The authors are deeply indebted to all the members of the scientific community working on pH and ion transport, who have originated and improved the measurements described here. They particularly thank Dr. Jacques Pouyss&#233;gur who originated the H+-killing selection technique used here. They acknowledge the University of Nice-Sophia Antipolis, the CNRS, the ANR (JCJC SVSE1 NHEint) and the ICST Labex for support.

1. H + Uccidere Selezione <ol><li> Linee cellulari <ol><li> Stabilmente trasfettare cellule NHE-deficienti (ad esempio, il PS120 linea cellulare CCL39-derivato 16) utilizzando qualsiasi combinazione di mammiferi vettore di espressione e di metodo di trasfezione che produrrà rendimenti trasfezione efficienti e selezione in una linea cellulare di fibroblasti. NOTA: Per molti anni fosfato di calcio precipitazioni 19 è stato utilizzato con buoni rendimenti trasfezione. Questo è stato sostituito più recentemente da reagenti commerciali come Lipofectamine2000, le trasfezioni essere eseguiti seguendo le istruzioni del produttore. </li></ol></li><li> Soluzioni <ol><li> Preparare tre soluzioni sterili descritti di seguito. Sterilizzare queste soluzioni per filtrazione (0,22 micron). <ol><li> Preparare una soluzione di NH 4 + Loading (pH 7,4) comprendente 50 mM NH 4 Cl, 70 mM di cloruro di colina, 5 mM KCl, 1 mM MgCl 2, 2 mM CaCl 2, GL 5 mMucose e 15 MOPS mm a pH 7.4. </li><li> Preparare una soluzione di risciacquo (pH 7,0) composto da 120 mM di cloruro di colina, 5 mM KCl, 1 mM MgCl 2, 2 mM CaCl 2, glucosio 5 mM e 15 mM HEPES a pH 7,0. </li><li> Preparare una soluzione di ripristino (pH 7,4) compreso di 120 mM NaCl, KCl 5 mM, 1 mM MgCl 2, 2 mM CaCl 2, 5 mM di glucosio e 15 mM HEPES a pH 7,4. </li></ol></li></ol></li><li> Prima di iniziare la selezione, ottenere un incubatore di coltura cellulare che può essere utilizzato a 37 ° C senza ulteriore CO 2 dal serbatoio esterno (chiamato CO 2 - incubatore &quot;libero&quot;), il 5% di CO 2 si tradurrà in buffer che possono compromettere la acidificazione. Amplifica la linea cellulare che esprime la NHE di interesse fino a circa 2 x 10 8 cellule (in genere circa 20 confluenti 100 piastre mm). </li><li> H + uccidendo procedura: <ol><li> Incubare le cellule che esprimono la intracellulare NHE di interesse per 1 ora in soluzione di caricamento, a 37 ° C in CO 2 incubatore -free. </li><li> Aspirare la soluzione di carico e quindi risciacquare due volte nella soluzione descritta sopra risciacquo. Eseguire questo passaggio efficiente per eliminare il residuo extracellulare NH 4 Cl che compromettano la creazione di una ripida NH 4 + gradiente che genererà l&#39;acidificazione. </li><li> Eliminare il mezzo risciacquo mediante aspirazione e incubare le cellule per un&#39;ora in soluzione di recupero di nuovo a 37 ° C in incubatore CO 2 libera. </li><li> Sostituire il mezzo di recupero con terreno di coltura regolare (tipicamente DMEM con 7,5% FCS) e far crescere le cellule in condizioni di coltura standard (37 ° C in atmosfera umidificata al 5% di CO 2 e il 95% di aria). </li></ol></li><li> Ripetere questo ciclo di selezione due volte a settimana fino cloni stabili emergono. NOTA: Prestare particolare attenzione per quanto riguarda la coltura cellulare e condizioni di selezione. Mesi di lavoro può essere rovinato da qualsiasi contaminazione che è più incline a oCCur dopo un lungo periodo di cultura e di manipolazioni cellulari ripetuti. <ol><li> Qui, scegliere se amplificare i cloni e caratterizzare singolarmente come descritto nelle sezioni 2-3, o in piscina insieme per generare una popolazione cellulare prima di caratterizzazione. </li></ol></li><li> Applicare selezione occasionali (circa una volta ogni due settimane) per trattenere le cellule acidificazione resistente by-counter selezionando quelle che possono riversati fenotipo di acido sensibile parentale (Figura 1B). </li></ol> 2. intracellulare pH Misurazioni 2.1) fluorescenza pH Imaging <ol><li> Utilizzare il raziometrico pH sensibile colorante fluorescente BCECF / AM, che è sensibile al pH quando eccitato a 490 nm e 445 nm possiede un punto isosbestic, seguendo i protocolli del produttore. <ol><li> In alternativa, utilizzare altre sonde pH-sensibili, seguendo i protocolli del produttore. </li></ol></li><li> Utilizzare un set con immaginiCONSISTONO di un microscopio invertito accoppiato ad una videocamera ad alta sensibilità. Dotare questo set con 450 nm e 490 nm filtri interferenza a banda stretta in coppia con quarzo opportuni filtri densità neutra per l&#39;eccitazione. <ol><li> Se possibile, utilizzare l&#39;insieme appropriato di filtri e delle condizioni di illuminazione a fluorescenza di impostazione valori confrontabili per λ1 e λ2 in modo che il rapporto si avvicina a 1. Evitare anche valori di fluorescenza basali impostati o saturare o troppo bassa, sia per λ1 e / o λ2. Questo può cedere manufatti importanti come allora il rapporto non può essere rilevabile anche se il pH intracellulare sta cambiando. NOTA: Il sistema deve consentire di perfusione rapida, time-lapse eccitazione a due lunghezze d&#39;onda di cui sopra e di acquisizione alla lunghezza d&#39;onda di emissione corretta (535 nm per BCECF). Dotare il sistema con software che consente l&#39;acquisizione automatica dei dati e di stoccaggio. </li></ol></li></ol> 2.2) Misura della NHE Forward Activity (Estrusione diI protoni dal citoplasma) <ol><li> Acidificare cellule da una incubazione di 1 ora in soluzione di NH 4 + Loading (vedere il punto 1.4) in assenza di CO 2. </li><li> Aggiungere BCECF-AM (concentrazione finale 5 mM) alla soluzione per gli ultimi cinque minuti e quindi risciacquare con NH 4 + soluzione tampone per eliminare la sonda extracellulare. </li><li> Montare le celle del microscopio e prendere più immagini per registrare una linea di base stabile. Profumato soluzione di risciacquo (vedi sopra). Ciò si traduce in una diminuzione della fluorescenza corrispondente al acidificazione. </li><li> Dopo la stabilizzazione del pH, profumato una delle soluzioni indicate qui di seguito per misurare i tassi di recupero pH mediati da Li + / H +, Na + / H + o K + / H + scambio. Ogni soluzione contiene un potenziale cationico accoppiamento da testare per H + scambio. Se uno di questi è trasportata, questo si tradurrà in un aumento della fluorescenza a 495 nm che correspond ad un aumento del pH intracellulare: , Glucosio LiCl 120 mM, KCl 5 mM, 1 mM MgCl 2, 2 mM CaCl 2 5 mM e 15 mM HEPES a pH 7.4 , Glucosio 120 mM NaCl, KCl 5 mM, 1 mM MgCl 2, 2 mM CaCl 2 5 mM e 15 mM HEPES a pH 7.4 , Glucosio 125 mM KCl, 1 mM MgCl 2, 2 mM CaCl 2 5 mM e 15 mM HEPES a pH 7.4 </li></ol> 2.3) Misura della NHE Activity Reverse NOTA: Il principio è quello di invertire i gradienti ionici transmembrana. <ol><li> Prima della misura, caricare le cellule con il catione intracellulare di interesse (vedi sotto). </li><li> Incubare per 5 minuti con BCECF / AM (vedi punto 2.2.2); sciacquarle, montarli sul microscopio video. </li><li> Profumato la soluzione di carico e prendere più immagini per registrare una linea di base stabile. </li><li> Dopo la stabilizzazione della linea di base, immagine le variazioni di pH quando le cellule vengono perfusi con soluzione acida extracellulare contenente 120 mM cloruro di colina, 5 mM KCl, 1 mM MgCl 2, 2 mM CaCl 2, il glucosio 5 mm e 15 mm a MES pH 6.5. </li><li> Per LiCl carico, incubare le cellule per 2 ore in una soluzione composta di 120 mM LiCl, 5 mM KCl, 1 mM MgCl 2, 2 mM CaCl 2, 5 mM di glucosio e 15 mM HEPES a pH 7,4. Litio intracellulare misurata mediante spettroscopia ad assorbimento atomico produce un valore di circa 60 mM. </li><li> Per Na + carico, incubare le cellule, per due ore in una soluzione composta di 120 mM NaCl, KCl 5 mM, 1 mM MgCl 2, 2 mM CaCl 2, 5 mM di glucosio e 15 mM HEPES a pH 7,4 in presenza di 1 mM ouabain per bloccare la Na / K ATPasi. In queste condizioni, la concentrazione intracellulare Na + è nella gamma di 40 mM. NOTA: K + caricamento non è necessaria in quanto K + citosolico concentrazione nell&#39;intervallo 140 mM e un gradiente K + verso l&#39;esterno diretto è quindi facile da realizzare. </li></ol> 2.4) Il trattamento dei dati e calibrazione: NOTA: Questo passaggio si applica sia alle misure avanti e retromarcia. <ol><li> Al termine di ogni esperimento, profumato cellule con una soluzione di 140 mM KCl, 20 mM HEPES e 5 mM nigericin regolata a valori di pH tra 6,5 ​​e 7,4. </li><li> Raccogliere dati come misure di fluorescenza dalle immagini (livelli di grigio che rappresentano i livelli di intensità) e l&#39;esportazione in formato testo e trattare come spiegato di seguito con un programma di foglio di calcolo. </li><li> Calcolare i valori di pH intracellulare per ogni singola cella o regione di interesse utilizzando la seguente equazione: pH = pKa + (Log (R-Rmin) / (R max -R)) x F min (λ2) / F max (λ2) </li><li> Utilizzare l&#39;min F (λ2) / F ma x (λ2) rapporto se c&#39;è sonda sbianca o perdite, con conseguente diminuzione della fluorescenza a 450 nm durante gli esperimenti. Questo è comunque molto raramente osservata nelle condizioni utilizzate nel desEsperimenti Cribed. </li><li> Poiché sono presenti alla fine di ogni misurazione pH i dati di calibrazione, verificare se i corrispondenti valori calcolati differiscono dai valori di pH utilizzati per la calibrazione. Se è il caso, quindi regolare tutti i valori di pH sperimentalmente determinato alla calibrazione utilizzando la seguente procedura: <ol><li> Calcolare il seguente quoziente (Q, la differenza tra il valore misurato / differenza tra i valori di calibrazione). Moltiplicare l&#39;intero set di dati da Q. Effettuare la regolazione finale per addizione o sottrazione in modo che i valori calcolati saranno pari ai valori di taratura corrispondenti. </li></ol></li></ol> 3. Misurazioni di iniziali Tassi di NHE7 di veloce Li + assorbimento <ol><li> Cellule seme su lastre multiple e (da 6 a 24 pozzetti) e acidificare loro utilizzando la tecnica di NH 4 + carico descritto sopra o alternativamente la Nigericina / albumina di siero bovino (BSA) acidificazione descritto in 20. </li><li&gt; piastre Sciacquare due volte rapidamente utilizzando la soluzione di risciacquo sopra descritta e poi incubare le cellule in soluzioni di assorbimento contenenti 1-10 litio mm (leggermente al di sotto dei valori Km) e le concentrazioni desiderate di cationi e / o inibitori di interesse per l&#39;importo desiderato di tempo. </li><li> Mantenere brevi e coerenti durata di assorbimento (in genere un minuto o anche oltre) per garantire che il trasporto opera in tassi di condizioni iniziali. Assicurarsi inoltre che tutte le soluzioni utilizzate per l&#39;assorbimento è isotonico. </li><li> Alla fine del tempo di assorbimento, eliminare accuratamente il mezzo assorbimento e lavare le cellule quattro volte con soluzione salina fosfato ghiacciato tamponata (PBS). Eseguire questi lavaggi più velocemente possibile (meno di 10 sec per quattro di essi è ideale) per impedire litio efflusso. </li><li> Lyse le cellule del 25% di acido nitrico. Applicare 250 microlitri per pozzetto di 25% di acido nitrico e lasciate riposare per almeno 1 ora, poi rottami ogni pozzetto con l&#39;estremità della punta della pipetta e trasferire l&#39;intera sospensione bene in una nuova mitubo crocentrifuge. </li><li> Trasferire il lisato in provette da 1,5 ml microcentrifuga, li centrifugare per 5 minuti a 15.000 xg (temperatura ambiente) per rimuovere i detriti cellulari. </li><li> Misurare il contenuto di litio dei sopranatanti mediante spettroscopia ad assorbimento atomico 21. NOTA: Diverse aziende forniscono spettrometri di assorbimento atomico, che devono essere dotati di una batteria agli lampada a catodo cavo. Seguire le istruzioni del produttore in quanto queste macchine sono molto precise, ma anche molto delicato. </li></ol>

<ol>
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The authors have nothing to disclose.

Questo protocollo descrive come selezionare le cellule che esprimono intracellulare Na + / H + scambiatori a livello della membrana plasmatica, senza alterarne la sequenza primaria per mutagenesi sito-specifica. Questi scambiatori possono essere caratterizzati. Questo metodo si basa sulla tossicità cellulare di protoni intracellulari per selezionare linee cellulari che esprimono vescicolare Na + / H + scambiatori a livello della membrana plasmatic...

La maggior parte dei compartimenti intracellulari mostrano un pH luminale acido, che è un parametro fondamentale per la maturazione, il traffico, il riciclaggio di proteine ​​o ormoni e neurotrasmettitori loading. E &#39;stato dimostrato che il gradiente di pH tra contenuto citosol e vescicolare è generato da vacuolar H + ATPasi 1, accoppiato ad vescicolari trasportatori cloruro ClC 2. Sia in knock-out (KO) topi e pazienti umani, l&#39;importanza di questi trasportatori è stata evidenziata dai fenotipi pesanti causate da mutazioni nei loro geni 3-6. I membri della famiglia SLC9A scambiatori sodio-idrogeno, anche chiamati NHEs per Na + / H + scambiatori, hanno dimostrato di essere effettori chiave pH intracellulare e regolazione del volume cellulare, così come nel trasporto vettoriale di equivalenti acido-base di tutti gli epiteli . Oltre alle NHEs membrana plasmatica, tre altamente conservati Na + / H + scambiatori, NHE 6, 7e 9 sono espressi in rete trans-Golgi e in endosomi precoci 7. Le mutazioni nei loro geni sono stati associati con l&#39;autismo basato sulla famiglia 10 e Attention Deficit Hyperactivity Disorder Angelman-like o Christianson Sindromi 8-9 11-12. Questi scambiatori sono stati coinvolti in problemi neurodegenerative quali la malattia di Alzheimer suscettibilità 13 e collegata a X ritardo mentale geni contigui SINDROMI 14. Presi insieme, questi studi sottolineano l&#39;importanza di questi NHEs intracellulari di sviluppo e / o la funzione del cervello. La localizzazione intracellulare di questi scambiatori impedisce misurazioni accurate delle loro selettività ionica, direzione di trasporto, i parametri cinetici e regolazione. Come è il caso per tutti i trasportatori espressi in compartimenti intracellulari, è estremamente difficile valutare le loro attività biochimiche e quindi di comprendere appieno i loro ruoli fisiologici e meccanismi sottostanti le loro implicazioni patologiche. Sulla base della concentrazione citosolica K +, l&#39;ipotesi più comunemente accettata era che stavano lavorando come K + accoppiati protoni trasportatori di efflusso. L&#39;esistenza di tale perdita protonica era stato ipotizzato, come può controbilanciare protonica pompaggio dalle V-ATPasi al fine di mantenere uno stato stazionario vescicolare pH. Lo scopo di questo articolo visiva è (i) un metodo per dimostrare che permette la selezione genetica di linee cellulari che esprimono tali trasportatori vescicolari al loro membrana plasmatica, e (ii) mostrano due approcci indipendenti per misurare le funzioni di questi trasportatori. Tre decenni fa, Pouysségur e Franchi hanno sperimentato un approccio genetico che ha permesso la clonazione molecolare e la caratterizzazione dei membri della famiglia NHE 15. Questa era basata sulla tossicità di protoni intracellulari come metodo di screening. Il primo passo è stato quello di ottenere linee di cellule deficientiin qualsiasi / H scambio Na + + espresso a livello della membrana plasmatica, utilizzando la reversibilità di questo trasportatore. I fibroblasti (linea cellulare CCL39) sono stati precaricati con Na + o Li + e poi poste in un mezzo extracellulare acido (pH 6,5) per 2 ore. Ciò ha portato alla morte di cellule esprimenti un funzionale Na + / H + scambio e la selezione di celle antiporter-deficienti (linea cellulare PS120) 16. Quando coltivate in terreno privo di bicarbonato, queste cellule sono molto sensibili all&#39;acidificazione intracellulare acuta. Di conseguenza, l&#39;espressione di un meccanismo di protoni efflusso funzionale a livello della membrana plasmatica sarà selezionato positivamente (vedi 17) se tali cellule sono sottoposte a acidificazioni intracellulari acute. Tali tecniche acidificazione possono essere utilizzati per isolare linee cellulari con traffico difetti che consentono l&#39;espressione forzata di NHEs intracellulari WT a livello della membrana plasmatica. Come eucarioti Na + / H+ Scambiatori sono elettricamente neutro, non sono misurabili dagli approcci elettrofisiologici che sono stati utilizzati con successo per misurare canali. Questo manoscritto dimostra quindi come misurare l&#39;attività di questo scambiatore per misure di pH intracellulari e rapidi cinetica di assorbimento di litio. Poiché i concetti di base sono gli stessi, è interessante notare che molti dei processi sviluppati per la sezione di selezione vengono anche utilizzate direttamente per misurazioni funzionali. È interessante notare, abbiamo osservato che il difetto traffico presente nelle linee cellulari selezionate utilizzando l&#39;approccio descritto in questo manoscritto porta ad una maggiore espressione di altre proteine ​​vescicolari alla membrana plasmatica, come il canale del potassio vescicolare TWIK1 18. Questo indica verso la selezione di un meccanismo di ritenzione difetto generale per proteine ​​transmembrana vescicolari. Quindi questa procedura di selezione e le cellule che genera maggiocostituiscono uno strumento promettente per la comunità scientifica lavorare sulle proteine ​​di membrana di compartimenti intracellulari. Come pure le tecniche di misurazione qui presentate potrebbero essere applicabili per studiare altri trasportatori non elettrogeniche.

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functional characterization of na+/h+ exchangers of intracellular compartments using proton-killing selection to express them at the plasma membrane

Acidificazione endosomali è critica per una vasta gamma di processi, quali il riciclaggio proteica e la degradazione, desensibilizzazione del recettore, e neurotrasmettitore caricamento in vescicole sinaptiche. Questo acidificazione è descritto per essere mediato da ATPasi protonica, insieme ai trasportatori di cloruro CLC. Altamente conservata protoni elettricamente trasportatori, la Na + / H + scambiatori (NHE) 6, 7 e 9 sono espresse anche in questi comparti. Le mutazioni nei loro geni sono stati collegati con le malattie cognitive e neurodegenerative umane. Paradossalmente, i loro ruoli rimangono elusivi, come la loro localizzazione intracellulare ha impedito dettagliata caratterizzazione funzionale. Questo manoscritto mostra un metodo per risolvere questo problema. Questo consiste nella selezione di linee cellulari mutanti, capaci di sopravvivere acidificazione citosolica acuta mantenendo NHEs intracellulari alla membrana plasmatica. Quindi raffigura due protocolli complementari per misurare la selettività e attività ionicadi questi scambiatori: (i) una basata su misurazioni di pH intracellulare mediante videomicroscopia a fluorescenza, e (ii) uno basato sulla cinetica veloci di litio assorbimento. Tali protocolli possono essere estrapolati per misurare altri trasportatori non elettrogeniche. Inoltre, la procedura di selezione qui presentata genera cellule con fenotipo ritenzione intracellulare difettoso. Pertanto queste cellule esprimono anche altre proteine ​​di membrana vescicolare a livello della membrana plasmatica. La strategia sperimentale descritta qui può quindi costituire uno strumento potenzialmente efficace per studiare altre proteine ​​intracellulari che saranno poi espressi a livello della membrana plasmatica insieme con la vescicolare Na + / H + scambiatori usati per la selezione.

Selezione: La selezione + uccidendo H si basa sulla diffusione della ammonio base debole, come illustrato nella Figura 1A. L&#39;effetto di basi deboli e acidi diffusione sul pH intracellulare è stata introdotta da Walter Boron e collaboratori 22. L&#39;elegante idea di utilizzare questo fenomeno per la produzione di una acidificazione letale per la selezione genetica positiva è stata poi sviluppata da Jacques Pouysségur 17. Sotto tale pro...

Watch this Scientific Journal Video about Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane at JoVE.c

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Caratterizzazione funzionale di Na<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt; Scambiatori di intracellulare compartimenti, utilizzando Proton-uccidere selezione di esprimerle livello della membrana plasmatica

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

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JoVE Journal

Biology

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

9.2K Views.  Université Nice-Sophia Antipolis, Laboratoire de Physiomédecine Moléculaire, CNRS UMR7370, and Laboratories of Excellence Ion Channel Science and Therapeutics. The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers. 

Video: Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

Misura Vicino membrana plasmatica e Global Dynamics calcio intracellulare negli astrociti

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Ricerca

Biologia

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Cristallizzare proteine ​​di membrana per la determinazione della struttura utilizzando Mesophases lipidica

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

Misurazione quantitativa della traslocazione GLUT4 sulla membrana plasmatica mediante citometria di flusso

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

Identificare gli effetti delle mutazioni BRCA1 sulla ricombinazione omologa utilizzando cellule che esprimono endogeno wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

L&#39;utilizzo di un robot per High-throughput cristallizzazione delle proteine ​​di membrana in mesofasi lipidici

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

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.

Cristalli di raccolta e di Cryo-raffreddamento delle proteine ​​di membrana Grown in mesofasi lipidici per la determinazione della struttura di cristallografia macromolecolare

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

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Intravitale Microscopia per le strutture Imaging subcellulari in topi vivi Esprimere proteine ​​fluorescenti

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

La base di Cell-L-Glutatione Protezione saggi per studiare endocitosi e riciclo dei Plasma proteine ​​di membrana

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

Imaging Plasma membrana Deformazioni Con pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Indagando i dannosi effetti di basso pressione sterilizzazione al Plasma sulla sopravvivenza di Bacillus subtilis spore utilizzando Live Cell microscopia

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...