Lavoro in laboratorio Montgomery sulle risposte fitocromo nelle piante è sostenuta dalla National Science Foundation (non concede. MCB-0919100 per BLM) e Scienze Chimiche, Scienze geologiche e Bioscienze Divisione, Ufficio di Basic Sciences Energy, Office of Science, US Department of energia (non concedere alcuna. DE FG02 91ER20021 a BLM). Ringraziamo Melissa Whitaker per l&#39;assistenza tecnica durante le riprese e la lettura critica del manoscritto, Stephanie Costigan sperimentale per l&#39;assistenza, il Dott. Luigi Re di assistenza sviluppare e ottimizzare le Fluorescence-Activated cella ordinamento protocolli per l&#39;ordinamento di protoplasti di Arabidopsis e il Dr. Telaio Melinda per assistenza confocale microscopia. Ringraziamo Marlene Cameron per l&#39;assistenza di progettazione grafica e Bird Karen per l&#39;assistenza editoriale.

1. Crescita delle piante <ol><li> Confermato UAS-BVR GAL4 X-GFP linea trappola enhancer isolato come descritto 4 (per sintesi vedi fig. 1) e le linee di wild-type o parentale vengono seminate sul terreno, ovvero ~ 2000 semi sterilizzati per riga. </li><li> Le piante sono coltivate per 5 settimane in terra sotto illuminazione bianca di 100 μmolm -2 s -1 a 22 ° C e 70% di umidità. </li></ol> 2. Foglia di protoplasti di isolamento (adattato da Denecke e Vitale 9) <ol><li> Per isolare protoplasti, preparare TEX buffer. Per buffer di 1 litro TEX, pesare i seguenti componenti: 3,1 g Gamborg di sali B5, 0,5 g di 2 - (N-morfolino) etano acido (MES, 2,56 mM), 0,75 diidrato g di calcio cloruro (CaCl 2 .2 H 2 O, 6,75 mM), 0,25 g di nitrato di ammonio (NH 4 NO 3, 3.12 mM), 136,9 g di saccarosio (0.4M). Sciogliere i componenti completamente in ~ 900 ml di acqua deionizzata, acqua distillata (DDH 2 O) e portare il pH a 5,7 con 1 M KOH. Portare il volume finale a 1 litro e sterilizzare con filtro da 0,2 micron per bottiglie con filtro collegato ad una pompa a vuoto. </li><li> Raccogliere verde, foglie di piante sane (~ 250 ml di foglie poco compresso in un bicchiere) e sciacquare con ~ 40 ml DDH 2 O 4 volte, seguita da risciacquo per due volte con sterili DDH 2 O. Usando un bisturi # 20, foglie tagliate a striscioline e si dividono equamente in due tessuti 50 ml, tubi di plastica sterile. Preparare 1x miscela digestione foglia aggiungendo 5 ml aliquota di 10x soluzione madre digestione foglia (10x digestione soluzione foglia magazzino: sciogliere 2% w / v Macerozyme R-10, 4% w / v Cellulase &quot;Onozuka&quot; R-10 nel TEX buffer, sterilizzare con filtro 0,2 micron filtro e blocco 5 aliquote ml a -80 ° C) a 45 ml di tampone fresco TEX. Aggiungi ~ 25 ml di 1x mix digestione foglia per tubo da 50 ml per coprire tutto il tessuto fogliare. </li><li> Vuoto infiltrarsi nel tessuto di foglie miscela digestione in tubi aperti per 1 ora a temperatura ambiente utilizzando un essiccatore a vuoto collegata ad una pompa d&#39;acqua seguita da 3-ore di incubazione a temperatura ambiente su una sedia a dondolo agitando delicatamente. Tubi ricoperto con foglia in tessuto misto 1x digestione foglia sono tenuti avvolti in carta stagnola durante questo processo per evitare l&#39;esposizione alla luce. </li><li> Dopo 3 ore, aumentare la velocità rocker per circa 2 minuti per rilasciare protoplasti. Filtrare la sospensione greggio protoplasti attraverso due strati di telo sterile per rimuovere i detriti e raccogliere il filtrato in un bicchiere di vetro sterile. Filtrare il filtrato attraverso una sterile da 100 micron mesh di nylon in una piastra di Petri sterile. Raccogliere il flusso attraverso e trasferirlo ad un nuovo sterile tubo da 50 ml. Usa circa 15-20 ml di tampone fresco TEX a lavare i piastra di Petri sterile e raccogliere qualsiasi protoplasti aderire alla superficie. </li><li> Centrifugare il flusso attraverso usando un rotore a rotazione 100xg a 10 ° C per 15 minuti (6 accelerazione, decelerazione 0). </li><li> Rimuovere ~ 25 - 30 ml di liquido al di sotto dello strato protoplasti fluttuante, che contiene residui e pellet, con una sterile 9 &quot;pipetta Pasteur di vetro collegata ad una pompa peristaltica senza disturbare il livello fluttuante protoplasti e lasciando ~ 10-15 ml. </li><li> Aggiungi nuovo tampone TEX per un volume finale di 40 ml, mentre delicatamente risospendere l&#39;protoplasti. </li><li> Ripetere i passaggi 2,5-2,7 due volte per rimuovere il più possibile di detriti cellulari. Il tempo di centrifugazione è ridotto a 10 minuti nella prima ripetizione ed a 5 minuti nella ripetizione finale. </li><li> Aspirare protoplasti galleggiante con un taglio, 1 pipetta sterile trasferire ml in un nuovo tubo 15 ml. Procedere direttamente alla cernita o conservare al buio per un massimo di 2 ore a 4 ° C fino ordinamento. </li></ol> 3. Ordinamento protoplasti da Fluorescence-Activated ordinamento Cell (FACS) <ol><li> Prima di smistamento, esaminare protoplasti da microscopia confocale a scansione laser (CLSM) utilizzando un laser a 488-nm per l&#39;eccitazione per confermare l&#39;integrità protoplasti e la presenza di fluorescenza GFP in piscina protoplasti. Detriti minimo è necessario per evitare l&#39;intasamento l&#39;ordinamento FACS ugello. </li><li> Protoplasti sorta isolato in TEX tampone tramite FACS (BD FACSVantage SE, BD Biosciences) con un 200-micron ugello su una testa tipo macro a tassi di eventi tra 6.000 e 15.000, con una pressione del sistema di circa 9 psi a seguito di un adeguato protocollo 10. </li><li> Wild-type non GFP protoplasti vengono utilizzati per determinare le soglie di autofluorescenza. </li><li> Per raccogliere GFP-positive protoplasti, le cellule utilizzando un tipo raffreddato ad aria, argon laser (Spectra Physics Modello 177, Newport Corporation, Irvine, CA), gestito a 100 mW a 488-nm linea argon per identificare GFP fluorescenza mediante una band 530/30 filtro passa. </li><li> Dopo la raccolta di GFP-positive protoplasti, esaminare protoplasti ordinati per CLSM come descritto al punto 3.1. </li><li> Estrarre l&#39;RNA totale da protoplasti ordinati utilizzando un impianto di Qiagen RNeasy Mini kit. cDNA possono quindi essere pronti per l&#39;ibridazione, come descritto nella Affymetrix GeneChip Expressione manuale analisi tecnica e poi ibridato a ATH1 Arabidopsis array Affymetrix intero genoma. </li></ol> Rappresentante Risultati Abbiamo rilevato un numero significativo di cellule GFP-positive nel canale GFP da FACS (Fig. 2). Nei test di ottimizzazione usando GFP enhancer-trap genitori, la GFP che esprimono costitutivamente linea, J0571, visualizzati ~ 17% al 24% GFP-positive protoplasti, mentre la linea con l&#39;espressione vascolare e cutaneo, J1071, aveva ~ 1,4% GFP-positive protoplasti (Tabella 1). Ordinamento di 3 x 500 microlitri (~ 1,5 ml di sospensione totale di protoplasti) di J0571 protoplasti per 1 ora ha dato ~ 100.000 GFP-positive protoplasti. Ordinamento di 3 x 500 microlitri (~ 1,5 ml sospensione di protoplasti totale) di J1071 protoplasti per 1,5 h dato ~ 3.000 GFP-positive protoplasti. Immagini confocale indicato un rendimento molto elevato di protoplasti per campioni sia J0571 e J1071 prima di ordinare è stata effettuata (Fig. 3C e 3E), e le frazioni ordinati conteneva solo luminoso fluorescente GFP-protoplasti (Fig. 3G e dati non riportati). Questa scoperta conferma che intatti GFP-positive protoplasti possono essere ordinati tramite FACS. La non-GFP protoplasti possono anche essere ordinati e raccolti in un canale separato. La non-GFP protoplasti servire come controllo negativo ideale per analisi microarray successive per rilevare i cambiamenti specifici nell&#39;espressione genica con la riduzione dei livelli holophytochrome. Estrazione di RNA da protoplasti isolati (1 ml) RNA produce in quantità sufficiente per il rilevamento da parte fluorospectrometry (Fig. 4). Isolato l&#39;RNA è stata valutata utilizzando uno strumento NanoDrop (NanoDrop 1000, Thermo Scientific) e quantificato NanoDrop 3.7.1 del software quantificazione RNA. Rendimenti RNA da pre-ordinati C24 wild-type protoplasti o FACS-ordinati, GFP-positive protoplasti trappola enhancer superato il minimo di 20 ng necessaria per l&#39;utilizzo in RNA di etichettatura per i test microarray (Tabella 2). <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Enhancer Trappola di linea </td><td> % Di GFP-positive protoplasti </td><td> Numero di protoplasti GFP ordinati </td></tr><tr><td> J0571 </td><td> 17,18% ~ 24,06% </td><td> 26.400 ~ 36.000 </td></tr><tr><td> J1071 </td><td> ~ 1,43% </td><td> 1000 ~ 1300 </td></tr></tbody></table> Tabella 1. Percentuale di GFP-positive protoplasti da due linee trappola potenziatore prima selezione e il numero di GFP-positive protoplasti raccolti eseguendo 500 microlitri di sospensione di protoplasti attraverso l&#39;Ordine fluorescenza cellulare attivato (FACSVantage, BD). <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Impianti di linea </td><td> RNA rendimento (ng) </td></tr><tr><td> C24 WT </td><td> 12486,5 </td></tr><tr><td> J0571 </td><td> 60 </td></tr><tr><td> J1071 </td><td> 71,5 </td></tr></tbody></table> Tabella 2. Resa dall&#39;isolamento RNA da protoplasti. L&#39;RNA è stato isolato da pre-ordinati C24 wild-type o ordinati GFP-positive protoplasti da due linee trappola potenziatore e quantificati. <img src="/files/ftp_upload/1925/1925fig1.jpg" alt="Figura 1" /> Figura 1. GAL4 enhancer-trap a base di induzione della biliverdina reduttasi (BVR) espressione in piante transgeniche di Arabidopsis thaliana. (A). Un individuo selezionato da una libreria di GAL4 trappola a base di linee enhancer, che contengono un GAL4-reattiva gene marcatore GFP, può essere attraversata con una linea contenente un GAL4-reattiva gene bersaglio di indurre l&#39;espressione del gene bersaglio in GAL4 contenenti cellule segnato fluorescenza di GFP. Sulla base di una figura del Dr. Jim Haseloff (http://www.plantsci.cam.ac.uk/Haseloff/geneControl/GAL4Frame.html). (B) Produzione di fitocromo cromoforo, phytochromobilin (PΦB) e holophytochrome in linee parentali. (C) Sinistra, Riduzione della biliverdina IXα (BV IXα) e PΦB da biliverdina reduttasi (BVR) attività di BR e PΦR, rispettivamente. Risultati dell&#39;attività BVR a esaurimento delle PΦB e porta ad una riduzione nella produzione di fotoattivi holophytochrome. A destra, la reazione catalizzata da BVR è mostrato. <img src="/files/ftp_upload/1925/1925fig2.jpg" alt="Figura 2" /> Figura 2. Fluorescenza separazione delle cellule attivate (FACS) appezzamenti punto di acquisizione. Confronto di celle protoplasti di ordinamento per C24 di tipo selvatico (A), J0571 (B) e J1071 (C). (A) Acquisizione di dot plot non-C24 fluorescente GFP wild-type protoplasti eccitati da un 488-nm argon laser e utilizzato per determinare la soglia autofluorescenza. (B) e (C) appezzamenti dot acquisizione mostrano proporzioni di protoplasti che sono GFP positive in risposta ad eccitazione di un 488-nm laser. R3 cancelli ordinamento in B e C delimitano la GFP-positive obiettivi che sono stati ordinati per Fluorescence-Activated cell sorter (FACSVantage, BD) e raccolti. Canale rosso indica vALORI per autofluorescenza clorofilla da protoplasti e il canale verde indica valori di fluorescenza GFP. <img src="/files/ftp_upload/1925/1925fig3.jpg" alt="Figura 3" /> Figura 3. Microscopia confocale di protoplasti pianta usata per l&#39;ordinamento delle cellule. Confocale a scansione laser immagini di protoplasti prima (A, C, E) e dopo (G) ordinamento tramite fluorescenza-Activated cell sorter (FACS). B, D, F e H sono immagini DIC. Protoplasti di C24 wild-type (A, B), J1071 (C, D) e J0571 (E, F, G, H) sono mostrati. Immagini C, E e G si fondono immagini di fluorescenza GFP (BP 505 nm - 575 nm) e autofluorescenza (LP 650 nm) ottenuti da eccitazione con un 488-nm laser. Da A ad H sono nella media di 4 scansioni sott&#39;olio 63x. Bar = 10 micron. <img src="/files/ftp_upload/1925/1925fig4.jpg" alt="Figura 4" /> Figura 4. Quantificazione di RNA isolati da protoplasti da fluorospectrometry. RNA estratto da (A) tipo C24 selvatico, (B) J0571, e (C) J1071. A indica RNA per la pre-ordinati wild-type protoplasti. B e C indicare RNA da GFP-positive protoplasti ordinati per Fluorescence-Activated ordinamento Cell (FACS). RNA software quantificazione, NanoDrop 3.7.1, (NanoDrop 1000, Thermo Scientific).

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Profili di espressione genica attraverso microarray (1) ha indicato che oltre il 30% dei geni in piantine di Arabidopsis sono regolati luce 11 e (2) ha identificato un vasto gruppo di geni che codificano le proteine ​​luce di trasduzione del segnale coinvolte nella cascata di segnalazione fitocromo 12, 13 . Tali esperimenti suggeriscono che la luce induce cambiamenti rapidi e lungo termine espressione genica. Ogni pool di fitocromi può controllare solo un sottoinsieme di risposte di sviluppo e...

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<td>Anti-BVR antibody</td>
<td>&nbsp;</td>
<td>QED Bioscience Inc.</td>
<td>56257-100</td>
<td>&nbsp;</td>
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<td>Cellulase &#x201C;Onozuka&#x201D; R-10</td>
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<td>SERVA Electrophoresis GmbH, Crescent Chemical Company</td>
<td>MSPC 0930</td>
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<td>Gamborg&#x2019;s B5 basal salt mixture</td>
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<td>Sigma</td>
<td>G5768</td>
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<td>Macerozyme R-10</td>
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<td>SERVA Electrophoresis GmbH, Crescent Chemical Company</td>
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<td>MES, low moisture content</td>
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<td>Murashige and Skoog salts</td>
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<td>Caisson Laboratories</td>
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<td>Phytablend</td>
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<td>Caisson Laboratories</td>
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<td>RNeasy Plant Minikit</td>
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<td>Qiagen</td>
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investigating tissue- and organ-specific phytochrome responses using facs-assisted cell-type specific expression profiling in arabidopsis thaliana

Media luce una serie di processi evolutivi e adattativi per tutto il ciclo di vita di una pianta. Le piante utilizzano la luce di assorbimento delle molecole chiamate fotorecettori di senso e di adattarsi alla luce. Il rosso / lontano-rosso che assorbono la luce fotorecettori fitocromo sono stati studiati ampiamente. Fitocromi esistere come una famiglia di proteine ​​con funzioni diverse e sovrapposte in tutti i sistemi vegetali superiori in cui sono stati studiati<sup&gt; 1</sup&gt;. Fitocromo-mediata risposte luce, che vanno dalla germinazione del seme attraverso la fioritura e la senescenza, sono spesso localizzate a specifici tessuti vegetali o organi<sup&gt; 2</sup&gt;. Nonostante la scoperta e la spiegazione delle funzioni individuali e fitocromo in esubero mediante analisi mutazionale, report conclusivi su siti distinti photoperception e dei meccanismi molecolari di piscine localizzata di fitocromi che mediano le risposte fitocromo spazio-specifici sono limitati. Abbiamo progettato esperimenti basati su ipotesi che i siti specifici di photoperception fitocromo regolano tessuti e organi specifici aspetti della fotomorfogenesi, e che le piscine localizzato fitocromo impegnarsi distinti sottoinsiemi di geni bersaglio a valle in cellula-segnalazione cellulare. Abbiamo sviluppato un approccio biochimico per ridurre selettivamente fitocromi funzionale in un organo o tessuto-specifica all&#39;interno di piante transgeniche. I nostri studi si basano su un bipartito enhancer-trap approccio che si traduce in transattivazione dell&#39;espressione di un gene sotto il controllo della sequenza di attivazione a monte (UAS) elemento dal attivatore trascrizionale GAL4<sup&gt; 3</sup&gt;. La biliverdina reduttasi (<em&gt; BVR</em&gt; Gene) sotto il controllo delle scuole universitarie professionali è mantenuto in silenzio, in assenza di GAL4 transattivazione nella UAS-BVR genitore<sup&gt; 4</sup&gt;. Incroci genetici tra un UAS-BVR linea transgenica e un GAL4-GFP risultato trappola enhancer linea in un&#39;espressione specifica del<em&gt; BVR</emGene&gt; nelle cellule segnato da<em&gt; GFP</em&gt; Espressione<sup&gt; 4</sup&gt;. Accumulo BVR in Arabidopsis risultati piante in carenza cromoforo fitocromo<em&gt; In planta</em<sup&gt; 5-7</sup&gt;. Così, piante transgeniche che abbiamo prodotto mostra GAL4-dipendente l&#39;attivazione del<em&gt; BVR</em&gt; Gene, con conseguente inattivazione della biochimica fitocromo, così come GAL4-dipendente<em&gt; GFP</em&gt; Espressione. Fotobiologiche analisi genetiche e molecolari<em&gt; BVR</em&gt; Linee transgeniche stanno dando comprensione tessuto e organo-specifiche fitocromo-mediata risposte che sono state associate con siti corrispondenti di photoperception<sup&gt; 4, 7, 8</sup&gt;. Fluorescenza separazione delle cellule attivate (FACS) di GFP-positive, enhancer-trap-indotta<em&gt; BVR</em&gt; Che esprimono protoplasti impianto accoppiato con cellula-tipo-specifici profili di espressione genica attraverso l&#39;analisi microarray viene utilizzato per identificare i putativi geni target a valle coinvolti nella mediazione risposte fitocromo spazio-specifiche. Questa ricerca sta ampliando la nostra comprensione dei siti di percezione della luce, i meccanismi attraverso i quali diversi tessuti o organi cooperare in luce regolata la crescita delle piante e lo sviluppo, e avanzando la dissezione molecolare dei complessi fitocromo-mediata cellula-cellula cascate di segnalazione.

Watch this Scientific Journal Video about Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana at JoVE.com

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Le basi molecolari delle risposte fitocromo spazio-specifica è indagato con le piante transgeniche che presentano tessuti e carenze fitocromo organo-specifiche. L&#39;isolamento di cellule specifiche esporre indotte esaurimento fitocromo cromoforo da Fluorescence-Activated cella Ordinamento seguita da analisi di microarray viene utilizzata per identificare i geni coinvolti nello spazio-risposte specifiche fitocromo.

Indagare tessuti e risposte fitocromo organo-specifici utilizzando FACS-Cell-assistita di tipo profili specifici di espressione in Arabidopsis thaliana</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

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Research

JoVE Journal

Biology

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

15.7K Views.  Michigan State University (MSU). Le basi molecolari delle risposte fitocromo spazio-specifica è indagato con le piante transgeniche che presentano tessuti e carenze fitocromo organo-specifiche. L'isolamento di cellule specifiche esporre indotte esaurimento fitocromo cromoforo da Fluorescence-Activated cella Ordinamento seguita da analisi di microarray viene utilizzata per identificare i geni coinvolti nello spazio-risposte specifiche fitocromo.

Video: Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

Floreale-dip Trasformazione Arabidopsis thaliana Per esaminare PTSO2:: β-glucuronidasi Reporter espressione genica

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Ricerca

Biologia

Heterokaryon Technique for Analysis of Cell Type-specific Localization

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling. These proteins display a diverse array of cellular functions including nuclear import/export of RNA and protein, transcriptional regulation, and apoptosis. Interestingly, major cellular reorganizations including cell division, differentiation and transformation, often involve such activities3,4,8,10. The detailed study of these proteins and their respective regulatory mechanisms can be challenging as the stimulation for these localization changes can be elusive, and the movements themselves can be quite dynamic and difficult to track. Studies involving cellular oncogenesis, for example, continue to benefit from understanding pathways and protein activities that differ between normal primary cells and transformed cells6,7,11,12. As many proteins show altered localization during transformation or as a result of transformation, methods to efficiently characterize these proteins and the pathways in which they participate stand to improve the understanding of oncogenesis and open new areas for drug targeting.
Here we present a method for the analysis of protein trafficking and shuttling activity between primary and transformed mammalian cells. This method combines the generation of heterokaryon fusions with fluorescence microscopy to provide a flexible protocol that can be used to detect steady-state or dynamic protein localizations. As shown in Figure 1, two separate cell types are transiently transfected with plasmid constructs bearing a fluoroprotein gene attached to the gene of interest. After expression, the cells are fused using polyethylene glycol, and protein localizations may then be imaged using a variety of methods. The protocol presented here is a fundamental approach to which specialized techniques may be added.

A flexible and efficient method for the characterization of cell type-specific protein localization and nucleocytoplasmic shuttling is described. This heterokaryon approach uses fluorescently-labeled fusion proteins to image protein localizations after cell fusion. The protocol is amenable to steady-state localizations or more dynamic determinations based on live cell imaging.

Tecnica Heterokaryon per l&#39;analisi di cellule tipo-specifica localizzazione

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling.  These proteins display a diverse array of ...

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

Arabidopsis thaliana Profiling Glycerolipid Polar Cromatografia su strato sottile (TLC) Accoppiato con cromatografia gas-liquido (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

Cella di analisi specifica di<em&gt; Arabidopsis</em&gt; Foglie usando fluorescenza activated cell sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

Isolamento efficiente e rapida di embrioni nelle prime fasi di<em&gt; Arabidopsis thaliana</em&gt; Semi

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

Colorazione istochimica di<em&gt; Arabidopsis thaliana</em&gt; Cellula secondaria Elementi da parete

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Risoluzione Quantificazione alta del cristallino cellulosa accumulo in<em&gt; Arabidopsis</em&gt; Roots a Monitor tessuto-specifica delle cellule parete Modifiche

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can &#8211; in contrast to fluorescent lights &#8211; be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Here, we present a protocol for studying plant growth behavior and especially phenotypes in a reproducible manner. We show how to provide variable and at the same time stable light conditions. Proper analyses depend on sufficient sample numbers and valid statistical evaluations.

Analisi del comportamento di crescita di Arabidopsis thaliana in diverse qualità di luce

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

TChIP-Seq: Cellula-tipo-specifico Epigenome profilatura

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Profiling dell'espressione genica specifica del tipo di cella nel fegato del mouse

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...