Il nostro lavoro è stato supportato anche da finanziamenti American Heart Association (Greater Southeast Affiliate) Inizio-grant-in-aiuto 0765185B, la Fondazione U. Pardee Elsa assegno di ricerca, e Wayne State University intramurale del fondo di avvio e Cardiovascular Research Institute premio Initiative Isis. Questo metodo di assemblaggio in vitro complesso macromolecolare CFTR è stata originariamente introdotta da Dr. AP Naren (University of Tennessee Health Science Center).

1. Espressione e purificazione di proteine ​​ricombinanti in batteri Fusion etichettate <ol><li> Amplifica le regioni definite del C-code (gli ultimi 50-100 aminoacidi contenenti i motivi PDZ a C-terminale) per CFTR, LPA 2, MRP2, MRP4, β 2 AR, e NHERFs (full-length o PDZ1 o PDZ2 domini ) con approccio PCR. </li><li> Clonare i prodotti di PCR in pGEX4T-1 vettore per le proteine ​​di fusione GST-(come GST-NHERFs, GST-MRP4 CT), pMAL-C2 vettore per MBP-fusione proteine ​​(come MBP-β 2 AR CT, MBP-CFTR CT ), e per i Suoi pET30-S-proteine ​​di fusione (come la sua-S-CFTR CT, His-S-PDZK1). </li><li> Trasforma in una proteasi carente di E. coli ceppo (BL21-DE3) per ridurre al minimo possibile degradazione della proteina ricombinante. </li><li> Crescere la coltura di una notte a 37 ° C in terreno Luria-Bertani (pH 7,0) contenente antibiotici adatti (come ampicillina o kanamicina). Diluire la coltura di una notte a 1:10 e crescere per altre 2 ore a 37 ° C. Indurre con 0,5-1 m M IPTG per i prossimi 4 ore a 30 ° C. Pellet delle cellule per centrifugazione a 8000 g per 10 min a 4 ° C. </li><li> Lisare le cellule in tampone saccarosio (50 m M Tris-HCl, pH 8,0, 1 mM EDTA, 1 mM PMSF, e 10% di saccarosio) contenente lisozima (1 mg / mL), 0,2% Triton X-100, e proteasi inibitori. Usare 20 mL per pellet di cellule proveniente da 1 L della cultura. </li><li> Mescolare su un agitatore rotante per 30 minuti a 4 ° C. </li><li> Spin a 20.000 xg per 30 min a 4 ° C. Raccogliere il surnatante chiaro. </li><li> Nel supernatante chiaro, aggiungere 1 ml delle seguenti resina / agarosio perline (50% slurry in tampone saccarosio): <ul><li> Glutatione perline di agarosio per proteine ​​di fusione GST. </li><li> Perle Talon per le Sue S-proteine ​​di fusione. </li><li> Resina amilosio per proteine ​​di fusione MBP. </li></ul></li><li> Mescolare per 4 ore a 4 ° C su un agitatore rotante. </li><li> Lavare le perle risospendendo in 1x PBS (15 ml), mescolare per 2 min, filatura a 800 g per 2 min, e scartando il supernatante. Ripetere questo passaggio per sei volte. </li><li> Eluire la proteina dalle perline mediante rispettivo buffer di eluizione (2 mL di tampone di eluizione per 1 mL di perline). <ul><li> Tampone di eluizione per proteine ​​di fusione GST: 25 m M Tris-HCl (pH 8,0), 140 m M NaCl, e 20 m M glutatione ridotto. </li><li> Tampone di eluizione per le sue proteine ​​di fusione: 20 m M Tris-HCl (pH 8,0), 500 m M NaCl, e 200 mM imidazolo. </li><li> Tampone di eluizione per proteine ​​di fusione MBP: 20 m M Tris-HCl (pH 8,0), 200 m M NaCl, 1 mM EDTA, 1 mM DTT, e 10 mM maltosio. </li></ul></li><li> Dializzare le proteine ​​eluite contro 2 L di 1x PBS a 4 ° C (per evitare la possibile degradazione delle proteine). Sostituire ogni 4 ore per quattro volte PBS. Concentrare la proteina utilizzando un filtro Centricon (10.000 taglio MW, Millipore), e memorizza come piccole aliquote a -80 ° C. </li><li> Determellino la concentrazione di proteina mediante il metodo Bradford. Valutare qualità delle proteine ​​mediante SDS-PAGE usando BSA come standard. Se l&#39;integrità della proteina non è soddisfacente, le procedure di purificazione secondarie, come la filtrazione su gel o scambio ionico possono essere utilizzati. (Ad esempio, abbiamo utilizzato una colonna G-75 Sepharose per purificare ulteriormente-proteina di fusione GST). </li></ol> 2. Di coltura cellulare e Preparazione del lisato cellulare <ol><li> Bambino Cultura renali di criceto (BHK) stabilmente cellule che over-esprimono CFTR-peso e CFTR-his10 o cellule BHK transitoriamente che over-esprimono Flag-LPA 2-peso o Flag-LPA 2-ΔSTL e Madin-Darby rene canino (MDCK stabilmente) cellule che sovra-esprimono MRP2 in mezzo minimo essenziale di Eagle (MEM) contenente 10% siero di vitello fetale e penicillina / streptomicina in fiasche di polistirene a 37 ° C con 5% CO 2. </li><li> Cultura embrionali umane di rene (293 HEK293) stabilmente cellule che over-esprimono CFTR-peso e CFTR-his10 in Dulbecco&#39;S mezzo Eagle modificato (DMEM) integrato con 10% siero fetale bovino e penicillina / streptomicina in fiasche di polistirene a 37 ° C con 5% CO 2. </li><li> Lisare le cellule in tampone di lisi (PBS - 0,2% Triton-X-100 integrato con una miscela di inibitori di proteasi contenenti 1 mM fenilmetilsolfonilfluoruro, 1 ug / ml di aprotinina, 1 ug / mL di leupeptina, 1 ug / ml di pepstatina); uso 500 tampone di lisi microlitri per ogni 60 mm piastra di Petri, e 1000 tampone di lisi microlitri per ogni 100 mm piastra di Petri. </li><li> Rock the lisati cellulari per 20 min a 4 ° C, e rimuovere il materiale insolubile mediante centrifugazione a 16.000 xg per 15 min a 4 ° C. Determinare la concentrazione proteica con il saggio Bradford. </li></ol> 3. In Assemblea in vitro di un CFTR contenente complesso macromolecolare (CFTR-PDZK1-MRP4) <ol><li> In 200 microlitri di tampone di lisi (PBS - 0,2% Triton X-100 + inibitori della proteasi), aggiungere 20 mcg purificato GST-MRP4 C-terminale50 bis (CT50), proteina di fusione. </li><li> Aggiungi vari importi (0-40 mg) di purificata His-S-PDZK1 proteina di fusione. </li><li> Mescolare le due proteine ​​su un agitatore rotante a 22 ° C per 1-2 h. </li><li> Aggiungi 20 perle del glutatione pl (50% slurry) nella miscela di proteine, e continuare a mescolare per un altro 1 h. Questa fase viene anche indicato come a coppie legante. </li><li> Durante questo tempo, preparare le HEK293 lisati cellulari che iperesprimono Flag-CFTR (wt) come sopra descritto al punto 2). </li><li> Lavare il complesso due volte con tampone di lisi. Spin a 800 xg per 1 minuto per ogni lavaggio e aspirare accuratamente il supernatante dopo ogni lavaggio. Prestare attenzione a non succhiare le perline in fondo. </li><li> Aggiungere preparato sopra lisati cellulari HEK293 alle perle, e mescolare delicatamente a 4 ° C per 3 h (o tutta la notte). </li><li> Lavare le perline estesamente tre volte con tampone di lisi, come descritte nella Fase 3,6). </li><li> Eluire le proteine ​​con 30 microlitri Sample Buffer (5 ×): 0,6 M<html> Tris-HCl (pH 6,8), 50% glicerolo, 2% SDS, e 0,1% blu di bromofenolo, contenente 5% di β-mercaptoetanolo. </li><li> Incubare in bagnomaria a 37 ° C per 10-15 min, e far girare a 5000 × g per 30 s per far precipitare le perline. </li><li> Caricare tutto l&#39;eluato su gel 4-15%. </li><li> Eseguire SDS-PAGE per circa 30-40 min. </li><li> Trasferimento bande proteiche alla membrana PVDF, per 1,5 ore. </li><li> Bloccare la membrana in TBS-Tween contenente 5% di latte non grasso. </li><li> Incubare la membrana con l&#39;anticorpo primario (ad esempio anti-IgG CFTR, R1104) a 4 ° C, una notte. </li><li> Lavare la membrana con TBS-Tween per 5 min, sei volte. </li><li> Incubare la membrana con anticorpo secondario (come capra anti-topo coniugato HRP-anticorpo 2 °) per 45 min a 22 ° C. </li><li> Lavare la membrana con TBS-Tween per 5 min, sei volte. </li><li> Visualizzare le bande proteiche tramite ECL. </li><li> I dati rappresentativi sono mostrati in Figura 1. </li></ol>jove_title &quot;&gt; 4. risultati rappresentativi Un esempio di CFTR contenente complesso macromolecolare segnalazione che è stata assemblata in vitro è illustrato nella figura 1. Un complesso macromolecolare è formata tra MRP4 C-terminale di 50 amminoacidi (MRP4-CT50), PDZK1 e full-length CFTR (Figura 1, in basso). La formazione del complesso aumento dose-dipendente con quantità crescenti di proteina intermediario, PDZK1 (Figura 1, in basso) 18. <img alt="Figura 1" src="/files/ftp_upload/4091/4091fig1.jpg" /> Figura 1. Una rappresentazione pittorica del saggio complesso macromolecolare (in alto). Un complesso macromolecolare è stato rilevato in vitro con tre proteine ​​(GST-MRP4-CT50, His-S-PDZK1, e Flag-CFTRwt) in un modo dipendente dalla dose (in basso) 18. <table border="1"><tbody><tr><td></td><td><strong&gt; CFTR-NHERF1-β 2 AR (rif. 14) </td><td> CFTR-NHERF2-LPA 2 (rif. 15) </td><td> CFTR-PDZ proteine ​​MRP-2 (rif. 17) </td><td> CFTR-PDZK1-MRP4 (rif. 16) </td></tr><tr><td> Affinity perline </td><td> Amilosio resina </td><td> S-proteina agarosio </td><td> Amilosio resina </td><td> Glutatione agarosio </td></tr><tr><td> Proteina purificata-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> Proteina purificata-2 </td><td> GST-NHERF1 </td><td> GST-NHERF2 </td><td> GST-proteine ​​PDZ </td><td> His-S-PDZK1 </td></tr><tr><td> Proteina purificata-3 (o lisati cellulari) </td><td> CFTR-peso o CFTR-his10 (BHK o lisati cellulari HEK) </td><td> BandieraLPA-2-peso o Flag-LPA 2-ΔSTL (lisati cellulari BHK) </td><td> MRP2 (lisati cellulari MDCK) </td><td> Purificata Flag-CFTR wt-o-CFTR his10 (o lisati cellulari) </td></tr><tr><td> Anticorpo </td><td> Anti-IgG CFTR </td><td> Anti-Flag HRP </td><td> Anti-IgG MRP2 </td><td> Anti-Flag HRP </td></tr></tbody></table> Tabella 1. Sintesi di vari CFTR contenenti complessi macromolecolari assemblati in vitro.

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In questo protocollo abbiamo dimostrato un metodo in vitro per l&#39;assemblaggio e il rilevamento di una CFTR contenente complesso macromolecolare segnalazione utilizzando proteine ​​purificate (o frammenti di proteine) e / o lisati di cellule come riportato precedentemente 16-19,29,30. Per ottenere i migliori risultati i seguenti punti critici durante il processo di preparazione richiedono una particolare attenzione: <ul><li> È importante che il pH del tampone di eluizione essere regolata ...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> pGEX4T-1 vettore </td><td> GE Healthcare </td><td> 28-9545-49 </td><td> già Amersham Biosciences </td></tr><tr><td> pMAL-C2 vettoriale </td><td> New England BioLabs </td><td></td><td></td></tr><tr><td> pET30 vettore </td><td> EMD Chemicals </td><td> 69077-3 </td><td> ex Novagen </td></tr><tr><td> Glutatione agarosio perline </td><td> BD Biosciences </td><td> 554780 </td><td></td></tr><tr><td> Amilosio resina </td><td> New England BioLabs </td><td> E8021S </td><td></td></tr><tr><td> Talon perline </td><td> Clontech </td><td> 635501 </td><td></td></tr><tr><td> glutatione ridotto </td><td> BD Biosciences </td><td> 554782 </td><td></td&gt; </tr><tr><td> imidazolo </td><td> Pescatore </td><td> BP305-50 </td><td></td></tr><tr><td> maltosio </td><td> Pescatore </td><td> BP684-500 </td><td></td></tr><tr><td> S-proteina agarosio </td><td> EMD Chemicals </td><td> 69704-3 </td><td> ex Novagen </td></tr><tr><td> Anti-Flag HRP </td><td> Sigma </td><td> A8592 </td><td></td></tr><tr><td> Anti-IgG CFTR </td><td> Su misura </td><td> R1104 </td><td> mAb riconoscendo CFTR epitopo al aa 722-734 </td></tr><tr><td> Anti-IgG MRP2 </td><td> Chemicon International </td><td> MAB4148 </td><td> Ora una parte di Millipore </td></tr></tbody></table> Tabella 2. Reagenti e attrezzature specifiche.

in vitro analysis of pdz-dependent cftr macromolecular signaling complexes

Transmembrana della fibrosi cistica regolatore della conduttanza (CFTR), un canale del cloro si trova soprattutto a livello delle membrane apicali delle cellule epiteliali, gioca un ruolo cruciale nella omeostasi del liquido transepiteliale 1-3. CFTR è stata implicata in due importanti malattie: fibrosi cistica (CF) 4 e 5 secretoria diarrea. In CF, la sintesi o attività funzionale della CFTR Cl-canale è ridotta. Questo disturbo colpisce circa 1 su 2500 caucasici negli Stati Uniti 6. Attività eccessiva CFTR è stata anche implicata in casi di tossina indotta diarrea secretoria (ad esempio, da tossina del colera e stabile al calore enterotossina E. coli) che stimola la produzione di cAMP o cGMP nell&#39;intestino 7. Accumulare le prove suggeriscono l&#39;esistenza di interazioni fisiche e funzionali tra CFTR e un numero crescente di altre proteine, tra trasportatori, canali ionici, recettori, chinasi, fosfatasi, segnalemolecole zione ed elementi del citoscheletro, e queste interazioni tra CFTR e le sue proteine ​​leganti hanno dimostrato di essere criticamente coinvolte nella regolazione CFTR trasporto mediato transepiteliale ionico in vitro e in vivo 8-19. In questo protocollo, ci concentriamo solo sui metodi che gli aiuti nello studio delle interazioni tra CFTR carbossile terminale della coda, che possiede una proteina di legame motivo [denominato PSD95/Dlg1/ZO-1 (PDZ) motivo], e un gruppo di proteine ​​scaffold, che contengono un modulo di legame specifico denominato domini PDZ. Finora, molte diverse proteine ​​PDZ ponteggi sono stati riportati da associare alla coda terminale carbossile di CFTR con affinità diverse, ad esempio, i NHERF1 NHERF2 e PDZK1 e PDZK2 e CAL (CFTR-associata ligando), Shank2, e GRASP 20-27. Il motivo PDZ all&#39;interno CFTR che è riconosciuto da PDZ proteine ​​scaffold è ultimi quattro amminoacidi al C terminale (cioè, 1477-DTRL-1480 in umano CFTR) 20. Interessante,CFTR può legare più di un dominio PDZ sia NHERFs e PDZK1, pur in diversi affinità 22. Questa polivalenza rispetto CFTR legame ha dimostrato di essere di importanza funzionale, suggerendo che le proteine ​​PDZ impalcatura può facilitare la formazione di complessi macromolecolari CFTR segnalazione per la segnalazione specifici / selettivo ed efficace in cellule 16-18. Più saggi biochimici sono stati sviluppati per studiare CFTR, che coinvolge interazioni proteiche, come la co-immunoprecipitazione, pull-down assay, pair-wise saggio di legame, colorimetrico pair-wise saggio di legame, e il dosaggio di assemblaggio macromolecolare complessa 16-19,28,29 . Qui ci concentriamo sulle procedure dettagliate di assemblaggio di un motivo PDZ CFTR-dipendente contenenti macromolecole complesse in vitro, che è ampiamente utilizzato dal nostro laboratorio per studiare proteina-proteina o di dominio nel dominio delle interazioni che coinvolgono CFTR 16-19,28,29.

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

Transmembrana della fibrosi cistica regolatore conduttanza (CFTR), un canale cloruro epiteliale, è stata riportata per interagire con varie proteine ​​e regolano importanti processi cellulari, tra i quali i CFTR PDZ motivo mediate da interazioni sono stati ben documentati. Questo protocollo descrive i metodi che abbiamo sviluppato per assemblare un PDZ-dipendente macromolecolare CFTR segnalazione complessa<em&gt; In vitro</em&gt;.

L&#39;analisi in vitro dei complessi CFTR PDZ-dipendenti macromolecolari di segnalazione

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

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Research

JoVE Journal

Biology

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

10.3K Views.  Wayne State University School of Medicine. Transmembrana della fibrosi cistica regolatore conduttanza (CFTR), un canale cloruro epiteliale, è stata riportata per interagire con varie proteine ​​e regolano importanti processi cellulari, tra i quali i CFTR PDZ motivo mediate da interazioni sono stati ben documentati. Questo protocollo descrive i metodi che abbiamo sviluppato per assemblare un PDZ-dipendente macromolecolare CFTR segnalazione complessa In vitro.

Video: 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.

Analisi di Schwann-astrociti Interazioni utilizzo In Vitro Saggi

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

Ricerca

Biologia

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.

Rilevazione di segnalazione Effettistica-Complessi valle di BMP4 utilizzo In situ PLA, una legatura di prossimità Assay

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

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.

Rapida analisi genetica di epitelio-mesenchimale segnalazione durante la rigenerazione dei capelli

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.

Un alto contenuto di flusso di lavoro di imaging per studiare GRB2 Centri di segnalazione di espressione clonazione

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.

La stimolazione meccanica indotta calcio Wave Propagation in monostrati cellulari: l&#39;esempio di bovini cellule endoteliali corneali

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.

Metabolic Etichettatura e con membrana frazionamento per proteomica comparativa Analisi del<em&gt; Arabidopsis thaliana</em&gt; Culture sospensione cellulare

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.

Isolamento e Cultura del topo adulto Cardiomyocytes per Cell Signaling e<em&gt; In vitro</em&gt; Cardiaca Ipertrofia

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.

Misurazione del tasso metabolico in<em&gt; Drosophila</em&gt; Utilizzando respirometria

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 Ricostituzione di Complessi Light-raccolta di piante e alghe verdi

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

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.