI+of+the+beta2+adrenoceptor.">Agonists induce conformational changes in transmembrane domains III and VI of the beta2 adrenoceptor.</a> The EMBO journal. 16, 6737-6747 (1997).</li><li>Hubbell, W. L., Altenbach, C., Hubbell, C. M., Khorana, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodopsin+structure,+dynamics,+and+activation:+A+perspective+from+crystallography,+site-directed+spin+labeling,+sulfhydryl+reactivity,+and+disulfide+cross-linking.">Rhodopsin structure, dynamics, and activation: A perspective from crystallography, site-directed spin labeling, sulfhydryl reactivity, and disulfide cross-linking.</a> Advances in Protein Chemistry; Membrane Proteins. 63, 243-290 (2003).</li><li>Cai, K., Itoh, Y., Khorana, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+contact+sites+in+complex+formation+between+transducin+and+light-activated+rhodopsin+by+covalent+crosslinking:+use+of+a+photoactivatable+reagent.">Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: use of a photoactivatable reagent.</a> Proceedings of the National Academy of Sciences of the United States of America. 98, 4877-4882 (2001).</li><li>Dorman, G., Prestwich, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benzophenone+photophores+in+biochemistry.">Benzophenone photophores in biochemistry.</a> Biochemistry. 33, 5661-5673 (1994).</li><li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+strategy+for+the+site-specific+modification+of+proteins+in+vivo.">A new strategy for the site-specific modification of proteins in vivo.</a> Biochemistry. 42, 6735-6746 (2003).</li><li>Dirksen, A., Hackeng, T. M., Dawson, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleophilic+catalysis+of+oxime+ligation.">Nucleophilic catalysis of oxime ligation.</a> Angew. Chem. Int. Ed. 45, 7581-7584 (2006).</li><li>Yanagisawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multistep+engineering+of+pyrrolysyl-tRNA+synthetase+to+genetically+encode+N(epsilon)-(o-azidobenzyloxycarbonyl)+lysine+for+site-specific+protein+modification.">Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification.</a> Chemistry &amp; biology. 15, 1187-1197 (2008).</li></ol>

Autoren haben nichts zu offenbaren.

Wir beschreiben hier eine robuste Methode zur ortsspezifischen Einbau eines reaktiven Sonde, AZF, in GPCRs und zeigen drei nützliche Anwendungen dieses Werkzeug, um die Struktur und Dynamik von GPCRs zu studieren. Unsere Methode zur ortsspezifisch integrieren UAAs umgeht ein grundsätzliches Problem mit einer alternativen Strategie, die auf chemisch Beschriftung 32, 33 oder Anbringen Photovernetzer 34 zu einem einzigen zugänglichen Cystein-Mutante. Obwohl die chemischen Cysteinthiolgruppen Ziel v...

Heptahelikalen G-Protein-gekoppelte Rezeptoren (GPCRs) eine Superfamilie von hochdynamischen Membranproteine, die wichtige und vielfältige extrazelluläre Signale zu vermitteln. In der klassischen Paradigma wird Rezeptor-Aktivierung mit Liganden-induzierter Konformationsänderungen gekoppelt. 1, 2 Jüngste Fortschritte in der Strukturbiologie von GPCRs haben bedeutende Einblicke in die molekularen Mechanismen der Transmembran-Signalisierung zur Verfügung gestellt. 3-5 jedoch mit größerer Präzision der chemischen verstehen Funktionsmechanismus und Strukturdynamik von GPCR-Signalisierung wird ein Toolkit Ansätze erforderlich, um informative molekulare und chemische Sonden enthalten, um aktive Signalkomplexe zu verhören. Zu diesem Zweck angepasst wir eine Methode, um ortsspezifisch einzuführen nicht oder minimal störenden Sonden in exprimierten Rezeptoren basierend auf unnatürliche Aminosäure (LF) Mutagenese mit Amber-Codon Unterdrückung Technologie zuvor von Schultz und c Pionieroworkers. 6 Wir optimierten die UAA-Mutagenese-Methode, um eine High-Yield-Expression und Mutagenese-System für Proteine, wie zB GPCRs, die nur schwer in anderen als den meisten Säugetierzellen heterologen Systemen zum Ausdruck zu erzielen. Mit einem orthogonalen entwickelt Suppressor-tRNA und entwickelte sich Aminoacyl-tRNA-Synthetase-Paar für eine bestimmte UAA, wir ortsspezifisch eingeführt UAAs ausgedrückt Ziel GPCRs. Der erfolgreiche Einbau UAAs, p-Acetyl-L-Phenylalanin (ACF), p-Benzoyl-L-phenylalanin (BzF) und p-Azido-L-phenylalanin (AZF) wurde in unserem Modell GPCRs nachgewiesen - Rhodopsin und Menschen CC-Chemokin-Rezeptor CCR5. 7, 8 Im Prinzip kann ein UAA genetisch an beliebigen Stellen innerhalb der Proteinsequenz codiert werden, und diese Eigenschaft ist ein unschätzbares Werkzeug, wie es biochemische ermöglicht Single-Codon Abtastung eines Ziel GPCR. Wir konzentrieren uns hier speziell auf die Vielseitigkeit der UAA, AZF, die eine reaktive azi hatEinheit zu tun. Zusätzlich zur Funktion als eine einzigartige Infrarot (IR)-Sonde, 8, 9 AZF kann auch als photoaktivierbare Vernetzer dienen, indem sie mit benachbarten primären Aminen oder aliphatische Wasserstoffe dienen. Zusätzlich kann das biologisch inert Azidogruppe als selektive chemische Griff in bioorthogonalen Markierungsreaktionen teilnehmen. Hier präsentieren wir Ihnen Beispiele, die die nützlichen Anwendungen von ortsspezifischen Einbau von AZF in GPCRs, wie gezielte Photofalle zu einem Rezeptor-Liganden-Komplex, und Modifikation von GPCRs durch bioorthogonale Epitop-Tagging und Fluoreszenzmarkierungsstrategien. Photoaktivierbare Reagenzien sind verwendet worden, um biologische Systeme untersuchen seit den 1960er Jahren. 10 In diesem Zeitraum sind eine Fülle von Rezeptor-Ligand-Vernetzungsexperimente wurde berichtet, GPCR-Komplexe zu untersuchen, von denen die meisten bei dem durch Verwendung von Photoaffinitätsliganden. 11, 12 jedoch, diese Anwendungen sind technisch begrenzt, da sie requirE Synthese von Liganden mit einer Vernetzungsgruppe. 13-15 Zudem mit dem Vernetzer-Einheit in dem Liganden, es ist eine Herausforderung, die Position der auf der Querverbindung GPCR identifiziert. Ortsspezifisch Einführen Photogruppen UAAs in Proteine ​​unter Verwendung der Amber-Codon Unterdrückungstechnik ist ein wertvoller Fortschritt. 16, 17. Wir entwickelten eine Phototechnik, um die Bindungsschnittstelle an einen Rezeptor, der in der Bildung eines Rezeptor-Ligand-Komplex beteiligt ist, zu identifizieren lebende Zellen durch die Einführung von photolabilen Gruppen in GPCRs. 18, 19 Hier beschreiben wir die Versuchsprotokoll und Methode der Datenanalyse für die Anwendung dieses gezielte Photo Technologie, um die Bindungsstelle eines niedermolekularen Liganden, tritiiertes Maraviroc, auf CCR5 identifizieren. Dieses Verfahren nutzt die genaue Quantifizierung der radioaktiven Griff auf dem Liganden, die neben Beibehaltung der nativen chemischen Struktur des Liganden. Fluoreszenz-basierte Techniken unterstützen die präzise Kenntnis der strukturellen Basis der Rezeptor-Aktivierung durch direkte Erforschung der Konformation des Rezeptors. 20, 21 jedoch ortsspezifisch sind Techniken, die über die Flexibilität, um Fluoreszenzmarkierungen in GPCRs vorstellen begrenzt. Wir interessieren uns für den Einsatz bioorthogonale chemische Modifikation von Strategien zur Einzelmoleküldetektion (SMD) der GPCR-Signalkomplexe zu erleichtern. 22. Die Azidogruppe in bioorthogonale Chemikalien wie Staudinger-Ligation Bertozzi, 23, 24 kupferfreien Stamm-Azid-Teilnahme Alkin-Cycloaddition (SPAAC) 25 und Kupfer-katalysierte Azid-Alkin-Cycloaddition (CuAAC) 26 Wir konzentrieren. hier auf der Staudinger-Bertozzi Ligationsreaktion, die die spezifische Reaktion zwischen einem Azid und einem Phosphin beinhaltet. Wir zeigen die Verwendung zweier unterschiedlicher Phosphin-Derivate, ein Peptid-Epitop (FLAG-Peptid) oder einer fluoreszierenden Markierung konjugiert(Fluorescein), um ortsspezifische Modifikation von GPCRs zu erreichen. Wir haben bereits optimiert die Bedingungen für die ortsspezifische Markierung von Rhodopsin AZF-Varianten mit Hilfe der Röntgenkristallstruktur und dynamische Simulationen, um Zielstellen, die Lösungsmittel ausgesetzt sind, zu wählen. 27, 28 Wir veranschaulicht auch die Möglichkeit, Hintergrundfreie Kennzeichnung zu erreichen durch Staudinger- Bertozzi Ligation. 29. Wir zeigen hier eingesetzt, um die Fluoreszenzmarkierung von einem Reinigungsmittel mit gelöstem Rezeptor, der auf einer Immun Matrix immobilisiert wird und anschließend durch in-Gel-Fluoreszenz sichtbar zu erzielen allgemeine Verfahren. Außerdem zeigen wir eine sinnvolle Erweiterung auf dieser Kennzeichnungsstrategie zu Positionen zugänglich Kennzeichnung auf einem Rezeptor unbekannter Struktur, CCR5 identifizieren. Dies wird unter Verwendung einer Zielpeptid-Epitop-Tagging-Strategie, die auf bioorthogonalen Modifikation AZF-GPCR-Varianten in einem zellbasierten semiHochDurchSatzFormat durchgeführt stützt. 30 DieserVerfahren nutzt die mehrstufige Detektionseigenschaften eines Zelloberflächen-ELISA zur Kennzeichnung Ereignisse überwachen. Die Methoden wir hier diskutieren, sind die allgemeinen und im Prinzip kann auf jeden GPCR mit AZF mit der Amber-Codon Unterdrückung Technologie integriert angewendet werden. In den Protokollen hier präsentierten wir detailliert die Schritte in Säugerzell Expression von Rezeptoren mit UAAs wie AZF mit der unnatürlichen Aminosäure-Mutagenese-Methode und ihre Folgeanträge auf strukturelle und dynamische Studien von GPCRs erleichtern eingebaut beteiligt.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmid pSVB. Yam</td>
			<td></td>
			<td></td>
			<td>(Ye et al., 2008)</td>
		</tr>
		<tr>
			<td>Plasmid pcDNA.RS for azF</td>
			<td></td>
			<td></td>
			<td>(Ye et al., 2009)</td>
		</tr>
		<tr>
			<td>Plasmids pMT4.Rho and pcDNA 3.1.CCR5</td>
			<td></td>
			<td></td>
			<td>Optionally contain the amber stop codon (TAG) at a desired position</td>
		</tr>
		<tr>
			<td>HEK 293T cells</td>
			<td></td>
			<td></td>
			<td>Adherent cells</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Gibco</td>
			<td>10566</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Gibco</td>
			<td>14200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gemini Bio-products</td>
			<td>100-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine Plus</td>
			<td>Invitrogen</td>
			<td>Lipofectamine: 18324-012 
			Plus: 11514-015</td>
			<td></td>
		</tr>
		<tr>
			<td>p-azido-L-phenylalanine</td>
			<td>Chem-Impex International</td>
			<td>6162</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. Site-specific genetic incorporation of unnatural amino acids into GPCRs materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>Maxima ML-3500S UV-A lamp</td>
			<td>Spectronics Corporation</td>
			<td></td>
			<td>azF is activated by 365-nm light</td>
		</tr>
		<tr>
			<td>Hank&#39;s Buffered Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14065</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Irvine Scientific</td>
			<td>9319</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Roche</td>
			<td>3117405001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tritium-labeled ligand</td>
			<td></td>
			<td></td>
			<td>From collaborator (Grunbeck et al., 2012)</td>
		</tr>
		<tr>
			<td>1% (w/v) n-dodecyl-&#946;-D-maltoside</td>
			<td>Anatrace</td>
			<td>D310LA</td>
			<td></td>
		</tr>
		<tr>
			<td>1D4-sepharose resin</td>
			<td></td>
			<td></td>
			<td>1D4 mAb immobilized on CNBr-activated sepharose 2B resin</td>
		</tr>
		<tr>
			<td>1% (w/v) sodium dodecyl sulfate (SDS)</td>
			<td>Fisher Scientific</td>
			<td>BP166</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4 - 12% SDS gels</td>
			<td>Invitrogen</td>
			<td>NP0322BOX</td>
			<td>SDS-PAGE performed on NuPAGE apparatus</td>
		</tr>
		<tr>
			<td>Trans-Blot SD apparatus</td>
			<td>Biorad</td>
			<td></td>
			<td>Apparatus for semi-dry transfer</td>
		</tr>
		<tr>
			<td>Immobilon polyvinylidene difluoride (PVDF) membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat powered milk</td>
			<td>Fisher Scientific</td>
			<td>NC9934262</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Aldrich</td>
			<td>274348</td>
			<td></td>
		</tr>
		<tr>
			<td>Ecoscint A</td>
			<td>National Diagnostics</td>
			<td>LS-273</td>
			<td>Scintillation fluid</td>
		</tr>
		<tr>
			<td>Scintillation vials</td>
			<td>Fisher Scientific</td>
			<td>333726</td>
			<td></td>
		</tr>
		<tr>
			<td>LKB Wallac 1209 Rackbeta Liquid Scintillation Counter</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>Beta-Scintillation counter</td>
		</tr>
		<tr>
			<td>Anti-rhodopsin 1D4 mAb</td>
			<td>National Cell Culture Center</td>
			<td>custom</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase (HRP)-conjugated anti-mouse IgG</td>
			<td>KPL, Inc.</td>
			<td>474-1806</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Thermo Scientific</td>
			<td>34080</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyblot CL AR film</td>
			<td>Denville</td>
			<td>E3018</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Targeted photocrosslinking materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>0.25% Trypsin</td>
			<td>Invitrogen</td>
			<td>15050065</td>
			<td></td>
		</tr>
		<tr>
			<td>FLAG-triarylphosphine</td>
			<td>Sigma</td>
			<td>GPHOS1</td>
			<td></td>
		</tr>
		<tr>
			<td>M2 FLAG mAb</td>
			<td>Sigma</td>
			<td>F1804</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CCR5 2D7 mAb</td>
			<td>BD Biosciences</td>
			<td>555990</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Costar</td>
			<td>3601</td>
			<td>Clear bottom, high binding EIA/RIA</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (Calcium, Magnesium)</td>
			<td>Gibco</td>
			<td>14040</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>EMS</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP-conjugated</td>
			<td>KPL, Inc.</td>
			<td>474-1516</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rabbit IgG</td>
			<td>KPL, Inc.</td>
			<td>474-1516</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex Red</td>
			<td>Invitrogen</td>
			<td>A12222</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>DE Healthcare Products</td>
			<td>97-93399</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFluor II fluorescence multi-well plate reader</td>
			<td>Perseptive Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 3. Targeted peptide-epitope tagging and cell surface ELISA materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>Fluorescein-phosphine</td>
			<td></td>
			<td></td>
			<td>(Huber et al., submitted 2012)</td>
		</tr>
		<tr>
			<td>Nonapeptide (C9 peptide)</td>
			<td>AnaSpec</td>
			<td>62190</td>
			<td>Peptide mimicking the 1D4-epitope NH2-TETSQVAPA-COOH</td>
		</tr>
		<tr>
			<td>1% (w/v) n-dodecyl-&#946;-D-maltoside</td>
			<td>Anatrace</td>
			<td>D310LA</td>
			<td></td>
		</tr>
		<tr>
			<td>1D4-sepharose resin</td>
			<td></td>
			<td></td>
			<td>1D4 mAb immobilized on CNBr-activated sepharose 2B resin</td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4 - 12% SDS gels</td>
			<td>Invitrogen</td>
			<td>NP0322BOX</td>
			<td>SDS-PAGE performed on NuPAGE apparatus</td>
		</tr>
		<tr>
			<td>Confocal Typhoon 9400 fluorescence scanner</td>
			<td>GE Healthcare</td>
			<td>discontinued</td>
			<td>Scanner with 488-nm wavelength laser</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 4. Fluorescent labeling materials</td>
		</tr>
	</tbody>
</table>

genetically-encoded molecular probes to study g protein-coupled receptors

Um strukturelle und dynamische Untersuchungen von G-Protein-gekoppelten Rezeptor (GPCR)-Signalkomplexe zu erleichtern, sind neue Ansätze erforderlich, um informative Sonden oder Etiketten in exprimierten Rezeptoren, die Rezeptor-Funktion nicht stören, nicht vorstellen. Wir verwendeten Amber-Codon Unterdrückungstechnologie genetisch codieren die unnatürlichen Aminosäure-, p-Azido-L-phenylalanin (AZF) an verschiedenen Positionen in gezielter GPCRs heterolog in Säugerzellen exprimiert. Die Vielseitigkeit der Azidogruppe ist hier in verschiedenen Anwendungen dargestellt, dass GPCRs in ihrer Mutterzellumgebung oder unter Reinigungsmittel gelösten Bedingungen zu studieren. Zuerst zeigen wir eine zellbasierte Zielphototechnik, um die Rückstände in den Liganden-Bindungstasche von GPCR wo ein Tritium-markierten niedermolekularen Liganden mit einem genetisch codierte Aminosäure Azido vernetzt identifizieren. Wir haben dann zeigen, ortsspezifische Modifikation von GPCRs durch die bioorthogonale Staudinger-Ligation Bertozzi Reaktionention, die die Azidogruppe zielt mit Phosphin-Derivate. Wir diskutieren eine allgemeine Strategie zur gezielten Peptid-Epitop-Tagging von exprimierten Membranproteinen in-Kultur und ihrer Erkennung mit Hilfe eines Ganzzell-ELISA-basierten Ansatz. Schließlich zeigen wir, dass AZF-GPCRs selektiv mit fluoreszierenden Sonden markiert werden. Die diskutierten Methoden sind die allgemeinen, in dass sie sich grundsätzlich an jede Aminosäureposition in jedem angewendet werden, ausgedrückt GPCR aktive Signalkomplexe zu verhören.

Wir beschäftigten die unnatürliche Aminosäure Mutagenese Methode zur ortsspezifisch molekulare Sonden einzuführen in ein GPCR mit der Amber-Codon Unterdrückung Technologie. Das Schema in Abbildung 1 skizziert die wesentlichen Schritte der Methodik und die verschiedenen Anwendungen der Einbeziehung der vielseitige UAA, p-Azido-L-Phenylalanin (AZF) in GPCRs. Die Expression von AZF-GPCRs in Säugerzellen ermöglicht eine gezielte Photoliganden und gezielte Peptid-Epitop-Tagging oder fluoreszi...

Watch this Scientific Journal Video about Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors at JoVE.com

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

Wir genetisch kodieren die unnatürliche Aminosäure, p-Azido-L-Phenylalanin in verschiedenen gezielten Positionen in GPCRs und zeigen die Vielseitigkeit der Azidogruppe in verschiedenen Anwendungen. Dazu gehören eine gezielte Photo Technologie, um Rückstände in der Ligandenbindungstasche eines GPCR identifizieren und ortsspezifische Modifikation von GPCRs bioorthogonale mit einem Peptid-Epitop-Tag oder fluoreszierende Sonde.

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 ...

genetically-encoded-molecular-probes-to-study-g-protein-coupled

Research

JoVE Journal

Biology

15.1K Aufrufe.  The Rockefeller University.  Wir genetisch kodieren die unnatürliche Aminosäure, p-Azido-L-Phenylalanin in verschiedenen gezielten Positionen in GPCRs und zeigen die Vielseitigkeit der Azidogruppe in verschiedenen Anwendungen. Dazu gehören eine gezielte Photo Technologie, um Rückstände in der Ligandenbindungstasche eines GPCR identifizieren und ortsspezifische Modifikation von GPCRs bioorthogonale mit einem Peptid-Epitop-Tag oder fluoreszierende Sonde. 

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

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testen visueller Sensibilität für die Geschwindigkeit und Richtung der Bewegung in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Forschung

Biologie

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Isolierung und Reinigung von Drosophila Peripheren Neuronen durch Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

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 ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Überwachung dynamischen Veränderungen in der mitochondrialen Kalzium während der Apoptose mit einem genetisch kodierten Kalzium-Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

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

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

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

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

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 ...

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 ...

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 ...

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

Hydroponics: ein vielseitiges System Nährstoff Allocation und Pflanzen Reaktionen auf die Nährstoffverfügbarkeit und Belastung durch toxische Elemente zu studieren

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

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 ...