Vorremmo ringraziare Andrea Kremsreiter per l&#39;eccellente assistenza tecnica. Il lavoro nel nostro laboratorio è supportato da NIH GM096088 e Vilas fiducia della University of Wisconsin - Madison.

1. Metodi di lievito luminometria Utilizzare ceppo BYYT di Saccharomyces cerevisiae. Si tratta di un yvc1-tok1-derivato di BY4741 (Mata, ura3D0, his3D1, leu2D0, lys2D0, yvc1 :: HIS3, tok1 :: kanMX4). Trasformare cellule con i-equorina esprimendo pEVP1/Aeq plasmide Leu-selezionabili e ura selezionabili rat--TRPV4 esprimendo plasmide p416GPDV4, come descritto in Batiza et al. 26 e Loukin et al. 24 Con metodi standard di costruzione plasmide e trasformazione 27 . Ceppo di lievito e plasmidi sono disponibili su richiesta. <ol><li> Cultura 2 ml di cellule di lievito durante la notte a fase post-logaritmica in uno shaker 30 ° C in leu-ura-&quot;DCD&quot; dropout medio 28 (una variante solfato-impoverito del mezzo CMD-leu-ura 29), integrato con 1M sorbitolo . Seminare 200 ml di queste culture in 1,8 ml di mezzo fresco integrato con 2 micron del Luciferin coelenterazina. Crescere al buio a temperatura ambiente per altre 24 ore senza agitare. </li><li> Misurare la osmolarità della cultura (tipicamente a 1.400 mOsm) e altre soluzioni (sotto) usando un osmometer pressione di vapore. </li><li> Aliquota 20 ml di coltura fresca in un tubo luminometro 12 mm per un singolo luminometro tubo. </li><li> Per lo shock ipotonico, aggiungere 200 ml di una soluzione contenente 25 NaEGTA mM (pH 7,2) o nomi (pH 7.2) e 500-100 mM NaCl, (osmolarità totale di 1,200-400 mOsm) a seconda del grado di shock desiderato. Monitorare costantemente la luminescenza prima e almeno 120 secondi dopo la downshock osmotica, interrotta solo dalla breve operazione di diluizione. Registrati uscita luminometer su un computer desktop come unità di luminescenza relativa (RUL). </li></ol> Anche se non per lo studio di TRPV4 transgenico, abbiamo utilizzato una variante del protocollo di cui sopra in un sistema automatizzato per esaminare le risposte di un gran numero di ceppi di lievito. Studio of risposte alle ipo-30 o iperosmotico 31 stimoli del deletome lievito (la raccolta di 4.906 lievito deletants un singolo gene) usando un luminometro micropiastre e analizzati con il software corrispondente ha avuto successo. 2. e 3. Ovociti Metodi di Elettrofisiologia Elettrofisiologia utilizza metodi di base 32. PCR amplifica la cornice open-lettura della wild-type o TRPV4 mutante con alta fedeltà PfuUtra polimerasi e integrato nel pGH19 33. Ciò comporta un inserimento preciso della ORF tra i 5 &#39;e 3&#39; UTR del Xenopus β-globina ed a valle di un promotore T7 RNA polimerasi. Linearizzare il costrutto valle UTR in 3 &#39;e usato come modelli in vitro un T7 RNA polimerasi in reazione. Utilizzare tecniche di biologia molecolare standard di 34. Utilizzare fase V-VI ovociti da X. laevis. Zootecnia, ovariecto parzialela mia, la rimozione busta defolliculation, e vitellina seguire procedure standard 32,33,35. Iniettare 2-40 ng di soluzione di cRNA per ovocita con un microinjector automatico Drummond Nanoinject II. Iniettare ~ 30 ovociti per ciascuna serie di esperimenti. Dopo l&#39;iniezione TRPV4-cRNA, incubare gli ovociti nel medio ND96 con gentamicina (100 ug / ml) e 1 mM rosso rutenio 14. 2. Due elettrodi di tensione morsetto <ol><li> Tirare borosilicato pipette di registrazione vetro dalla precisione monouso Micropipette 100 microlitri individualmente con un estrattore pipetta. </li><li> Pull sia la tensione di misura ed elettrodi pipette correnti-iniezione sono tirato avere un&#39;apertura punta di circa 1 micron di diametro. </li><li> Elettrodi recupero informazioni con 2 M KCl conseguente 0,1-0,2 resistenza mW. Li montano sul headstages HS2A, che insieme ad un virtuale di messa a terra clamp bagno VG-2Ax100, sono collegati ad una interfaccia amplificatore GeneClamp 500 attraverso un Digidata 1440 digitalizzatore. Acquisire<html> dati utilizzando il software pClamp10. </li><li> Mettere l&#39;ovocita da testare in un bagno di 1 ml in una camera di plastica fabbricato montato sul palco di un microscopio da dissezione con 20X di ingrandimento. La soluzione del bagno è virtuale a terra da un ponte agar 3 M KCl e un filo di argento clorurato collocati in prossimità del ovocita e collegato ad una fase terra virtuale VG-2A. </li><li> Costruire la camera di perfusione continua con tubi di plastica come soluzione di ingresso e di uscita. Collegare il tubo di ingresso è collegato ad un sistema di perfusione fabbricato, che è un collettore di sei conchiglie siringa 50 ml, riempiti ognuno con una soluzione differente. Velocità di avanzamento di gravità di qualsiasi soluzione particolare è controllato da ~ 2-3 ml / min con fascette di rampa &quot;Keck&quot; sui tubi. </li><li> Montare entrambi gli elettrodi con i loro headstages su micromanipolatori. Eseguire penetrazioni degli elettrodi l&#39;ovocita da micromanipolazione. </li></ol> 3. Patch Clamp Esame della diretta mechanosensitivity &quot;&gt; I metodi di base di patch clamp compresa la preparazione pipetta, formazione GΩ-sigillo, e la formazione di on-cella o modalità escisse sono quelli in Hamill et al. Conn 36 e 32. <ol><li> Riempire pipette in vetro borosilicato con una ~ 1 micron apertura del diametro in punta (numero bolla 5-6) con 98 mM KCl, 1 mM MgCl 2, e 10 mM K +-HEPES, pH 7.2. </li><li> Fissare la pipetta patch clamp attraverso un tubo di plastica per una siringa da 5 ml e attraverso un giunto a T, anche ad un manometro. Utilizzare un computer per gestire sia l&#39;elettrodo e le uscite manometro. Acquisire dati a 10 kHz, e quindi riprodurre attraverso un filtro a otto poli Bessel a 1 kHz per l&#39;analisi. </li><li> Patch differenti possono essere asportati dallo stesso ovocita ripetutamente. Questa pratica rende l&#39;esame delle attività molecolare di TRPV4 più efficiente e affidabile. </li></ol>

<ol>
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Gli autori dichiarano di non avere interessi finanziari in competizione.

Come indicato nella Premessa, i metodi comunemente usati per studiare le funzioni TRPV4 talvolta portato a incongruenze e controversie. Le due serie di metodi qui descritti offrono alcuni vantaggi e possono integrare i metodi esistenti. Mentre si descrivono solo gli studi sulla TRPV4, i metodi possono essere estesi a studiare altri canali ionici pure. 1. Metodi di lievito luminometria Lievito in erba ha un nativo di Ca 2 +-risposta afflusso allo shock ip...

TRP (potenziali recettori transitori) comprendono sette sottofamiglie di canali di comunicazione che servono funzioni sensoriali 1,2. Nei mammiferi, TRPV, sottofamiglia vanilloid del TRP, ha sei varietà. TRPV4 (tipo 4) 3,4 risponde al calore, alcune sostanze chimiche, gonfiore osmotico, o shear stress. Il gene TRPV4 stato ripetutamente isolato mediante candidato-gene e / o espressione clonazione 5-8. Quest&#39;ultimo metodo seguito la risposta del prodotto genico di ipo-osmolarità. TRPV4 è espresso in quasi tutti gli organi e le funzioni per lo sviluppo, fisiologia, patologia o di molti tipi di cellule disparate 3,4. Striking sono le autosomiche dominanti umano mutazioni TRPV4&gt; 50, causando neuropatie periferiche e / o displasie scheletriche (anomalie nello sviluppo scheletrico) 9-11. La gamma di displasie scheletriche da lievi tipo brachyomia 3 (tipo 3 nanismo), displasia spondilometafisaria tipo Kozlowski, a Severe displasie, alcuni causando la morte neonatale o embrionale. Anche se tutte le maniere di meccanismi sembrano possibili, nessuno spiega la diversità, la complessità, la variabilità e sovrapposizioni occasionali di queste malattie 4. Come altri canali TRP 1, TRPV4 è un tetramero. Nel ratto o TRPV4 umana, ogni subunità è costituita da 871 residui. Il suo elemento centrale è il sei transmembrana una eliche (S1-S6), che sono probabilmente disposti in un modo simile a K + canali voltaggio-dipendenti. Lì, S1 a S4 formare un dominio periferico e la S5 e S6 formano il dominio permeazione poro. Tra S5 e S6 di TRPV4 è un&#39;elica poro breve seguito dal LDLFKLTIGMGDL sequenza, quattro delle quali presumibilmente convergono per formare il filtro catione. Il 470-residuo N-terminale del segmento citoplasmatico contiene 6 ripetizioni ankyrin, noti per legare le proteine ​​o piccoli ligandi. Il 152-residuo segmento citoplasmatica C-terminale include una sequenza-calmodulina legame tra gli altri possibili siti che legano oti suoi elementi 3. TRPV4 è un canale cationico che esclude essenzialmente anioni 1. Mentre la sua funzione fisiologica è per trasdurre stimoli a Ca 2 + afflusso, è anche permeabile ai cationi attraverso una sequenza permeabilità Eisenman IV, favorendo bivalente a P Ca: P Na ~ 7 12. Conduttanza monocanale rettifica a ~ 90 pS verso l&#39;esterno e verso l&#39;interno ~ 40 pS 6,13,14. Eterologamente espressa corrente (in basso) può essere attivato da ipo-osmotico gonfiore, sforzo di taglio, o di calore 15. Inoltre è stato attivato da acidi grassi polinsaturi 16,17 e l&#39;estere sintetico phorbal 4αPDD 18. Allo stato attuale, il più potente agonista è GSK1016790A 19 e antagonista è GSK2193874, efficace nel 10 -9 a 10 -8 M 20, entrambi scoperti da alta velocità, schermo piccolo-molecolare. Due settori chiave della ricerca TRPV4 rimangono confuse: (1) Anche se TRPV4 è ampiamente studiato per la sua mechanosensitivity, la sua base molecolare è controversa. Un modello descrive ipo-osmolarità qualche modo attiva la fosfolipasi A2 (PLA2) per produrre l&#39;acido grasso polinsaturo (PUFA) acido arachidonico (AA), che viene convertito in acido epoxyeicosatrienoic (TEE) da un epoxygenase, e il legame di EET attiva TRPV4 16, 17. Eppure, TRPV4 si è dimostrato di rispondere direttamente ai membrana tratto 14 (sotto), fornendo una spiegazione più semplice. (2) Le patologie mutanti TRPV4 sono sconcertanti. Alla base, si ha la necessità di sapere se le malattie sono dovute alla perdita, la riduzione o l&#39;aumento di attività di canale. Anche qui, la letteratura è tutt&#39;altro che chiara. Mentre alleli multipli scheletrico-displasia sono stati segnalati per avere attività superiori, (guadagno-di-funzione, GOF) 4,21, vari sono stati segnalati per aver ridotto le attività (perdita-di-funzione, LOF) 10,22. Uno studio sistematico di 14 alleli li ha trovati pertutte le mutazioni essere GOF (sotto) 23. L&#39;affermazione che alcuni sono LOF sembra contraddire il fenotipo di TRPV4-/ - topo knockout, che sono vitali o fertili, con solo piccoli difetti, nonostante una perdita completa della funzione TRPV4. Una parte di queste polemiche ha origine metodologica. I laboratori usano metodi diversi, o varianti di un metodo, e utilizzano diversi standard di giudizio. Più comunemente, TRPV4 viene transitoriamente espresso in cellule di mammifero in coltura (HEK, CHO, HeLa) e l&#39;ascesa di interno [Ca 2 +] sulla agonista o stimolazione ipotonico è registrato con il colorante fluorescente + sensibile Ca 2, Fura-2. L&#39;eccessivo affidamento su questo test fluorimetrico è stato criticato 1. Il livello di espressione in differenti popolazioni, la distribuzione in esso, nonché la concentrazione di colorante efficace, deve essere controllato e documentato. Più attendibile è esami elettrofisiologici diretti. Anche questo, come commonly praticato, inoltre, non è senza problemi. Poiché i livelli di espressione in cellule coltivate individuali sono difficili da controllare, correnti di cellule intere hanno grandi variazioni. Inoltre, perché le correnti sono piccole, statistiche affidabili dovranno fare affidamento su campioni di grandi dimensioni, spesso non è pratico. Raramente sono stati effettuati gli esami di patch-clamp. Alcune di queste registrazioni mostrano gruppi di scoppi di attività che rendono la valutazione statistica impegnativo 16,17. Per capire meglio il mechanosensitivity molecolare, abbiamo sviluppato due sistemi aggiuntivi per esaminare TRPV4. (1) Per isolare TRPV4 lontano dai suoi abituali partner di mammiferi, abbiamo espresso ratto TRPV4 in erba lievito 24. Espressione funzionale di TRPV4 in questo contesto evolutivamente distante ha dimostrato che potrebbe ancora rispondere alla forza osmotica senza i suoi abituali partner. Perché il lievito non rilascia acidi grassi polinsaturi come AA o EET, e il suo genoma non ha PLA2 o epoxygenase geni, questa espresSsion dimostra anche che non sono necessari per TRPV4 di percepire la forza. Avere TRPV4 in eucarioti biologicamente più suscettibili molecolari permette anche manipolazioni efficiente avanti o indietro genetici 25. (2) Per le analisi biofisiche di TRPV4 in profondità, abbiamo espresso TRPV4 in ovociti di Xenopus. A differenza delle cellule coltivate, che producono sub-nA a nA (10 -10 a 10 -9 A) correnti, un ovocita esprime correnti μA di (10 -6 a 10 -4 A). Molto più grande segnale su rumore consente una migliore quantificazione e comparazione più sicuri. TRPV4, così espresso, può anche essere esaminata come singole molecole utilizzando una patch clamp. Un singolo ovocita permette il campionamento di patch ripetuto, ancora una volta rendendo quantificazione più attendibile. Tali studi hanno dimostrato che canale TRPV4 stessa può essere attivata direttamente con la forza membrana tratto 14. Le analisi hanno inoltre dimostrato che 14 rappresentative alleli scheletrico-displasia sono tutte le mutazioni guadagno-di-funzione. Inoltre, il Degree di questa costitutiva Ca 2 + perdite parallelo alla gravità delle malattie scheletriche 23. A causa della loro novità e l&#39;utilità, le modalità dettagliate di questi due metodi sono riuniti qui per consentire repliche in futuro la ricerca sulle TRPV4 o canali simili.

<table border="0" cellpadding="0" cellspacing="0" width="595">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vapor Pressure Osmometer</td>
			<td style="width:93px;">Wescor</td>
			<td style="width:119px;">Wescor 5500</td>
			<td style="width:167px;">Logan, UT, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sirius Single Tube Luminometer </td>
			<td style="width:93px;">Titertek-Berhold</td>
			<td style="width:119px;">Sirius </td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microplate luminometer</td>
			<td style="width:93px;">Titertek-Berthold </td>
			<td style="width:119px;">Mithras LB940</td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Luminometry software</td>
			<td style="width:93px;">Titertek-Berthold</td>
			<td style="width:119px;">MikroWin2000</td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:216px;">Patch clamp and software</td>
			<td style="width:93px;">Axon Instrument</td>
			<td style="width:119px;">HS2A headstage, VG-2Ax100 virtual grounded bath clamp, GeneClamp 500 amplifier, digidata 1440 digitizer, pClamp10 software </td>
			<td>Union City, CA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">manometer</td>
			<td style="width:93px;">Bio-Tec</td>
			<td style="width:119px;"> </td>
			<td>Winooski, VT, USA</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pipet puller</td>
			<td style="width:93px;">Sutter Instrument Co</td>
			<td style="width:119px;">Model P-97</td>
			<td>Novata, CA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microinjector</td>
			<td style="width:93px;">Drummond Scientific</td>
			<td style="width:119px;">Nanoinject II</td>
			<td>Broomall, PA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Material:</td>
			<td style="width:93px;"> </td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">luciferin coelenterazine</td>
			<td style="width:93px;">Invitrogen</td>
			<td style="width:119px;"> </td>
			<td>Carlsbad, CA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">T7 RNA polymerase reaction</td>
			<td style="width:93px;">Ambion</td>
			<td style="width:119px;">mMessage mMachine</td>
			<td>Austin, TX, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gentamicin</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ruthenium red </td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">micropipets </td>
			<td style="width:93px;">Drummond</td>
			<td style="width:119px;">100 microliter micropipets</td>
			<td>Broomall, PA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">high-fidelity PfuUtra polymerase </td>
			<td style="width:93px;">Stratagene</td>
			<td style="width:119px;"> </td>
			<td>La Jolla, CA, USA</td>
		</tr>
	</tbody>
</table>

yeast luminometric and xenopus oocyte electrophysiological examinations of the molecular mechanosensitivity of trpv4

TRPV4 (Transient Receptor Potenziali, famiglia vanilloid, tipo 4) è ampiamente espressa nei tessuti vertebrati ed è attivato da diversi stimoli, tra cui da forze meccaniche. Alcune mutazioni TRPV4 causano complesso osso ereditaria o patologie neuronali umano. Wild-type o mutante TRPV4 transgeni sono comunemente espresse in cellule di mammifero in coltura ed esaminati dalla Fura-2 fluorometria e da elettrodi. In termini di meccanismo mechanosensitivity e le basi molecolari delle malattie, la letteratura attuale è confusa e controversa. Per completare metodi esistenti, si descrivono due metodi aggiuntivi per esaminare le proprietà molecolari di TRPV4. (1) Rat TRPV4 e un transgene aequorina si trasformano in lievito in erba. Uno shock ipo-osmtic della popolazione trasformante produce un segnale luminometrici causa della combinazione di equorina con Ca 2 +, rilasciato attraverso il canale TRPV4. Qui TRPV4 è isolato dai suoi abituali partner di proteine ​​di mammiferie rivela la propria mechanosensitivity. (2) cRNA di TRPV4 viene iniettato in oociti di Xenopus. Dopo un adeguato periodo di incubazione, la corrente TRPV4 macroscopico viene esaminato con un morsetto di tensione a due elettrodi. L&#39;attuale aumento dopo la rimozione di osmoticum inerti dal bagno ovocita è indicativo di mechanosensitivity. I microampere (10 -6 a 10 -4 A) correnti da ovociti sono molto più grandi rispetto alle subnano-to nA (10 -10 a 10 -9 A) correnti da cellule in coltura, producendo quantificazioni più chiare e valutazioni più sicuri. Correnti microscopiche che riflettono le attività delle proteine ​​dei singoli canali possono anche essere direttamente registrati sotto una patch clamp, in on-cella o in modalità asportato. Lo stesso ovocita fornisce più campioni di patch, consentendo la replica dei dati migliori. Aspirazioni applicate le patch possono attivare TRPV4 per valutare direttamente mechanosensitivity. Tali metodi dovrebbero anche essere utile nello studio di altri tipi di canali TRP.

In un tipico esperimento luminometria, una serie di 400-1,200 mOsm shock ipotonici non si raggiunge con l&#39;aggiunta di 200 ml di soluzione d&#39;urto di diversa bassa osmolarità a 20 ml di cultura a 1.400 mOsm. L&#39;attività TRPV4 è evidente quando il RLU della disposizione sperimentale è confrontata con quella di due controlli negativi: cellule di lievito trasformate con il plasmide p416GPDV4 vuoto o uno che contiene il gene TRPV4 con una mutazione nel filtro di ioni (Figura 1) 24...

Watch this Scientific Journal Video about Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4 at JoVE.com

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

Per completare i metodi comunemente usati per studiare TRPV4 di, sono descritti due metodi: la sua mechanosensitivity può essere studiato da Ca 2 + luminometria-aequorina in lievito transgenico upon sfida ipo-osmotico. Può pure esaminato TRPV4-RNA oociti di Xenopus iniettati da cellula intera clamp a due elettrodi tensione o Patch Clamp on-cella o modalità asportato.

Lievito luminometrici e<em&gt; Xenopus</em&gt; ovocita elettrofisiologiche Esami del mechanosensitivity molecolare del TRPV4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...

yeast-luminometric-xenopus-oocyte-electrophysiological-examinations

Research

JoVE Journal

Biology

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

10.0K Views.  University of Wisconsin – Madison.  Per completare i metodi comunemente usati per studiare TRPV4 di, sono descritti due metodi: la sua mechanosensitivity può essere studiato da Ca 2 + luminometria-aequorina in lievito transgenico upon sfida ipo-osmotico. Può pure esaminato TRPV4-RNA oociti di Xenopus iniettati da cellula intera clamp a due elettrodi tensione o Patch Clamp on-cella o modalità asportato. 

Video: Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

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

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Patch di registrazione pinza dei canali ionici Espressa in ovociti di Xenopus

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

Visualizzazione RNA localizzazione in Xenopus Gli oociti

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Un saggio microscopia a fluorescenza per Mitophagy monitoraggio nel lievito Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Microiniezione degli ovociti mouse, Maturazione e Ploidia valutazione

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Schermi competitivo genomica delle Biblioteche di lievito con codice a barre

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Purificazione cromatografica dei ribosomi di lievito Highly Active

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

Il processo di Green Monster per la generazione di ceppi di lieviti trasporto gene delezioni multiple

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

Il<em&gt; Xenopus</em&gt; Ovociti Cut-open vaselina Gap Tecnica Voltage-clamp Con fluorometria

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

Diffondere superfici e Immunostaining di cromosomi del lievito

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...