Questo lavoro è stato finanziato da borse di DMH e TDM (NHMRC sovvenzioni per progetti). DMH è membro Grimwade, finanziato dal Trust Miegunyah.

1. Preparazione delle cellule per l&#39;etichettatura con ReAsH / Flash <ol><li> Usando metodi standard di coltura cellulare per la linea cellulare di interesse, preparare una coltura di cellule aderenti direttamente in una diapositiva dal vivo imaging cellulare pronto per la trasfezione. </li><li> Trasfezione vostro plasmide contenente TC-tagged gene di interesse in base al metodo di trasfezione di scelta. </li></ol> Nota è importante utilizzare i controlli positivi e negativi per valutare l&#39;entità del legame specifico ai tag TC e per valutare bleedthrough di fluorescenza tra i canali al momento del ritiro microscopio confocale. Quindi, per due colori (per esempio Flash / Cherry o ReAsH / o CFP ReAsH / GFP combinazioni), assicurano i campioni sono preparati per singoli colori (es. proteina fluorescente da soli o, se possibile, una proteina TC-tagged legato a Flash / ReAsH ma senza fluorescente proteine) <ol start="3"><li> Uno o due giorni dopo la transfezione, risciacquare delicatamente le cellule con 300 ml di pre-riscaldato (a 37 ° C) HBSS. </li><li> Immergere delicatamente le cellule con 1 mM Flash (o ReAsH) in 300 ml di HBSS preriscaldata e 10 mM 1,2-ethanedithiol (EDT). </li></ol> E &#39;importante aggiungere EDT prima di aggiungere Flash / ReAsH e fare il tampone appena prima di aggiungerlo alle cellule. Incubare per esattamente 30 minuti a 37 ° C nel tessuto culturale incubatore. Nella nostra esperienza i tempi di incubazione più lungo aumenta significativamente la fluorescenza di fondo. Nuovi costrutti dovrebbe essere ottimizzato per l&#39;etichettatura di tempo e Flash / ReAsH concentrazione (0.5-2 mM). <ol start="5"><li> Aspirare delicatamente etichettatura soluzione dalle cellule e quindi sostituire con 300 microlitri HBSS preriscaldata + 250 mM 2,3-dimercaptopropanol (BAL) per 15 minuti a 37 ° C. </li><li> Rimuovere soluzione di lavaggio mediante aspirazione dolce e sostituire con 300 microlitri HBSS preriscaldata. </li></ol> Dopo questo lavaggio, le cellule possono essere fissati con paraformaldeide (15 min con il 3,2% di soluzione), anche se abbiamo trovato che questo aumenta la fluorescenza non specifica biarsenical colorante. Quindi di solito l&#39;immagine cellule vive a temperatura ambiente. (Si noti che la fissazione delle cellule prima dell&#39;etichettatura previene il legame biarsenical colorante.) 2. Imaging le cellule su un microscopio confocale <ol><li> Al microscopio confocale, impostare i parametri per l&#39;imaging del fluorofori individuali (vedi tabella 1) e assicurarsi che non vi è bleedthrough trascurabile tra i canali. Ciò può essere ottenuto controllando fluorofori individuale (in campioni di controllo) contro ogni diversa impostazione di acquisizione fluorescenti per fluorescenza. Regolazione della lunghezza d&#39;onda di emissione può aiutare a minimizzare bleedthrough (anche se questo può anche ridurre il rapporto segnale / rumore). </li><li> Anche regolare le impostazioni del fotomoltiplicatore in modo che la fluorescenza massima nel campione non saturare il rivelatore. (Questo può essere rilevato con il Q-LUT impostazione sul confocale Leica SP2). Una volta che le migliori impostazioni di imaging sono determinati, non modificare nessuno di loro tra i campioni. </li><li> Altre impostazioni si utilizzano in genere (anche se questi possono essere ottimizzati) sono una velocità di scansione di 200 Hz e raccogliere 4 medie di linea e un diametro foro stenopeico di 1 unità Airy. Il diametro del foro può essere ampliata in caso di segnale / rumore è un problema, tuttavia, questo può causare una perdita di imaging. </li><li> Raccogliere le immagini confocale a 12-bit (o superiore) formato, se possibile. 12-bit in formato cattura una più ampia gamma dinamica di valori (0-4095) per ogni pixel l&#39;intensità di 8-bit (0-255). Questo è importante per garantire il più ricco set di dati vengono registrati, che massimizza la qualità di analisi quantitativa dei dati. </li><li> Raccogliere le immagini per il canale proteina fluorescente (Cherry, GFP o CFP) e anche per il canale biarsenical colorante (Flash o ReAsH) per tutti i campioni. </li></ol> 3. Analisi dei dati <ol><li> Installare il software ImageJ sul computer 8. <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a> </li><li> Assicurarsi che la versione di ImageJ ha i seguenti plugin: <ul><li> <a href="http://rsbweb.nih.gov/ij/plugins/file-opener.html">http://rsbweb.nih.gov/ij/plugins/file-opener.html</a> </li><li> <a href="http://rsbweb.nih.gov/ij/plugins/image_correlator.html">http://rsbweb.nih.gov/ij/plugins/image_correlator.html</a> </li><li> <a href="http://rsbweb.nih.gov/ij/plugins/lut-editor.html">http://rsbweb.nih.gov/ij/plugins/lut-editor.html</a> </li></ul></li><li> ImageJ aperto, e se si utilizza una versione precedente alla v1.32c, fare clic sulle seguenti opzioni per abilitare più immagini da aprire in una sola volta (che può essere fatto tenendo premuto il tasto di controllo mentre si fa clic su file diversi): <img src="/files/ftp_upload/2857/2857step1.jpg" alt="Figura 1" /></li><li> Aprire le immagini di interesse in ImageJ. ImageJ automaticamente definire le intensità dei pixel massimi e minimi che vengono visualizzati sullo schermo per ogni immagine separatamente. Dato che le diverse immagini avranno dintensità dei pixel ifferent, questo significa che le immagini visualizzate non sono paragonabili come appaiono. </li><li> Per garantire le immagini aperte sono tutti sulla stessa scala, è possibile definire fisicamente superiore e più bassa intensità di pixel da visualizzare (questo non cambierà il contenuto effettivo dei dati delle immagini come sarebbe il caso di qualche altro software). Questi valori possono essere impostati con il seguente menu: <img src="/files/ftp_upload/2857/2857step2.jpg" alt="Figura 2" /></li><li> Un approccio alternativo è quello di lavorare con gli stack, che mette insieme tutte le immagini in un file e verrà automaticamente scala (per la visualizzazione), tutte le immagini in stack per la stessa scala. Il modo più semplice per lavorare con i dati è quello di convertire ogni canale ad una pila. Così, per il canale proteina fluorescente convertire tutti a stack come mostrato. È possibile selezionare facilmente un canale sovrapponendo tutte le immagini contenenti un nome comune (ad esempio &quot;ch00&quot;) nel titolo. <img src="/files/ftp_upload/2857/2857step3.jpg" alt="Figura 3" /></li><li> Ora convertire tutte le immagini aperte ad 8-bit per l&#39;analisi. Questo effettivamente riscalare la gamma visto in una serie 0-255 (che definisce 8-bit). IMPORTANTE: Non salvare sopra l&#39;originale a 12-bit (o superiore) formato o si perderanno contenuto informativo. Si noti che alcuni pacchetti software in grado di visualizzare solo immagini a 8 bit in modo da questo formato è utile per creare figure, ecc <img src="/files/ftp_upload/2857/2857step4.jpg" alt="Figura 4" /></li><li> Salva una copia in una nuova cartella chiamata &quot;8-bit convertito&quot; utilizzando la funzione &quot;Salva con nome ...&quot; opzione. </li><li> Un metodo per esaminare l&#39;entità del legame colorante biarsenical in una intera immagine è quella di eseguire un pixel tracciato di correlazione intensità. Questo complotti ogni valore di pixel in un canale rispetto al valore di pixel corrispondente in un secondo canale. Quindi una posizione di pixel ad alta nel PCP fluorescenza sarà anche ad alto contenuto di fluorescenza ReAsH se c&#39;è alta vincolante. </li><li> Per analizzare il pixel co-correlazione, assicurarsi che la 8-bit ReAsH e Cerulean / GFP immagini sono aperti in ImageJ. </li><li> Aprire il &quot;Correlatore Immagine&quot; plugin come di seguito indicato. Selezionare &quot;Image1&quot;, come lo stack ReAsH o Flash e &quot;Image2&quot;, come lo stack proteina fluorescente. <img src="/files/ftp_upload/2857/2857step5.jpg" alt="Figura 5" /></li><li> Lo stack risultante non può visualizzare informazioni dettagliate - questo è normale. Salva la pila in una nuova cartella chiamata &quot;dispersione&quot; e dargli lo stesso nome di file come il campione si riferisce (ad esempio &quot;Flash-ciliegia tracciato di correlazione&quot;) </li><li> Per visualizzare i dati significativamente si può fare una delle due opzioni. In primo luogo trasformare i dati in modo che sia in formato log. Poi ridimensionare visivamente i dati come segue: <img src="/files/ftp_upload/2857/2857step6.jpg" alt="Figura 6" /></li><li> In alternativa, è possibile ridimensionare visivamente i dati per mostrare solo valori bassi (ad esempio 1-255) e visualizzare i dati utilizzando una LUT speciale. Una LUT (Look Up Table) rappresenta una tabella di colori assegnati ad ogni valore di pixel in un&#39;immagine . Questo può essere usato per definire uno schema pseudocolori per un&#39;immagine ed è utile per definire alcune caratteristiche in un&#39;immagine. Per creare un semplice clic personalizzati LUT su &quot;editor di LUT&quot; e fare uno nuovo come quello mostrato (che possono essere salvati e utilizzati successivamente). <img src="/files/ftp_upload/2857/2857step7.jpg" alt="Figura 7" /></li><li> Impostare la luminosità / contrasto vanno da 0-255 (come descritto al punto 13 della presente sentenza). Per &quot;bloccare&quot; l&#39;immagine, come a quella visualizzata sullo schermo, l&#39;immagine può essere convertita in formato RGB che salva un valore a 8 bit per ognuno dei colori rosso, verde e blu di ogni pixel. <img src="/files/ftp_upload/2857/2857step8.jpg" alt="Figura 8" /> Per copiare e incollare le immagini ad altri programmi per prima cosa aprire le immagini a 8-bit o 16-bit. Come descritto al punto 14 della presente sentenza, assegnare un sistema di colori LUT all&#39;immagine. Per PCP, assegnare la LUT &quot;Cyan&quot;, come descritto di seguito ... <img src="/files/ftp_upload/2857/2857step9.jpg" alt="Figura 9" /></li><li> Selezionare l&#39;immagine e copiarlo negli appunti, selezionare ... <img src="/files/ftp_upload/2857/2857step10.jpg" /></li></ol> 4. Rappresentante dei risultati: Il successo di cellule etichettatura con coloranti biarsenical dipende da alcuni parametri chiave. In primo luogo, i tempi della etichettatura con coloranti è cruciale. Abbiamo scoperto che lunghi periodi di etichettatura (più di 30 minuti) si traduce in un elevato livello di colorazione aspecifica di fondo. Fig. 1 mostra un tipico risultato di una wild-type forma di frammento huntingtina (25Q) unito alla Cerulean PCP derivato contenente un tag TC come descritto in precedenza 7. Questo campione è stato macchiato per 30 minuti con ReAsH e non c&#39;è sfondo minimo nel campione manca il tag di TC. Abbiamo scoperto che la fissazione delle cellule con sfondo paraformaldeide aumenta mentre fissaggio con metanolo abroga la fluorescenza del tag proteina fluorescente. Quindi dove possibile, l&#39;immagine del cellule vive. E &#39;importante notare inoltre che la fissazione preventival&#39;etichettatura con coloranti biarsenical impedisce loro legame, presumibilmente a causa di modifiche del TC motivo. Un altro fattore critico per risultati costanti è la densità delle cellule. Abbiamo trovato critiche a celle di immagine che vengono liberamente distribuiti e, inoltre, che ampia aggregazione può portare a colorazione irregolare dei coloranti biarsenical in celle diverse. <img src="/files/ftp_upload/2857/2857fig1.jpg" alt="Figura 1" /> Figura 1. Tetracysteine ​​tag e colorazione ReAsH in cellule vive transfettate con huntingtina (exon1-25Q)-Cerulean fusioni. Il tag TC si trova all&#39;incrocio tra la huntingtina Cerulean-fusione (come descritto in 7). Il tracciato di correlazione di intensità dei pixel consente una valutazione delle differenze di ReAsH vincolanti per tutta la cellula e può essere utilizzato per mappare i cambiamenti in ReAsH vincolante a causa di cambiamento conformazionale o interazioni ligando.

<ol>
	<li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+fluorescent+protein.">The green fluorescent protein.</a> Ann. Rev. Biochem. 67, 509-544 (1998).</li><li>Griffin, B. A., Adams, S. R., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+covalent+labeling+of+recombinant+protein+molecules+inside+live+cells.">Specific covalent labeling of recombinant protein molecules inside live cells.</a> Science. 281, 269-2672 (1998).</li><li>Uttamapinant, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorophore+ligase+for+site-specific+protein+labeling+inside+living+cells.">A fluorophore ligase for site-specific protein labeling inside living cells.</a> Proc. Natl. Acad. Sci. USA. 107, 10914-10919 (2010).</li><li>Ignatova, Z., Gierasch, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+protein+stability+and+aggregation+in+vivo+by+real-time+fluorescent+labeling.">Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling.</a> Proc. Natl. Acad. Sci. USA. 101, 523-528 (2004).</li><li>Coleman, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+detection+of+prion+protein+with+biarsenical+labeling+and+FlAsH+fluorescence.">Conformational detection of prion protein with biarsenical labeling and FlAsH fluorescence.</a> Biochem. Biophys. Res. Commun. 380, 564-568 (2009).</li><li>Luedtke, N. W., Dexter, R. J., Fried, D. B., Schepartz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surveying+polypeptide+and+protein+domain+conformation+and+association+with+FlASH+and+ReAsH.">Surveying polypeptide and protein domain conformation and association with FlASH and ReAsH.</a> Nat. Chem. Biol. 3, 779-784 (2007).</li><li>Ramdzan, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformation+sensors+that+distinguish+monomeric+proteins+from+oligomers+in+live+cells.">Conformation sensors that distinguish monomeric proteins from oligomers in live cells.</a> Chem. Biol. 17, 371-379 (2010).</li><li>Abramoff, M. a. g. e. l. h. a. e. s., PJ, S. J. R. a. m. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+processing+with+ImageJ.">Image processing with ImageJ.</a> Biophotonics International. 11, 36-42 (2004).</li><li>Hearps, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biarsenical+dye+Lumio+exhibits+a+reduced+ability+to+specifically+detect+tetracysteine-containing+proteins+within+live+cells.">The biarsenical dye Lumio exhibits a reduced ability to specifically detect tetracysteine-containing proteins within live cells.</a> J. Fluor. 17, 593-597 (2007).</li><li>Adams, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+biarsenical+ligands+and+tetracysteine+motifs+for+protein+labeling+in+vitro+and+in+vivo:+Synthesis+and+biological+applications.">New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: Synthesis and biological applications.</a> J. Am. Chem. Soc. 124, 6063-6076 (2002).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat. Meth. 2, 905-909 (2005).</li></ol>

Nessun conflitto di interessi dichiarati.

L&#39;approccio alla localizzazione etichetta proteina con una proteina fluorescente e le proprietà conformazionali con un colorante secondo offre un grande potenziale per la mappatura in cui diverse conformazioni delle proteine ​​nelle cellule maturano e gli eventi che cambiano le dinamiche della conformazione delle proteine. ReAsH / Flash per la prima volta utilizzato come in-cell sensore per folding delle proteine ​​dei mammiferi cellulari retinoico-binding protein I 4. In questo esempio, il Flash...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> 8-ben μ-slide </td><td> Ibidi </td><td> 80826 </td><td> Troviamo queste camera di diapositive di essere particolarmente utile per le celle di coltura per l&#39;imaging. </td></tr><tr><td> TC-Flash II nella cella di rilevamento Tag Tetracysteine ​​Kit fluorescenza verde * * * per il live-cell imaging </td><td> Invitrogen </td><td> T34561 (Flash) o T34562 (ReAsH) </td><td></td></tr><tr><td> Sale Hanks &#39;soluzione equilibrata </td><td> Invitrogen </td><td> 14175-103 </td><td></td></tr><tr><td> 2,3-Dimercapto-1-propanolo </td><td> Sigma-Aldrich </td><td> D1129-5ML </td><td></td></tr><tr><td> 1,2-Ethanedithiol </td><td> Sigma-Aldrich </td><td> 02390-25ML </td><td></td></tr></tbody></table>

reash/flash labeling and image analysis of tetracysteine sensor proteins in cells

Proteine ​​fluorescenti e coloranti sono strumenti essenziali per lo studio del traffico di proteine, la localizzazione e la funzione delle cellule. Mentre le proteine ​​fluorescenti come il verde proteina fluorescente (GFP) sono stati ampiamente utilizzati come partner di fusione di proteine ​​per monitorare le proprietà di una proteina di interesse 1, i recenti sviluppi con piccole etichette consentono nuove funzionalità di proteine ​​da esaminare nelle cellule, come il cambiamento conformazionale e proteine-associazione 2, 3. Un sistema di tag piccola comporta un motivo tetracysteine ​​(ccxxcc) geneticamente inserito in una proteina bersaglio, che si lega ai coloranti biarsenical, ReAsH (rosso fluorescente) e Flash (verde fluorescente), con elevata specificità anche nelle cellule vive 2. Il TC / biarsenical sistema colorante offre vincoli molto meno sterico alla proteina ospite di proteine ​​fluorescenti che ha permesso a numerosi nuovi approcci per misurare il cambiamento conformazionale e interazioni proteina-proteina 4-7. Abbiamo recentemente sviluppato una nuova applicazione di tag TC come sensori di oligomerizzazione nelle cellule che esprimono huntingtina mutata, che se mutato aggregati nei neuroni a Huntington malattia 7. Huntingtina era taggati con due coloranti fluorescenti, una una proteina fluorescente per tenere traccia posizione di proteine, e la seconda un tag TC che lega solo coloranti biarsenical in monomeri. Quindi, i cambiamenti colocalizzazione tra proteine ​​e biarsenical reattività colorante abilitato contenuto submicroscopiche oligomero di essere mappate spazialmente all&#39;interno delle cellule. Qui, descriviamo come etichettare TC-tagged proteine ​​fuse ad una proteina fluorescente (Cherry, GFP o CFP) con il flash o ReAsH in vivo le cellule di mammifero e come quantificare i due fluorescenza di colore (ciliegio / Flash, CFP / Flash o GFP / combinazioni ReAsH).

Watch this Scientific Journal Video about ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells at JoVE.com

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Il biarsenical coloranti Flash e ReAsH legano specificamente ai motivi tetracysteine ​​di proteine ​​e in modo selettivo le proteine ​​etichetta in cellule vive. Recentemente questa strategia di etichettatura è stato utilizzato per sviluppare sensori per le diverse conformazioni delle proteine ​​o stati oligomerici. Abbiamo descritto l&#39;approccio di etichettatura e di metodi per analizzare quantitativamente vincolanti.

ReAsH / Flash Etichettatura e Image Analysis di proteine ​​sensore Tetracysteine ​​nelle celle

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells.  While fluorescent 
proteins ...

reashflash-labeling-image-analysis-tetracysteine-sensor-proteins

Research

JoVE Journal

Biology

16.9K Views.  Bio21 Molecular Science and Biotechnology Institute. Il biarsenical coloranti Flash e ReAsH legano specificamente ai motivi tetracysteine ​​di proteine ​​e in modo selettivo le proteine ​​etichetta in cellule vive. Recentemente questa strategia di etichettatura è stato utilizzato per sviluppare sensori per le diverse conformazioni delle proteine ​​o stati oligomerici. Abbiamo descritto l'approccio di etichettatura e di metodi per analizzare quantitativamente vincolanti.

Video: ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Robotica e Image Analysis dinamica per gli Studi di espressione genica nei tessuti vegetali

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Ricerca

Biologia

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Esaminando la dinamica conformazionale delle proteine ​​di membrana In situ Con il sito-diretta Etichettatura fluorescenza

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

L&#39;analisi fenotipica e l&#39;isolamento di murini cellule staminali ematopoietiche e Lineage-committed progenitori

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Analisi automatizzata del Dynamic Ca<sup&gt; 2 +</sup&gt; Segnali in sequenze di immagini

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Specifici Sequenza Etichettatura degli acidi nucleici e proteine ​​con metiltransferasi e cofattore Analoghi

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

Le proteine ​​immunofluorescenza di endogeni ed esogeni Centromere-cinetocoro

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Saggi per la degradazione delle proteine ​​nelle cellule mal ripiegate

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

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 ...

Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are highly efficient at virus transmission plays a key role in virus epidemics in fields. Studying virus-insect vector interactions can advance our understanding of virus transmission and epidemics with the aim of designing novel strategies to control plant viruses and their vector insects. Immunofluorescence labeling has been widely used to analyze interactions between pathogens and hosts and is used here in the white-backed planthopper (WBPH, Sogatella furcifera), which efficiently transmits the southern rice black streaked dwarf virus (SRBSDV, genus Fijivirus, family Reoviridae), to locate the virions and insect proteins in the midgut epithelial cells. Using laser scanning confocal microscopy, we studied the morphological characteristics of midgut epithelial cells, cellular localization of insect proteins, and the colocalization of virions and an insect protein. This protocol can be used to study virus activities in insects, functions of insect proteins, and interactions between virus and vector insect.

This protocol for immunofluorescent labeling of both plant virus proteins and vector insect proteins in excised insect guts can be used to study interactions among virus and vector insects, insect protein functions and molecular mechanisms underlying virus transmission.

Marcatura immunofluorescente di virus vegetali e proteine di insetti vettori nell'intestino degli emitteri

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are ...

ReAsH / Flash Etichettatura e Image Analysis di proteine ​​sensore Tetracysteine ​​nelle celle

ReAsH / Flash Etichettatura e Image Analysis di proteine sensore Tetracysteine nelle celle