Ringraziamo il National Institutes of Health (1R21CA138373 e Stand Up to Cancer (SU2C) per supporti finanziari di questo lavoro. HY è grato per la Award 2009 Elion dalla American Association of Cancer Research, un Kimmel Scholar Award dalla Fondazione per la Sidney Kimmel Ricerca sul Cancro (SKF-08-101), e la National Science Foundation Facoltà primi Premio alla Carriera (NSF0954819).

1. Considerazioni sulla clonazione <ol><li> Oligonucleotidi commercialmente preparato che rappresenta il TMD di interesse affiancato da NheI e siti di restrizione BamHI e 5&#39;-fosforilato può essere legata in pTox7 (modificato nel nostro laboratorio mediante inserimento di una coppia di basi direttamente dopo il sito di restrizione BamHI 14) (Figura 3) digerito in sequenza con BamHI e NheI. Un oligonucleotide esempio è mostrato sotto: 5&#39;ctagcTMDSEQUENCEg3 &#39; 3 &#39;gTMDSEQUENCEcctag5&#39; </li></ol> La sequenza TMD dovrebbe essere 12-24 residui (brevi sequenze presumibilmente sarà allungata da residui idrofobici vettore di codifica). Al fine di indagare l&#39;interfaccia, quattro varianti del design TMD dovrebbero essere creati dove inserimenti residui sequenziale e concomitante risultato residui delezioni nel rotazione del relativo TMD a ToxR. 15,16, infine, la concentrazione arabinosio dovrebbe essere variato tra 0,001 e 0,01% (w / v) per identificare la concentrazione di cui si rilevino differenze massima β-galattosidasi segnali tra le diverse sequenze TMD, alla verifica dei livelli di espressione differenti si raccomanda di identificare condizioni di affinità diverse si possono distinguere meglio. Oltre a arabinosio e antibiotici, 0,4 mM IPTG possono essere utilizzate per migliorare le differenze di affinità tra TMD diverse. La misura ToxR dovrebbe essere eseguito almeno in quattro esemplari. Tutta la procedura dovrebbe essere ripetuta almeno tre volte con diverse trasformazioni plasmide. 2. La crescita di colture batteriche <ol><li> Delicatamente disgelo FHK12 cellule competenti (200 microlitri) su ghiaccio e trasferire in una provetta da 15 ml cultura. Aggiungi DNA plasmidico (200 ng) e incubare le cellule in ghiaccio per 30 min. </li><li> Heat-shock cellule mediante incubazione per 90 s a 42 ° C, seguita da incubazione in ghiaccio per 2 minuti. </li><li> Aggiungi SOC mezzi di comunicazione (800 microlitri) e incubare i campioni a 37 ° C con agitazione (300 giri) per un h. </li><li> Inoculare 5 media LB ml con cloramfenicolo (30 mg / ml) e arabinosio (0,0025% w / v) con 50 microlitri della miscela trasformazione in provette da 15 ml cultura in triplice copia. Incubare i campioni a 37 ° C con agitazione (300 rpm) per 20 h. (In alternativa 5 ml di cultura può essere utilizzato per inoculare 100 l di media in una piastra da 96 pozzetti. Questo metodo è utile quando si tratta di un gran numero di campioni, anche se gli errori saranno leggermente più alto. Al fine di evitare l&#39;evaporazione, il che porterebbe per errore, riempire i pozzi più esterna con i media, ma non li usano per i campioni. Infine, doppio avvolgere la giunzione tra il coperchio e il piatto con parafilm). </li></ol> 3. Misura della β-galattosidasi <ol><li> Preriscaldare il piatto-reader a 28 ° C. </li><li> Trasferire la Z-buffer in un serbatoio con un puntale di grandi dimensioni, facendo attenzione a prendere solo la parte superiore (acquoso) strato. Trasferire 100 ml di Z-buffer/chloroform preparati al momento ai pozzetti di una piastra da 96 pozzetti. Trasferire 5 ml di ciascuna cultura nei pozzetti della piastra in quattro esemplari. Omettere la cultura da quattro pozzi che servirà come il vuoto. </li><li> Misurare il diametro esterno 595 del piatto per determinare la densità delle cellule. </li><li> Aggiungere 50 ml di Z-buffer/SDS a tutti i pozzetti della piastra. Agitare la piastra sulla piastra-reader per 10 minuti a Lyze le cellule. Assicurarsi che le sospensioni cellulari sono chiare dopo la lisi e ripetere il passo tremante, se necessario. Lisi incompleta suggerisce il Z-buffer/chloroform non era preparato fresco. </li><li> Aggiungere 50 microlitri preparati al momento Z-buffer/ONPG a tutti i pozzetti e tornare la piastra per il lettore di piastre e misurare OD 405 ogni 30 s per 20 min. </li><li> Calcolare β-galattosidasi utilizzando la seguente equazione (ricordandosi di sottrarre il vuoto). Il rapporto di OD 405 / min deve essere calcolata utilizzando tutti i punti dati nel campo 405 OD 0,0-1,0 usando un adattamento lineare modello. <img src="files/ftp_upload/2721/2721eq1.jpg" alt="Equazione 1" /> Unità Miller differiscono a volte quando registrate in giorni diversi. Quindi, un costrutto di riferimento come AAP dovrebbe essere misurata in ciascuna prova. I suoi valori possono essere utilizzati per la normalizzazione dei valori ToxR. </li></ol> 4. Controllo per l&#39;espressione della proteina <ol><li> Eseguire Western blotting per verificare anche l&#39;espressione della proteina tra i costrutti. Unire 50 ml di culture triplice copia e centrifuga (2000 rpm, 4 min) in una microcentrifuga. Rimuovere il surnatante pipettando e risospendere il pellet residuo in 2 tampone di caricamento del campione x. </li><li> Carico di 7,5 microlitri su un gel standard 8% ed effettuare elettroforesi a 125 V per 1 ora 5 min. Dopo il trasferimento, incubare con anticorpi anti-MBP HRP-anticorpo coniugato e visualizzare, la proteina chimerica è osservata a circa 70 kDa con alcuni prodotti di degradazione a volte visto circa 48 kDa. MBP endogena è osservato anche a 45 kDa (Figura 5). </li></ol>5. Controllo per l&#39;inserimento corretto di membrana Una linea di cellule carenti di proteina legante il maltosio viene utilizzato per valutare il corretto inserimento della membrana del costrutto chimerico TMD. Quando coltivate su terreno minimo con il maltosio come unica fonte di carbonio, solo le cellule che esprimono una membrana integrale prodotto espressione di proteine ​​maltosio legame correttamente situato a periplasma sono in grado di crescere. <ol><li> PD28 trasformare cellule (come descritto per FHK12 cellule) e inoculare 2 ml di LB medium. Crescere le cellule a 37 ° C con agitazione (300 rpm) durante la notte. </li><li> Agglomerare le cellule per centrifugazione a 3500 rpm, 10 min, 4 ° C e lavare da risospensione in PBS (2 ml) pipettando dolce con una punta di grandi dimensioni o vortexando gentilmente. Agglomerare le cellule (come sopra), lavare con PBS per la seconda volta, pellet e risospendere infine, in PBS (1 ml). </li><li> Usare 25 ml di cellule risospese per inoculare 5 Mezzi ml minimo in triplice copia e incubare a 37 ° C con agitazione (300 giri). Prendere OD 595 letture tra 15-25 h, circa ogni 2 ore con il trasferimento di 200 ul di ciascun campione in una piastra da 96 pozzetti e la lettura con la piastra-reader. </li></ol> 6. Rappresentante dei risultati: Un esempio di utilizzo del test ToxR giornalista trascrizionale di analizzare la propensione oligomerizzazione di domini transmembrana in Figura 4. In precedenza abbiamo studiato la oligomerizzazione di domini transmembrana dal multispanning membrana integrante latente della membrana proteica proteine-1 (LMP-1) con varie tecniche, tra cui ToxR 14 domini transmembrana cinque (TM5) è stato dimostrato che mostra una forte propensione a oligomerize.; ciò è dimostrato da alte Unità Miller, paragonabile al controllo positivo, GPA, una solida sequenza dimerizing. Una mutazione deleteria in TM5, D150A, riduce la capacità della sequenza di oligomerize. LMP-1 TM1 non influisce significativamente oligomerize e presenta un bassissimo segnale Unità Miller, appena sopra il segnale per il valore vuoto, non trasformato FHK12 cellule. <img src="/files/ftp_upload/2721/2721fig1.jpg" alt="Figura 1" /> Cartoon Figura 1. Raffigurante il saggio giornalista ToxR. Dominio transmembrana (TMD) guidato risultati oligomerizzazione in dimerizzazione di ToxR e l&#39;attivazione della trascrizione LacZ. Il prodotto del gene di LacZ, β-galattosidasi può essere quantificato in una misura della propensione di un TMD a oligomerize. <img src="/files/ftp_upload/2721/2721fig2.jpg" alt="Figura 2" /> Figura 2. La scissione idrolitica di ONPG da β-galattosidasi risultati nella produzione di specie che assorbono la luce, o-nitrofenolato (ONP). <img src="/files/ftp_upload/2721/2721fig3.jpg" alt="Figura 3" /> Figura 3. Plasmidi mappa di pToxR7. <img src="/files/ftp_upload/2721/2721fig4.jpg" alt="Figura 4" /> Figura 4. Rappresentante saggio giornalista ToxR trascrizionale analizzare la propensione oligomerizzazione di membrana latente proteina-1 domini transmembrana. Dominio transmembrana 5 (TM5) oligomerizes fortemente, mentre il dominio transmembrana 1 (TM1) presenta solo una interazione debole. D150A mutazione in TM5 riduce in modo significativo la sua capacità di oligomerize. GPA è incluso come una sequenza di controllo positivo per dimerizzazione forte. Vuoto rappresenta FHK12 cellule non trasformate. <img src="/files/ftp_upload/2721/2721fig5.jpg" alt="Figura 5" /> Figura 5. Western Blot per l&#39;espressione delle proteine. <img src="/files/ftp_upload/2721/2721fig6.jpg" alt="Figura 6" /> Figura 6. PD28 saggio di complementazione di controllo per l&#39;inserimento della membrana corrette al periplasma. Controllo negativo rappresenta un costrutto carente di maltosio legame proteico.

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Nessun conflitto di interessi dichiarati.

La trascrizione saggio giornalista ToxR è un modo facile per identificare sequenze transmembrana con la possibilità di oligomerize. Dato che le interazioni avvengono all&#39;interno della membrana batterica interna, questo saggio aggira i problemi associati con la validità dei sistemi di studiare in ambienti di membrana-mimetici. Dato che la clonazione di TMD multipli in un singolo plasmide può facilmente essere fatto in parallelo e il test completo può essere effettuata in 96-ben formato piatto, questo test può e...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> Enzima di restrizione BamHI </td><td> Invitrogen </td><td> 15201023 </td><td> Invitrogen enzimi sono stati trovati per essere più efficiente di altri fornitori </td></tr><tr><td> NheI enzima di restrizione </td><td> Invitrogen </td><td> 15444011 </td><td> Invitrogen enzimi sono stati trovati per essere più efficiente di altri fornitori </td></tr><tr><td> Provette da 15 ml cultura </td><td> Fisher Scientific </td><td> 14-956-1J </td><td></td></tr><tr><td> SOC mezzi di comunicazione </td><td> Teknova </td><td> S0225 </td><td> Composto al volume appropriato e sterilizzati in autoclave. </td></tr><tr><td> LB mezzi di comunicazione </td><td> Sigma-Aldrich </td><td> L7275 </td><td> Composto al volume appropriato e sterilizzati in autoclave. </td></tr><tr><td> Cloramfenicolo </td><td> Sigma-Aldrich </td><td> CO 3 78 </td><td> Soluzione madre di 30 mg / ml in etanolo conservati in congelatore </td></tr><tr><td> Arabinosio </td><td> Fluka </td><td> 10839 </td><td> Soluzione madre di 2,5% (w / v) in acqua immagazzinata nel congelatore </td></tr><tr><td> Na 2 HPO 4 </td><td> Sigma-Aldrich </td><td> S9390 </td><td></td></tr><tr><td> NaH 2 PO 4 </td><td> Sigma-Aldrich </td><td> S9638 </td><td></td></tr><tr><td> KCl </td><td> Mallinckrodt chimici </td><td> 6858-06 </td><td></td></tr><tr><td> MgSO 4 0,7 H 2 O </td><td> Sigma-Aldrich </td><td> 63138 </td><td></td></tr><tr><td> Dodecylsulfate sodio (SDS) </td><td> Sigma-Aldrich </td><td> L6026 </td><td></td></tr><tr><td> 2-Nitrophenyl β-D-galattopiranoside (ONPG) </td><td> Sigma-Aldrich </td><td> 73660 </td><td></td></tr><tr><td> Z-buffer </td><td></td><td></td><td> 16,1 g di Na 2 HPO 4 5.5g NaH 2 PO 4 0,75 g di KCl 0.246g MgSO 4 Portare a 1 l, pH 7,0 </td></tr><tr><td> Z-buffer/chloroform </td><td></td><td></td><td> 200 mL β-mercaptoetanolo, 2 ml di cloroformio, portare a 20 ml con Z-buffer. Vortex per 1 min, centrifugare per 1 min a 800 giri al minuto. Fai fresco per ogni piatto. </td></tr><tr><td> Z-buffer/SDS </td><td></td><td></td><td> 160 mg SDS sciolto in 10 mL di Z-buffer </td></tr><tr><td> Z-buffer/ONPG </td><td></td><td></td><td> ONPG 40 mg in 10 ml di Z-buffer. Fai fresco per ogni piatto </td></tr><tr><td> β-mercaptoetanolo </td><td> Calbiochem </td><td> 444203 </td><td></td></tr><tr><td> Anti-MBP anticorpi monoclonali (HRP coniugata) </td><td> NEB </td><td> E8038S </td><td></td></tr><tr><td> Terreno minimo con maltosio </td><td></td><td></td><td> 1 x M9 sali, 0,4% maltosio, 1 mg / mL tiamina, 2 mM MgSO 4 </td></tr><tr><td> 96 pozzetti fondo piatto </td><td> Sarstedt </td><td> 83.1835.300 </td><td></td></tr><tr><td> Piastra-reader </td><td> Beckman Coulter </td><td> DTX880 Detector Multimode </td><td></td></tr><tr><td> Bagnomaria </td><td> VWRI </td><td> 89032-204 </td><td></td></tr><tr><td> Scuotendo incubatore </td><td> FormaScientific </td><td></td><td></td></tr></tbody></table>

transmembrane domain oligomerization propensity determined by toxr assay

La visione semplicistica di domini di proteine ​​transmembrana semplicemente come ancore in doppi strati di fosfolipidi è stata da tempo confutata. In molti casi, attraversa la membrana le proteine ​​si sono evoluti meccanismi altamente sofisticate di azione. 1-3 Un modo in cui le proteine ​​di membrana in grado di modulare le loro strutture e delle funzioni avviene attraverso il contatto diretto e specifico di eliche idrofobiche, formando oligomeri transmembrana strutturato. 4,5 recente molto lavoro si è concentrato sulla distribuzione degli aminoacidi preferenzialmente trovato nell&#39;ambiente della membrana rispetto alla soluzione acquosa e le forze intermolecolari diverse proteine ​​associazione unità. 6,7 Tuttavia, gli studi di riconoscimento molecolare al dominio transmembrana di proteine ​​ancora in ritardo rispetto quelli di solubili in acqua le regioni. Un grande ostacolo rimane: nonostante la notevole specificità e affinità che oligomerizzazione transmembrana può raggiungere, 8 misura diretta della loro associazione è impegnativo. Metodologie tradizionali applicati allo studio della funzione integrale proteina di membrana può essere ostacolato dalla insolubilità intrinseca delle sequenze in esame. Conoscenze acquisite studiando biofisici peptidi sintetici che rappresentano domini transmembrana in grado di fornire utili visione strutturale. Tuttavia, la rilevanza biologica del micellare detergente o sistemi di liposomi utilizzati in questi studi per imitare le membrane cellulari è spesso messa in discussione; fare peptidi adottare un nativo struttura simile in queste condizioni e che il loro comportamento funzionale rispecchiano le reali modalità di azione all&#39;interno di una membrana nativa ? Al fine di studiare le interazioni delle sequenze transmembrana in doppi strati di fosfolipidi naturali, il laboratorio Langosch sviluppato test giornalista trascrizionale ToxR. 9 Il dominio transmembrana di interesse è espresso come una proteina chimerica con maltosio legame con le proteine ​​per la posizione al periplasma e ToxR a fornire una relazione del livello di oligomerizzazione (Figura 1). Negli ultimi dieci anni, diversi altri gruppi (ad esempio Engelman, DeGrado, Shai), ulteriormente ottimizzato e applicato questo saggio giornalista ToxR. 10-13 I saggi ToxR vari sono diventati un gold standard per testare interazioni proteina-proteina nelle membrane cellulari. Siamo qui dimostrare una tipica operazione sperimentale condotto nel nostro laboratorio che segue principalmente i protocolli sviluppati da Langosch. Questo metodo generalmente applicabile è utile per l&#39;analisi di dominio transmembrana auto-associazione in E. coli, in cui viene utilizzato β-galattosidasi produzione per valutare la propensione TMD oligomerizzazione. Su TMD indotta dimerizzazione, ToxR si lega al promotore ctx causando up-regolazione del gene LacZ per β-galattosidasi. Una lettura colorimetrica ottenuto mediante aggiunta di ONPG alle cellule lyzed. Scissione idrolitica di ONPG da β-galattosidasi risultati nella produzione di specie che assorbono la luce, o-nitrofenolato (ONP) (Figura 2).

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Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

Una procedura efficace per valutare la propensione oligomerizzazione del single-pass domini transmembrana (TMD) è descritta. Proteine ​​chimeriche costituito dalla TMD fusa ToxR sono espressi in un ceppo di E. coli giornalista. TMD indotta oligomerizzazione cause dimerizzazione di ToxR, l&#39;attivazione della trascrizione e la produzione della proteina reporter-galattosidasi.

Transmembrana Oligomerizzazione Propensione dominio determinato dal saggio ToxR

The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven.  In many cases ...

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15.1K Views.  University of Colorado at Boulder. Una procedura efficace per valutare la propensione oligomerizzazione del single-pass domini transmembrana (TMD) è descritta. Proteine ​​chimeriche costituito dalla TMD fusa ToxR sono espressi in un ceppo di E. coli giornalista. TMD indotta oligomerizzazione cause dimerizzazione di ToxR, l'attivazione della trascrizione e la produzione della proteina reporter-galattosidasi.

Video: Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

Espressione e purificazione della proteina transmembrana cistica regolatore fibrosi conduttanza in Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

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Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

Purificazione della fibrosi cistica regolatore della conduttanza transmembrana proteina espressa in<em&gt; Saccharomyces cerevisiae</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

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

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

Genome-wide proteina-proteina interazione Screening per Protein-frammento Complementation Assay (PCA) in cellule viventi

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Ricostituzione di una proteina transmembrana, la tensione gated canali ionici, KvAP, in Giant unilamellari vescicole per la microscopia e Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

Mappatura ottica di Intra-reticolo sarcoplasmatico Ca<sup&gt; 2+</sup&gt; E transmembrana potenziale nel Rabbit Heart Langendorff-perfuso

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecale Analisi glucocorticoidi: non invasivo di monitoraggio surrenale in equidi

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Approcci genetici e biochimici per<em&gt; In Vivo</em&gt; e<em&gt; In Vitro</em&gt; La valutazione di proteine ​​Oligomerizzazione: Il rianodina Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

Studio del reticolo endoplasmatico e mitocondri interazioni<em&gt; In Situ</em&gt; Proximity Assay legatura in cellule fissate

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...