Unsere Arbeit wurde durch Zuwendungen von der American Heart Association (Großraum Südost Affiliate) Beginning-Grant-in-Hilfe-0765185B, der Elsa U. Pardee Foundation Research Grant und der Wayne State University intramuralen Gründerfonds und Cardiovascular Research Institute Isis-Initiative ausgezeichnet unterstützt worden. Diese Methode der in vitro CFTR makromolekularen Komplex Montage wurde ursprünglich Pionierarbeit von Dr. AP Naren (University of Tennessee Health Science Center).

1. Expression und Aufreinigung von rekombinanten Tagged Fusionsproteine ​​in Bakterien <ol><li> Amplify definierte Bereiche des C-Schwänze (die letzten 50 bis 100 Aminosäuren mit den PDZ Motive am C-Terminus) für CFTR, LPA 2, MRP2, MRP4, β 2 AR und NHERFs (in voller Länge oder PDZ1 oder PDZ2 Domains ) durch PCR-Ansatz. </li><li> Klonen der PCR-Produkte in pGEX4T-1-Vektor für GST-Fusionsproteine ​​(wie GST-NHERFs, GST-MRP4 CT), pMAL-c2-Vektor für MBP-Fusionsproteine ​​(wie MBP-β 2 AR CT, CT MBP-CFTR ) und pET30 für His-S-Fusionsproteine ​​(z. B. His-S-CFTR-CT, His-S-PDZK1). </li><li> Verwandeln Sie in einem Protease-defizienten E. coli-Stamm (BL21-DE3), um mögliche rekombinanten Proteins zu minimieren. </li><li> Die Kultur über Nacht bei 37 ° C in Luria-Bertani-Medium (pH 7,0) mit entsprechenden Antibiotika (wie Ampicillin oder Kanamycin). Verdünnen Sie die Übernacht-Kultur bei 1:10 und wachsen für weitere 2 h bei 37 ° C InDuce mit 0,5-1 m M IPTG für die nächsten 4 Stunden bei 30 ° C Pellet die Zellen durch Zentrifugation bei 8000 × g für 10 min bei 4 ° C </li><li> Lyse der Zellen in Saccharose-Puffer (50 mM Tris-HCl, pH 8,0, 1 mM EDTA, 1 mM PMSF und 10% Saccharose) mit Lysozym (1 mg / ml), 0,2% Triton X-100, und Protease Inhibitoren. Verwenden Sie für 20 ml Zellpellet Ursprung aus 1 l Kultur. </li><li> Mit Hilfe eines rotierenden Schüttler für 30 min bei 4 ° C </li><li> Zentrifugieren bei 20.000 × g für 30 min bei 4 ° C Sammeln Sie die klare Überstand. </li><li> In den klaren Überstand, fügen Sie 1 mL der folgenden Harz / Agaroseperlen (50% Gülle in Saccharose-Puffer): <ul><li> Glutathion-Agarose-Beads für GST-Fusionsproteine. </li><li> Talon Perlen für His-S-Fusionsproteine. </li><li> Amylose-Harz für MBP-Fusionsproteine. </li></ul></li><li> Mischung für 4 h bei 4 ° C auf einem Rotationsschüttler. </li><li> Waschen der Kügelchen durch Resuspendieren in 1 × PBS (15 ml), Mischen für 2 Meilenn, Schleudern bei 800 × g für 2 min, und der Überstand verworfen. Wiederholen Sie diesen Schritt für das Sechsfache. </li><li> Eluieren des Proteins aus den Kügelchen unter Verwendung der jeweiligen Elutionspuffer (2 ml Elutionspuffer pro 1 ml Kügelchen). <ul><li> Elutionspuffer für GST-Fusionsproteine: 25 m M Tris-HCl (pH 8,0), 140 mM NaCl und 20 mM reduziertes Glutathion. </li><li> Elutionspuffer für seine Fusionsproteine: 20 m M Tris-HCl (pH 8,0), 500 mM NaCl und 200 mM Imidazol. </li><li> Elutionspuffer für MBP-Fusionsproteine: 20 m M Tris-HCl (pH 8,0), 200 mM NaCl, 1 mM EDTA, 1 mM DTT und 10 mM Maltose. </li></ul></li><li> Dialysieren der eluierten Proteine ​​gegen 2 l 1 × PBS bei 4 ° C (um die Protein-Abbau zu vermeiden). Das PBS wechseln alle 4 Stunden für das Vierfache. Konzentrieren Sie sich das Protein mit einem Centricon-Filter (Cutoff 10.000 MW, Millipore) und speichern als kleinen Aliquots bei -80 ° C </li><li> DetHermelin die Proteinkonzentration durch das Bradford-Verfahren. Bewerten Proteinqualität durch SDS-PAGE unter Verwendung von BSA als Standard. Wenn die Integrität des Proteins nicht zufriedenstellend ist, kann die sekundäre Reinigungsverfahren, wie Gelfiltration oder Ionenaustausch verwendet werden. (ZB haben wir eine G-75-Sepharose-Säule verwendet werden, um weiter zu reinigen GST-Fusionsprotein). </li></ol> 2. Cell Culture und Zelllysat Vorbereitung <ol><li> Kultur Babyhamsternierenzellen (BHK)-Zellen, die stabil überexprimieren CFTR-wt und CFTR-His10 oder BHK-Zellen, die vorübergehend über-Express-Flag-LPA 2-wt oder Flag-LPA 2-ΔSTL und Madin-Darby Canine Kidney (MDCK Zellen), die stabil überexprimieren MRP2 in Minimum Essential Eagle-Medium (MEM) 10% fötalem Kälberserum und Penicillin / Streptomycin in Polystyrol-Kolben bei 37 ° C, die mit 5% CO 2. </li><li> Kultur humanen embryonalen Nieren-293 (HEK293), die stabil überexprimieren CFTR-wt und CFTR-His10 in Dulbecco&#39;S modified Eagle-Medium (DMEM) mit 10% fötalem Rinderserum und Penicillin / Streptomycin in Polystyrol-Kolben bei 37 ° C mit 5% CO 2. </li><li> Lyse der Zellen in Lysepuffer (PBS - 0,2% Triton-X-100 mit einer Mischung von Protease-Inhibitoren, die 1 m M Phenylmethylsulfonylfluorid, 1 pg / ml Aprotinin, 1 ug / ml Leupeptin, 1 pg / ml Pepstatin ergänzt); Einsatz 500 ul Lysepuffer für jeden 60-mm-Petrischale, und 1000 ul Lysispuffer für jeden 100-mm-Petrischale. </li><li> Rock the Zelllysaten für 20 min bei 4 ° C, und Entfernen des unlöslichen Materials durch Zentrifugation bei 16.000 × g für 15 min bei 4 ° C Bestimmung der Proteinkonzentration nach der Bradford-Assay. </li></ol> 3. In-vitro-Montage eines CFTR-haltigen Makromolekulare Komplexe (CFTR-PDZK1-MRP4) <ol><li> In 200 &amp;mgr; l Lysepuffer (PBS - 0,2% Triton X-100 + Protease-Inhibitoren), fügen Sie 20 ug gereinigtes GST-MRP4-C Terminal50 aa (CT50) Fusionsprotein. </li><li> In unterschiedlichen Mengen (0-40 ug) von gereinigtem His-S-PDZK1 Fusionsprotein. </li><li> Mischen der beiden Proteine ​​auf einem Schüttler bei 22 ° C für 1-2 h. </li><li> In 20 uL Glutathion-Kügelchen (50% Gülle) in die Protein-Mischung, und weiterhin noch 1 h zu mischen. Dieser Schritt wird auch als paarweise Bindung bezeichnet. </li><li> Während dieser Zeit bereitet die HEK293 Zelllysaten überexprimieren Flag-CFTR (wt), wie oben beschrieben in Schritt 2). </li><li> Waschen Sie den Komplex zweimal mit Lysepuffer. Spin bei 800 × g für 1 min bei jeder Wäsche und vorsichtig den Überstand aspirieren nach jeder Wäsche. Seien Sie vorsichtig, nicht zum Absaugen der Perlen in der unteren. </li><li> In der oben hergestellten HEK293 Zelllysaten an die Kügelchen und sanft bei 4 ° C für 3 h gemischt (oder über Nacht). </li><li> Waschen der Kügelchen umfassend dreimal mit Lysepuffer, wie in Schritt 3.6) beschrieben. </li><li> Eluieren die Proteine ​​mit 30 ul Probenpuffer (5 ×): 0,6 m<html> Tris-HCl (pH 6,8), 50% Glycerin, 2% SDS und 0,1% Bromphenolblau, enthaltend 5% β-Mercaptoethanol. </li><li> Inkubieren bei 37 ° C Wasserbad für 10-15 min und Spin bei 5.000 × g für 30 s, die gebildeten Kügelchen. </li><li> Laden Sie alle das Eluat auf 4-15% Gel. </li><li> SDS-PAGE etwa 30-40 Minuten. </li><li> Transfer-Protein-Bands auf die PVDF-Membran, für 1,5 Stunden. </li><li> Blockieren Sie die Membran in TBS-Tween mit 5% fettfreie Milch. </li><li> Inkubieren der Membran mit primärem Antikörper (wie anti-IgG CFTR, R1104) bei 4 ° C über Nacht. </li><li> Waschen der Membran mit TBS-Tween für 5 min, sechsmal. </li><li> Inkubieren der Membran mit sekundärem Antikörper (z. B. Ziegen-Anti-Maus-HRP-konjugierten 2. Antikörper) für 45 min bei 22 ° C </li><li> Waschen der Membran mit TBS-Tween für 5 min, sechsmal. </li><li> Visualisieren Sie die Proteinbanden mittels ECL. </li><li> Repräsentative Daten sind in Abbildung 1 dargestellt. </li></ol>jove_title &quot;&gt; 4. repräsentative Ergebnisse Ein Beispiel von CFTR-enthaltenden makromolekularen Signalkomplexes, die in vitro zusammengebaut wurde, wird in 1 gezeigt. Ein makromolekularen Komplex wurde zwischen MRP4 C-terminalen 50 Aminosäuren (MRP4-CT50), PDZK1, und in voller Länge CFTR (Abbildung 1, unten) gebildet. Die Komplexbildung erhöht dosisabhängig mit steigenden Mengen des Proteins Überträger, PDZK1 (Abbildung 1, unten) 18. <img alt="1" src="/files/ftp_upload/4091/4091fig1.jpg" /> Abbildung 1. Eine bildliche Darstellung der makromolekularen Komplex-Assay (oben). Ein makromolekularen Komplex wurde in vitro mit drei (GST-MRP4-CT50, His-S-PDZK1, und Flag-CFTRwt) in einer Dosis-abhängigen Weise (unten) 18 erfasst. <table border="1"><tbody><tr><td></td><td><strong&gt; CFTR-NHERF1-β 2 AR (Code 14) </td><td> CFTR-NHERF2 LPA-2 (Ref. 15) </td><td> CFTR-Proteine ​​PDZ-MRP 2 (Ref. 17) </td><td> CFTR-PDZK1-MRP4 (Ref. 16) </td></tr><tr><td> Affinitätsbeads </td><td> Amyloseharz </td><td> S-Protein-Agarose </td><td> Amyloseharz </td><td> Glutathion-Agarose </td></tr><tr><td> Gereinigtes Protein-1 </td><td> MBP-β 2 AR CT </td><td> His-S-CFTR-CT </td><td> MBP-CFTR CT </td><td> GST-MRP4 CT </td></tr><tr><td> Gereinigtes Protein-2 </td><td> GST-NHERF1 </td><td> GST-NHERF2 </td><td> GST-PDZ-Proteine </td><td> His-S-PDZK1 </td></tr><tr><td> Gereinigtes Protein-3 (oder Zelllysaten) </td><td> CFTR-wt oder CFTR-His10 (BHK-oder HEK-Zelllysaten) </td><td> FlaggeLPA-2-wt oder Flag-LPA 2-ΔSTL (BHK Zelllysaten) </td><td> MRP2 (MDCK Zelllysaten) </td><td> Gereinigtes Flag-CFTR-wt oder CFTR-His10 (oder Zelllysaten) </td></tr><tr><td> Antikörper </td><td> Anti-CFTR-IgG </td><td> Anti-Flag HRP </td><td> Anti-MRP2-IgG </td><td> Anti-Flag HRP </td></tr></tbody></table> Tabelle 1. Zusammenfassung verschiedener CFTR-enthaltende makromolekulare Komplexe in vitro montiert.

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+CFTR+Interactome+in+the+Macromolecular+Complexes.">Analysis of CFTR Interactome in the Macromolecular Complexes.</a> Met. Molecul. Med. 741, 255-270 (2011).</li><li>Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemokine+receptor+CXCR2+macromolecular+complex+regulates+neutrophil+functions+in+inflammatory+diseases.">A chemokine receptor CXCR2 macromolecular complex regulates neutrophil functions in inflammatory diseases.</a> J. Biol. Chem. , (2011).</li></ol>

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In diesem Protokoll haben wir gezeigt, ein Verfahren zur In-vitro-Nachweis von Montage und ein CFTR-enthaltenden makromolekularen Signalkomplexes mit gereinigten Proteine ​​(oder Proteinfragmente) und / oder Zell-Lysate, wie zuvor berichtet 16-19,29,30. Um die besten Ergebnisse erzielt die folgenden kritischen Punkte während des Herstellungsverfahrens erfordern besondere Aufmerksamkeit: <ul><li> Es ist wichtig, dass der pH des Elutionspuffers auf 8,0 nach Zugabe reduziertem Glutathion bei de...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> pGEX4T-1-Vektor </td><td> GE Healthcare </td><td> 28-9545-49 </td><td> früher Amersham Biosciences </td></tr><tr><td> pMAL-c2-Vektor </td><td> New England Biolabs </td><td></td><td></td></tr><tr><td> pET30 Vektor </td><td> EMD Chemicals </td><td> 69077-3 </td><td> früher Novagen </td></tr><tr><td> Glutathion-Agarose-Kügelchen </td><td> BD Biosciences </td><td> 554780 </td><td></td></tr><tr><td> Amyloseharz </td><td> New England Biolabs </td><td> E8021S </td><td></td></tr><tr><td> Talon Perlen </td><td> Clontech </td><td> 635501 </td><td></td></tr><tr><td> reduziertem Glutathion </td><td> BD Biosciences </td><td> 554782 </td><td></td&gt; </tr><tr><td> Imidazol </td><td> Fischer </td><td> BP305-50 </td><td></td></tr><tr><td> Maltose </td><td> Fischer </td><td> BP684-500 </td><td></td></tr><tr><td> S-Protein-Agarose </td><td> EMD Chemicals </td><td> 69704-3 </td><td> früher Novagen </td></tr><tr><td> Anti-Flag HRP </td><td> Sigma </td><td> A8592 </td><td></td></tr><tr><td> Anti-CFTR-IgG </td><td> Maßgeschneidert </td><td> R1104 </td><td> mAb erkennt CFTR-Epitop aa 722-734 bei </td></tr><tr><td> Anti-MRP2-IgG </td><td> Chemicon International </td><td> MAB4148 </td><td> Jetzt ein Teil von Millipore </td></tr></tbody></table> Tabelle 2. Spezifischen Reagenzien und Geräte.

in vitro analysis of pdz-dependent cftr macromolecular signaling complexes

Zystische Fibrose-Transmembran-Regulator (CFTR), ein Chlorid-Kanal in erster Linie auf den apikalen Membranen der Epithelzellen befindet, spielt eine entscheidende Rolle bei der transepithelialen Homöostase 1-3. Zystischer Fibrose (CF) 4 und sekretorische Diarrhoe 5: CFTR wurde in zwei der wichtigsten Krankheiten in Verbindung gebracht. Bei CF wird die Synthese oder die funktionelle Aktivität des CFTR-Cl-Kanal reduziert. Diese Erkrankung betrifft etwa 1 von 2500 Kaukasiern in den Vereinigten Staaten sechs. Übermäßige CFTR-Aktivität wurde auch in Fällen von Toxin-induzierte sekretorische Diarrhöe (z. B. durch Cholera-Toxin und hitzestabil E. coli Enterotoxin), die cAMP oder cGMP-Produktion im Darm 7 stimuliert in Verbindung gebracht. Es gibt zunehmend Hinweise auf die Existenz der physischen und funktionellen Interaktionen zwischen CFTR und einer wachsenden Zahl von anderen Proteinen, einschließlich Transporter, Ionenkanäle, Rezeptoren, Kinasen, Phosphatasen, SignalIng. Moleküle und Zytoskelett-Elemente, und diese Interaktionen zwischen CFTR und ihre bindenden Proteinen wurde gezeigt, dass kritisch bei der Regulierung CFTR-vermittelten transepithelialen Ionentransport in vitro als auch in vivo 8-19 einbezogen werden. In diesem Protokoll, konzentrieren wir uns nur auf die Methoden, die Hilfe bei der Untersuchung der Wechselwirkungen zwischen CFTR carboxyterminalen Schwanz, der ein Protein-Bindungsmotiv besitzt [bezeichnet als PSD95/Dlg1/ZO-1 (PDZ) Motiv], und einer Gruppe von Gerüst-Proteine, die eine spezifische Bindung Modul enthalten, bezeichnet als PDZ-Domänen. Bisher wurden verschiedene PDZ Gerüstproteine ​​wurde berichtet, dass die Carboxyl-terminalen Schwanz des CFTR mit verschiedenen Affinitäten wie NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-assoziierten Liganden), SHANK2 binden und GRASP 20-27. Die PDZ-Motivs innerhalb CFTR, die von PDZ Gerüstproteine ​​erkannt wird, ist die letzten vier Aminosäuren am C-Terminus (dh 1477-DTRL-1480 in humanen CFTR) 20. InteressanterweiseCFTR binden können mehr als eine PDZ-Domäne beider NHERFs und PDZK1, wenn auch mit unterschiedlicher Affinitäten 22. Diese Multivalenz bezüglich CFTR Bindung wurde gezeigt, dass von funktionaler Bedeutung sein, was darauf hindeutet, dass PDZ Gerüstproteine ​​kann die Bildung von CFTR makromolekularen Komplexen ermöglichen Signalisierung für spezifische / selektive und effiziente Signalisierung in Zellen 16-18. Mehrere biochemische Assays wurden entwickelt, um zu studieren CFTR-Protein-Interaktionen beteiligt, wie z. B. Co-Immunopräzipitation, Pull-Down-Assay, paarweise Bindungsassay, kolorimetrische paarweise Bindungsassay und makromolekulare komplexe Montage-Assay 16-19,28,29 . Hier haben wir auf die genauen Verfahren zum Zusammenbau eines PDZ-Motiv-abhängige CFTR-haltigen makromolekularen Komplex in vitro, die ausgiebig in unserem Labor zu Protein-Protein-oder Domain-Domain-Wechselwirkungen unter Beteiligung von CFTR 16-19,28,29 Studie konzentrieren.

Watch this Scientific Journal Video about In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes at JoVE.com

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Zystische Fibrose-Transmembran-Regulator (CFTR), eine epitheliale Chlorid-Kanal, wurde berichtet, dass mit verschiedenen Proteinen zu interagieren und zu regulieren wichtige zelluläre Prozesse, darunter die CFTR PDZ-Motiv-vermittelte Wechselwirkungen sind gut dokumentiert. Dieses Protokoll beschreibt Methoden, die wir entwickelt, um eine PDZ-abhängigen CFTR makromolekularen Signalkomplexes montieren<em&gt; In-vitro-</em&gt;.

In-vitro-Analyse der PDZ-abhängigen CFTR Makromolekulare Signalkomplexe

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

in-vitro-analysis-pdz-dependent-cftr-macromolecular-signaling

Research

JoVE Journal

Biology

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

10.3K Aufrufe.  Wayne State University School of Medicine. Zystische Fibrose-Transmembran-Regulator (CFTR), eine epitheliale Chlorid-Kanal, wurde berichtet, dass mit verschiedenen Proteinen zu interagieren und zu regulieren wichtige zelluläre Prozesse, darunter die CFTR PDZ-Motiv-vermittelte Wechselwirkungen sind gut dokumentiert. Dieses Protokoll beschreibt Methoden, die wir entwickelt, um eine PDZ-abhängigen CFTR makromolekularen Signalkomplexes montieren In-vitro-.

Serie: In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Analyse von Schwann-Astrozyten Wechselwirkungen mit In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Forschung

Biologie

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects 1. Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription.
In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity 2-4. This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. The number of signals can be counted and compared providing a measurement. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. in a complex, which occurs only with signaling activation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation.

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.

Erkennung von Signaling Effektor-Komplexe Downstream von BMP4 Mit In situ PLA, einem Proximity Ligation Assay

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Ernte-und Cryo-Kühlung Kristalle von Membranproteinen in Lipidische Mesophasen zur Strukturaufklärung von Makromolekulare Kristallographie Grown

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Schnelle Genetische Analyse von epithelial-mesenchymale Signaling Während Haarregeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

Ein High-Content-Imaging Workflow Grb2 Signaling-Komplexe durch Expression Cloning Studie

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Mechanische Stimulation induzierte Calcium Wellenausbreitung in Zellmonoschichten: Das Beispiel Bovine Endothelzellen

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Metabolische Markierung und Membran Fraktionierung für Vergleichende Proteomanalyse von<em&gt; Arabidopsis thaliana</em&gt; Fahrwerk Zellkulturen

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Isolation und Kultur der erwachsenen Maus Kardiomyozyten für Cell Signaling und<em&gt; In-vitro-</em&gt; Herzhypertrophie

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Messung der Stoffwechselrate in<em&gt; Drosophila</em&gt; Mit Respirationstest

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

In-vitro-Rekonstitution der Lichtsammelkomplexe der Pflanzen und Grünalgen

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...