Gli autori desiderano ringraziare la piattaforma Montpellier Rio Imaging (MRI) in cui sono state effettuate le acquisizioni. Ringraziano Isabel Cristina Lopez Mejia, Alexandra Metz, Irina Lassot, Solange Desagher, Fabien Loustalot, e Virginie Georget per il loro aiuto in diverse parti del protocollo. Questo lavoro è stato sostenuto dalla Fondation pour la Recherche Médicale (FRM) (Equipe FRM 2011-n ° DEQ20111223745).

Tutte le procedure sugli animali in questo protocollo sono in stretta aderenza con le linee guida della Direttiva del Consiglio della Comunità Europea del 24 novembre 1986 (86-609/EEC). Casa dei topi in gruppo, permettendo cibo e acqua ad libitum. Mantenerle in un ambiente controllato (22 ± 1 ° C, 55 ± 5% di umidità), con una 12 ore: 12 ore di luce: ciclo di buio (luce alle 07:00). 1 Culture di cellule murine primari:. Fibroblasti embrionali di topo (MEF) <ol><li> Autoclave pinze sottili, così come pinze curve e forbici dissezione. Conservare in un contenitore sterile. Utilizzare sterile D-PBS (Dulbecco Phosphate-Buffered Saline). Preparare mezzo completo MEF: modificata mezzo Eagle Dulbecco (DMEM) / F12 supplementato con 10% di siero fetale bovino (FBS), 1 mM L-glutammina, 1 mM piruvato di sodio, 1% di amminoacidi non essenziali, e 0,5 mM 2-mercaptoetanolo e caldo a 37 ° C. </li><li> Euthanize un topo in stato di gravidanza (a 13,5 giorni post-coitum (DPC)) Per dislocazione cervicale. Rimuovere le corna uterine dal mouse igienizzato con il 90% EtOH. Sotto una cappa sterile e pulito, mettere le corna con gli embrioni nel 37 ° C preriscaldata D-PBS. Aprire la corna, posto gli embrioni in una plastica sterile capsula di Petri 100 mm, li ripulire dal materiale cordone ombelicale e lavarli con acqua calda D-PBS per rimuovere l&#39;eccesso di sangue. Sotto un binocolo, rimuovere gli arti dell&#39;embrione, gli organi interni e la parte superiore della testa contiene il cervello. </li><li> In un nuovo piatto di Petri, tritare il embrioni in pezzi molto piccoli con un chirurgico sterile lama o forbici (almeno per 1 min). Nel caso di embrioni di diversi genotipi, stare attenti a mettere ogni embrione nelle singole piastre di Petri e mantenere la coda o la testa superiore per la genotipizzazione. </li><li> Coprire l&#39;embrione con 1x tripsina-EDTA (0,25% tripsina, 1 mM EDTA). Incubare per 30 minuti a 1 ora a 37 ° C incubatore. Aggiungere 10 ml di completoMEF mezzo per fermare la reazione tripsina. Pipettare su e giù per rimuovere tutti gli aggregati. Lasciare riposare la sospensione cellulare in una provetta 50 ml (riempito con mezzo completo) per 10 min, e centrifugare il surnatante per 5 minuti a 300 xg a temperatura ambiente. Risospendere il pellet in mezzo completo (6 ml per 1 embrione). Cellule nucleate vitali possono essere contati utilizzando trypan blu (Circa 5 x 10 6 cellule può aspettare da un embrione). Piatto 3 ml per 60 millimetri piatto di Petri. </li><li> Cambiare il mezzo il giorno successivo e permettono alle cellule di crescere fino a quando i piatti sono confluenti. Molti tipi di cellule possono essere visti, ma solo fibroblasti sopravviverà sottocultura. </li><li> Dividere le celle e permettono loro di crescere in 35 piatti mm con fondo di vetro (importante per esperimento lasso di tempo), fino al 50-70% di confluenza per la trasfezione. Test per Mycoplasma e agenti patogeni mouse. </li></ol> 2. Culture Adaptations nel caso di neuroni <ol><li> Il giorno prima la cultura, cappotto di 35 millimetri di vetro fondo piastre di Petri con poli-L-lisina (200 ml di 0,1 mg / ml) sotto una cappa sterile e pulito, e lasciare tutta la notte. </li><li> La mattina successiva, sciacquare con acqua pura sterile, due volte per 5 min e una volta per 45 minuti a 1 ora. Sostituire con 2 ml di DMEM + 10% FBS medie e tenere in un incubatore a 37 ° C. </li><li> Sezionare embrioni a 18,5 dpc. Sotto una cappa sterile e pulito, mettere le corna con gli embrioni in freddo sterile HBSS (soluzione salina bilanciata di Hank) a 100 millimetri piatto di Petri. Cuccioli neonatali possono anche essere utilizzati al posto di embrioni al fine di preservare la durata della diga e consentirgli di produrre più discendenti, soprattutto nel caso di animali transgenici che possono essere difficili da ottenere. Nei singoli piatti Petri, prendere ogni embrione o neonato e tagliare la testa con le forbici. Tenere la testa inserendo pinze curve negli occhi, tagliare la pelle e aprire con cautela il cranio molle dal retrodella testa fino agli occhi, su ciascun lato della testa. Tagliare i nervi ottici ed il tronco cerebrale, rimuovere il cervello, e metterlo in un nuovo piatto di Petri contenente HBSS. Sotto uno stereoscopio, rimuovere tutte le meningi con due pinze sottili. Separare ippocampi, la corteccia o qualsiasi altra parte del cervello in funzione della struttura di studiare. </li><li> Immergere la struttura del cervello sezionato in 4,5 ml di HBSS freddo preparato in precedenza in provette da 15 ml e tenere in ghiaccio fino digestione con tripsina. Aggiungere 0,5 ml di 2,5% tripsina e incubare a 37 ° C per 15 min a 20 min. Sciacquare il 3x tripsina con HBSS, essere estremamente attenti a non scartare le parti del cervello digeriti. </li><li> Risospendere in 500 microlitri (ippocampo) a 1 ml (corteccia) DMEM + 10% FBS e pipettare su e giù parecchie volte con un ml micropipetta 1 dotato di una punta di 1 ml, quindi dotato di 1 ml più 200 consigli microlitri, finché non ci sarà nessun aggregato visibile. </li><li> Distribuire 100-200 ml di sospensione cellulare ad ogni botto vetro 35 millimetrim piatto contenente DMEM + 10% FBS e lasciare che i neuroni aderiscono a 37 ° C per almeno 3 ore. Sostituire con neurone preriscaldata mezzo completo (Neurobasal mezzo supplementato con 250 mM L-glutammina e NS21, preparato come descritto in Chen et al. 19) e lasciato a 37 ° C per permettere la crescita neuronale. Transfettare i neuroni a 5 a 14 giorni in vitro (div) (l&#39;efficienza di trasfezione è più elevato dopo un paio di connessioni sinaptiche div, ma sono meglio stabilito 7-10 div). </li></ol> 3. Trasfezione di EGFP-G3BP1 Construct Trasfezione delle cellule con un vettore contenente il cDNA della proteina di interesse (qualsiasi componente di SGS) fuso con un marcatore fluorescente (GFP, YFP, ecc), utilizzando 3 pg di plasmide purificato per piastra da 35 mm. <ol><li> Transfettare il MEF utilizzando un metodo commerciale, seguendo il protocollo del produttore (Vedi Tabella dei materiali / reagenti). </li><li> Transfettare i neuroni con un calcioMetodo di fosfato adattato da Xia et al 20 brevemente.: <ol><li> Preparare le soluzioni: DMEM-wash: DMEM contenente 25 mM KCl; soluzione di trasfezione: DMEM-lavaggio contenente 1x DMKY (HEPES 5 mM, MgCl2 10 mM, rosso fenolo); e la soluzione choc: HeBS 1x, DMKY 1x, e DMSO 2% (v / v); e tenerli a 37 ° C. </li><li> Rimuovere il supporto dai neuroni, filtrato, e la tiene a 37 ° C. Lavare con DMEM-wash quindi sostituire con terreno di trasfezione e mantenere a 37 ° C durante la preparazione dei calcio fosfato plasmide precipitati DNA. </li><li> In una provetta da 1,5 ml, aggiungere (in questo ordine) Braun acqua (volume finale di 50 ml), 5 ml di CaCl 2 2,5 M e 3 microgrammi di plasmide DNA. Cadere questo mix su 50 ml di HeBS 2x già introdotti in un tubo tondo fondo in polipropilene. Mescolare il tubo dalla rotazione insieme con l&#39;eliminazione. Lasciare la forma precipitato a temperatura ambiente durante 30 min. </li><li> Aggiungere il precipitate goccia a goccia sui neuroni e lasciare a 37 ° C durante 30 a 50 min. </li><li> Sostituire con una soluzione shock per 1 minuto, quindi risciacquare con DMEM-wash e, infine, reintrodurre il mezzo precondizionato. Conservare le cellule a 37 ° C. </li></ol></li></ol> 4. Visualizzazione G3BP contenenti SGs Montaggio e Dynamics <ol><li> Sostituire il medium delle cellule che contiene rosso fenolo da terreno privo di rosso fenolo. </li><li> Utilizzare un microscopio confocale equipaggiato con fluorescenza per le acquisizioni. Accendere i sistemi microscopio: lampada a mercurio, computer e laser. Riscaldare la camera a 37 ° C per almeno 20 minuti prima dell&#39;inizio delle acquisizioni. </li><li> L&#39;espressione sopra del G3BP induce la formazione spontanea di un tipo di SGS. Le cellule possono quindi essere osservati per l&#39;assemblaggio di SGs 24 ore dopo la trasfezione, senza induzione di stress. In alternativa, lo stress può essere indotto a indurre ulteriormente l&#39;assemblaggio della SGS. Aggiungere 0,5 mM arsenito di sodio e di stellet dell&#39;acquisizione subito dopo l&#39;aggiunta del composto (granuli saranno ben formate entro 1 ora). </li><li> Aggiungere olio l&#39;obiettivo ad immersione (usare 40X o 63X obiettivi) e installare la piastra di Petri rispetto all&#39;obiettivo nel supporto microscopio 35 millimetri adattato, stabilizzato sul supporto palco. Verificare che il piatto è piatto altrimenti le acquisizioni verranno alterati. Accendere la luce di fluorescenza secondo il fluoroforo rilevante e visualizzare le cellule trasfettate. Spegnere fluorescenza volta che una cellula di interesse nel settore al fine di minimizzare il candeggio e citotossicità. </li><li> Nella &quot;acquisire scheda&quot;, selezionare i laser in funzione della lunghezza d&#39;onda di eccitazione del fluoroforo tag (488 nm nel nostro protocollo che utilizza G3BP EGFP-tag) e seleziona la lunghezza emissione adattato per la PMT (tubo fotomoltiplicatore). Regolare la potenza del laser (di solito non più del 10%) per ridurre il rumore e sovrasaturazione nonché tossicità, e impostare il guadagno e offset di modificare il rapporto segnale-rumore.Scansione veloce (1,000 Hz), al fine di minimizzare la durata di esposizione laser (line media 2, media telaio 1). Se lo si desidera, impostare i parametri per la pila Z per poter ricostruire l&#39;immagine in 3D (una fase Z di 1 micron di solito va bene con le cellule). Determinare l&#39;intervallo di tempo tra ogni acquisizione: intervalli più piccoli permettono di ottenere un film più coerente ma questo può indurre fotoscolorimento e tossicità (un intervallo di 20 secondi è stato usato negli esperimenti descritti). Durata di acquisizione totale può variare a seconda del tipo di stress. Molti SGS-G3BP contenente si formano molto rapidamente sotto trattamento arsenito e sono ben visibili dopo 1 ora di trattamento: in tal caso, scegliere rapidamente le cellule per filmare e avviare le acquisizioni a destra dopo lo stress, con una durata complessiva di acquisizioni di 15 - 20 min ampiamente sufficiente per osservare l&#39;assemblaggio di diversi SGS. </li><li> Salvare le immagini e ricostruire le pile e film utilizzando il software ImageJ. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

I passaggi più critici del protocollo riguardano la trasfezione e più in particolare le acquisizioni lasso di tempo, che devono essere attentamente monitorati al fine di abbassare la citotossicità. Coltura di cellule primarie non è una parte difficile, finché le condizioni sterili sono mantenute e cautela è presa per evitare danni durante le fasi di dissezione e dissociazione cellulare. MEF può essere mantenuto congelato alle brevi passaggi. Neuron trasfezione con fosfato di calcio ha...

Granuli di stress (SGS) sono non-membranosa foci citoplasmatica formata come una risposta protettiva cellulare a stress ambientali, come la temperatura elevata, stress ossidativo, ipossia, shock osmotico, irradiazione UV, deprivazione di glucosio, o infezione virale 1. Essi possono essere indotti chimicamente mediante trattamento con composti come arsenito di sodio, che innesca lo stress ossidativo. SG accumulano in fase di stallo traduzione arrestato messaggero ribonucleoproteina (mRNPs) Complessi 2, sequestrando mRNA dal macchinario traslazionale, e il loro montaggio può essere attivato dalla fosforilazione di eIF2α (fattore di iniziazione eucariotica 2 α). SG sono strutture dinamiche che scambiano i componenti con le polisomi e altri granuli come il P-corpi. Essi costituiscono un &quot;centro di smistamento&quot;, dove mRNA sono ordinati ed elaborati sia per la traduzione, reinitiation, il degrado o l&#39;imballaggio in stabili mRNPs non polisomi 3. L&#39;assemblea di SGS è veloce but è un processo graduale, con iniziali numerosi piccoli aggregati che si coagulano in granuli più grandi. L&#39;uso di inibitori chimici che interrompono o stabilizzano i microtubuli mostra che è necessaria rete di microtubuli per dinamiche SG comprendenti processi di assemblaggio, di coalescenza e smontaggio. L&#39;assembly dinamico di SGS è anche favorito dalla aggregazione di specifici RNA-binding proteins (RNA-BP) come TIA-1 (T-cellulare interna antigene-1) e TIAR (proteina TIA-1-correlati), che sono in grado di dimerize e promuovere polisoma smontaggio e l&#39;instradamento di mRNA in SG 4. G3BP (RasGAP SH3 dominio di binding protein) è tale un RNA-BP che localizza a SGS quando le cellule sono stressati con arsenito o ad alta temperatura e sovraespressione di G3BP defosforilato può indurre SG gruppo 5. G3BP è evolutivamente conservata RNA-BP che è stato inizialmente caratterizzato attraverso la sua interazione con una proteina attivazione Ras-GTPasi (RasGAP p120 6); ma questa interazione è stato recentemente rivisitato 7. La famiglia G3BP comprende due membri nei mammiferi, G3BP1 (denominato G3BP) e G3BP2 8. Entrambe le proteine ​​colocalize in SGS, quando le cellule sono sottoposte a stress 9. G3BPs comprendono un dominio NTF2 N-terminale suggerito di influenzare la loro localizzazione e oligomerizzazione, seguiti da prolina ricchi (PxxP) motivi, quindi motivi C-terminale associato con RNA vincolante: il canonico RNA-Riconoscimento Motif (RRM) con RNP1 e RNP2 motivi conservati , seguita da un ricco casella arginina-glicina (RGG). È interessante notare che l&#39;analisi delle diverse parti della proteina creando diversi domini fuse ad EGFP dimostrato che il dominio NTF2-simili e il dominio legante l&#39;RNA erano i più efficientemente assunto per SGS, suggerendo l&#39;importanza delle proprietà di dimerizzazione e RNA vincolante l&#39;assemblea di SGS. Diversi modelli hanno rivelato diverse funzioni delle proteine ​​G3BP in vitro 10-13.Interruzione del G3BP nei topi hanno dimostrato l&#39;importanza di questa proteina in crescita dello sviluppo e sopravvivenza 14, così come un ruolo importante per G3BP nel Sistema Nervoso Centrale (SNC), caratterizzata da atassia e difetti nella memoria di lavoro spaziale 15. Carenza G3BP porta ad alterata plasticità neuronale e calcio omeostasi, stabilendo un collegamento diretto tra la formazione di SG e le malattie neurodegenerative 15. È quindi importante poter studiare la dinamica dello SGS in cellule primarie come neuroni. Questo protocollo fornisce un modo semplice per osservare l&#39;assemblaggio di-G3BP1 contenente SG in cellule primarie in trattamento arsenite. Può essere usato per studiare assembly SGs in condizioni diverse, ad esempio diversi tipi di stress. Può anche essere adattato ad altri granuli o altri costituenti del SGS. Infatti, questo protocollo si concentra sulla G3BP1, ma ci sono altri granuli di stress marcatori come TIA-1 / R, TTP (tristetraprolin) 2,FMRP (fragile X ritardo mentale Sindrome di proteine) 16, TDP-43 (DNA risposta transattivo binding protein 43) 17 o Staufen 18. In particolare, proteine ​​come TIA-1 / R sono, come G3BP, nucleazione RNA proteine ​​leganti che possono indurre assembly SGs quando overexpressed, anche se le varie SGs formate possono differire in funzione, regolazione e trascrizioni associati. Trasfezione versione fluoroforo-tag di una di queste componenti chiave o nucleatori di SG può essere eseguita per l&#39;immagine particolare assemblaggio e la dinamica SG.

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			<td height="21" style="height:21px;width:297px;">Name of Material/ Equipment</td>
			<td style="width:153px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:312px;">Comments/Description</td>
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			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:297px;">DMEM/F12</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">21331</td>
			<td>Pre-warm at 37 &#176;C</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Sodium Pyruvate</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">11360</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Non essential amino acids</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">11140</td>
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			<td height="21" style="height:21px;width:297px;">L-Glutamine</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">25030</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Trypsin</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">15096</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Glass bottom B-35</td>
			<td style="width:153px;">Greiner</td>
			<td style="width:119px;">627860/627861</td>
			<td>Treated or not (treated: increases attachment of adherent cells)</td>
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			<td height="21" style="height:21px;width:297px;">Poly-L-lysine</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2636</td>
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			<td height="21" style="height:21px;width:297px;">DMEM</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">31966</td>
			<td>Pre-warm at 37 &#176;C</td>
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			<td height="21" style="height:21px;width:297px;">HeBS</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">51558</td>
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			<td height="21" style="height:21px;width:297px;">Neurobasal</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">21103</td>
			<td>Pre-warm at 37 &#176;C</td>
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			<td height="21" style="height:21px;width:297px;">2-mercaptoethanol</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">31350</td>
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			<td height="21" style="height:21px;width:297px;">Forceps</td>
			<td style="width:153px;">Biotek</td>
			<td style="width:119px;">DU-110-A</td>
			<td>Very thin, tips 0,1 mm (useful to remove meninges)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Curved forceps</td>
			<td style="width:153px;">Biotek</td>
			<td style="width:119px;">P-110-BUF</td>
			<td>Very thin, tips 0,1 mm&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Small scissors</td>
			<td style="width:153px;">Biotek</td>
			<td style="width:119px;">CM-85-BS</td>
			<td>Can be useful to remove hippocampi</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Polyplus transfection JetPEI reagent&#160;</td>
			<td style="width:153px;">Ozyme</td>
			<td style="width:119px;">101-10</td>
			<td>MEFs transfection, follow the forward protocol</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Inverted laser scanning confocal microscope</td>
			<td style="width:153px;">Leica&#160;</td>
			<td style="width:119px;">SP5</td>
			<td></td>
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visualization of g3bp stress granules dynamics in live primary cells

SGs può essere visualizzato in cellule mediante immunocolorazione delle componenti proteici specifici o polyA + mRNA. SG sono altamente dinamico e lo studio del loro assemblaggio e il destino è importante per capire la risposta cellulare allo stress. Il deficit dei fattori chiave della SG come G3BP (RasGAP SH3 dominio Binding Protein) porta a difetti dello sviluppo in topi e alterazioni del Sistema Nervoso Centrale. Per studiare la dinamica di SG in cellule di un organismo, si può cellule primarie di cultura e seguire la localizzazione di un componente etichettato transfettate di SGS. Descriviamo esperimento di time-lapse per osservare-G3BP1 contenente SG in fibroblasti embrionali di topo (MEF). Questa tecnica può anche essere usato per studiare SGS-G3BP contenente neuroni in vivo, che è cruciale come è stato recentemente dimostrato che questi SGs si formano alla insorgenza di malattie neurodegenerative come il morbo di Alzheimer. Questo approccio può essere adattato a qualsiasi altro componente corpo cellulare e proteine ​​granuli, ed eseguitocon animali transgenici, permettendo lo studio vivo di granulati dinamiche per esempio in assenza di un fattore specifico di questi granuli.

Formazione stress granulo è importante nella risposta delle cellule allo stress, permettendo un adattamento cellulare con traduzione in stallo, finché lo stress ha autorizzato, associata alla prevenzione dell&#39;apoptosi. Permette questo test per studiare SGS in cellule primarie, seguendo la localizzazione delle principali componenti proteiche SGS (Figura 1). G3BP, un fattore chiave di montaggio SGS, è presente piuttosto diffusamente nel citoplasma di MEF coltivate o neuroni (Figura 2A A</st...

Watch this Scientific Journal Video about Visualization of G3BP Stress Granules Dynamics in Live Primary Cells at JoVE.com

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

Granuli di stress (SGS) sono granuli citoplasmatici RNA contenenti particelle in fase di stallo ribonucleoproteiche (RNP), e importante nella risposta cellulare alle varie sollecitazioni. Dinamica di SG possono essere seguite in cellule vive visualizzando la localizzazione di un componente taggato di SG in cellule primarie transfettate dopo stress.

Visualizzazione dei G3BP stress Granuli Dynamics in cellule primarie in diretta

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly ...

visualization-of-g3bp-stress-granules-dynamics-in-live-primary-cells

Research

JoVE Journal

Biology

18.2K Views.  Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535.  Granuli di stress (SGS) sono granuli citoplasmatici RNA contenenti particelle in fase di stallo ribonucleoproteiche (RNP), e importante nella risposta cellulare alle varie sollecitazioni. Dinamica di SG possono essere seguite in cellule vive visualizzando la localizzazione di un componente taggato di SG in cellule primarie transfettate dopo stress. 

Video: Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

Una tecnica rapida per la visualizzazione di Live cellule di lievito immobilizzato

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

Ricerca

Biologia

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

Immagini dal vivo di Dense-core Vescicole nella Primaria neuroni coltivati ​​ippocampale

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Isolamento e la coltura primaria di cellule epatiche di ratto

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

Visualizzazione di mRNA reticolo endoplasmatico localizzate in cellule di mammifero

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

4D Imaging di aggregazione proteica in cellule vive

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Visualizzazione del reticolo endoplasmatico sottodomini in cellule in coltura

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Un&#39;interfaccia utente grafica per il monitoraggio assistito della concentrazione di proteine ​​in protrusioni cellulari dinamiche

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to stress conditions, are implicated in various aspects of cell metabolism and survival. SGs modulate cellular signaling pathways, post-transcriptional gene expression, and stress response programs. The formation of these mRNA-containing granules is directly connected to cellular translation. SG assembly is triggered by inhibited translation initiation, and SG disassembly is promoted by translation activation or by inhibited translation elongation. This relationship is further highlighted by SG composition. Core SG components are stalled translation pre-initiation complexes, mRNA, and selected RNA-binding Proteins (RBPs). The purpose of SG assembly is to conserve cellular energy by sequestering translationally stalled housekeeping mRNAs, allowing for the enhanced translation of stress-responsive proteins. In addition to the core constituents, such as stalled translation preinitiation complexes, SGs contain a plethora of other proteins and signaling molecules. Defects in SG formation can impair cellular adaptation to stress and can thus promote cell death. SGs and similar RNA-containing granules have been linked to a number of human diseases, including neurodegenerative disorders and cancer, leading to the recent interest in classifying and defining RNA granule subtypes. This protocol describes assays to characterize and quantify mammalian SGs.

Stress Granules (SGs) are nonmembranous cytoplasmic structures that form in cells exposed to a variety of stresses. SGs contain mRNAs, RNA-binding proteins, small ribosomal subunits, translation-related factors, and various cell signaling proteins. This protocol describes a workflow that uses several experimental approaches to detect, characterize, and quantify bona fide SGs.

Metodi per classificare i foci citoplasmatici come granuli di stress di mammiferi

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to ...

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.

Recently we mapped the three-dimensional (3D) spatial locations of transport routes for various proteins translocating inside primary cilia in live cells. Here this paper details the experimental setup, the process of biological samples and the data analyses for the 3D super-resolution fluorescence imaging approach newly applied in live primary cilia.

Applicazione di microscopia di velocità ad alta velocità super-risoluzione in Live cilio primario

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear positioning occurs during skeletal muscle differentiation. Muscle fibers (myofibers) are multinucleated cells formed by the fusion of muscle precursor cells (myoblasts) derived from muscle stem cells (satellite cells) that undergo proliferation and differentiation. Correct nuclear positioning within myofibers is required for the proper muscle regeneration and function. The common procedure to assess myoblast differentiation and myofiber formation relies on fixed cells analyzed by immunofluorescence, which impedes the study of nuclear movement and cell behavior over time. Here, we describe a method for the analysis of myoblast differentiation and myofiber formation by live cell imaging. We provide a software for automated nuclear tracking to obtain a high-throughput quantitative characterization of nuclear dynamics and myoblast behavior (i.e., the trajectory) during differentiation and fusion.

Skeletal muscle differentiation is a highly dynamic process, which particularly relies on nuclear positioning. Here, we describe a method to track nuclei movements by live cell imaging during myoblast differentiation and myotube formation and to perform a quantitative characterization of nuclei dynamics by extracting information from automatic tracking.

Sfruttando Live Imaging ai Nuclei di pista durante il differenziamento dei mioblasti e Fusion

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear ...