Il lavoro portato a questa pubblicazione ha ricevuto finanziamenti dal programma Marie Curie COFUND &quot;U-Mobility&quot;, cofinanziato dall&#39;Università di Malaga e il programma dell&#39;Unione europea 7 ° programma quadro (FP7/2007-2013) sotto GA No. 246550. Il lavoro nel nostro laboratorio è sostenuto da sovvenzioni dal NIH, USDA / NIFA, NSF, BARD, e BSF a VC

1. Espressione della proteina <ol><li> Scelta del sistema di espressione Selezionare il sistema, cioè, vettori e vettore metodo di consegna, più adatto per l&#39;espressione della proteina di interesse nella organismo modello / cella specifica. Si noti che il nostro test richiede l&#39;espressione delle proteine ​​testate in quantità facilmente rintracciabile, che è meglio ottenere con la trasformazione temporanea di un gran numero di cellule. Nelle piante, per esempio, questo è migliore, utilizzare plasmidi binari come vettori di espressione e Agrobacterium come sistema di consegna. </li><li> Costruzione di vettori di espressione binari Clonare la sequenza codificante (s) della proteina (s) di interesse in un vettore di espressione da soli o in fusione traslazionale di un epitopo. Uso vettori adatti per il sistema di espressione scelto e procedure di biologia molecolare standard per la clonazione del gene. Per la consegna del gene mediata da Agrobacterium, impiegare vettori binari e far loro conoscere, utilizzando anche Standaprotocolli rd, in un ceppo Agrobacterium, come EHA105, per la successiva inoculazione di tessuti vegetali. </li><li> La scelta delle specie vegetali Selezionare le specie vegetali in cui verrà espressa la proteina (s) di interesse. Si noti che, mentre è possibile usare qualsiasi specie di piante sensibili alla trasformazione genetica mediata da Agrobacterium, la nostra specie vegetali di scelta è Nicotiana benthamiana, che è facilmente coltivata, altamente suscettibile Agrobacterium, e ha foglie grandi che sono facilmente inoculati. </li><li> La crescita delle piante Crescere un impianto da 4 a 6 settimane in una pentola con Pro-Mix BX in una pentola (20 centimetri x 20 centimetri x 20 cm) in condizioni eco-controllati (es. camera di crescita) di giorno lungo (cioè 16 h 130 -150 μE m-2 s-1 luce a 23 ° C e 8 h al buio a 20 ° C) e 40-65% di umidità relativa. 1.5.2 Concimazione tanto in tanto con i prodotti disponibili in commercio per le istruzioni del produttore. Una volta che la pianta viene coltivata, selezionarefoglie con le dimensioni di 50 mm x 70 mm o più grandi (queste misure di lunghezza non includono il picciolo) per Agrobacterium inoculazione. </li><li> Inoculazione con Agrobacterium Crescere ceppo Agrobacterium ospitare il costrutto binario esprimente la proteina (s) testato dal punto 1.3 notte a 28 ° C in mezzo YEP (1% peptone, estratto di lievito 1%, e 0,5% NaCl) supplementato con antibiotici appropriati (per esempio, 100 mg / L streptomicina e 10 mg / L rifampicina per vettori basati pPZP-RSC2 24-25). 1.6.2 Centrifugare le cellule, risospese a OD 600 = 0.5 in tampone infiltrazione [10 mM MgCl 2, 10 Mes mM (pH 5,6), 100 mM acetosyringone], e incubare per 2 ore a temperatura ambiente. Infiltrarsi nella cultura nel lato abaxial di una foglia, utilizzando una siringa senz&#39;ago 1-mL; notare che le foglie vengono inoculati in situ, mentre è attaccato alla pianta. 1.6.3 Dopo l&#39;infiltrazione, coltivare la pianta per 72 ore sotto il regime luce di 16 h 130-150 &amp;# 181, E m-2 s-1 luce a 23 ° C / 8 h al buio a 20 ° C prima della raccolta. </li><li> Applicazione di inibitori del proteasoma Per sostenere l&#39;idea che la proteina (s) testato è degradato attraverso una via del proteasoma, utilizzare specifici inibitori del proteasoma MG132 e lactacistina 28-29, che dovrebbe ridurre o addirittura bloccare il degrado. Quattro ore prima della raccolta (cfr. punto 2.1), infiltrarsi nelle zone foglia Agrobacterium-inoculati con 10 pM MG132 (EMD Millipore) o 10 micron lactacistina (Sigma-Aldrich) o mock-trattare la foglia con le rispettive solventi, cioè lo 0,1% DMSO o acqua distillata. </li></ol> 2. Preparazione di estratti cell-free <ol><li> Leaf raccolta Raccogliere le zone inoculati della foglia, di solito ca. 200-400 mg di peso fresco, e li macinare in polvere fine in azoto liquido. In alternativa, bead-battere i tessuti, utilizzando qualsiasi bead-beater (ad esempio, Biospec) o amalgamator dentale (ad esempio, TPC avanzata<html> Technologies) direttamente nel buffer di degradazione (cfr. punto 2.2). </li><li> Estrazione delle proteine Preparare l&#39;estratto proteico totale posizionando il tessuto macinato in 500 ml di tampone degradazione [50 mM Tris-HCl (pH 7,5), NaCl 100 mM, 10 mM MgCl 2, 5 mM DTT, adenosina 5 mM 5&#39;-trifosfato, e inibitore della proteasi 1x cocktail (Sigma-Aldrich)]. Si noti che il cocktail di inibitori delle proteasi colpisce principalmente serina, cisteina, aspartico, e metalloproteasi e non interferisce con la proteasi 26S. Chiarire l&#39;estratto da due centrifugazioni sequenziali a 12.000 xg per 5 min. </li><li> Reazione di degradazione delle proteine Trasferire volumi uguali di estratti, di solito 20 microlitri, per microcentrifuga tubi e incubare a temperatura ambiente per periodi di tempo crescente. Tipicamente, il tempo di campionamento pari a zero, e 5, 10, 15, 20, e 30 min punti temporali. Smettere di reazioni facendo bollire in SDS tampone di gel-sample, e analizzarli mediante western blotting. </li></ol>jove_title &quot;&gt; 3. Rilevamento degradazione della proteina mediante immunoblotting <ol><li> Elettroforesi su gel 3.1.1 risolvere i campioni di proteine ​​mediante elettroforesi su gel di SDS-poliacrilammide e electrotransfer le proteine ​​deliberato di una membrana di nitrocellulosa secondo il protocollo standard 30. 3.1.2 Determinare concentrazione proteica utilizzando il metodo di Bradford (Bio-Rad), e caricare 50-80 mg di proteine ​​totali per corsia. Assicurarsi che tutti i campioni sono stati caricati allo stesso modo. Per i controlli di carico, confrontare le intensità di una specie di proteine ​​ubiquitarie, come putativo Rubisco grandi catene che migrano come una band importante con una mobilità elettroforetica relativa di circa 50 kDa, possono essere rilevati Coomassie gel-blu macchiato o Ponceau S-o fluorescente SYPRO Ruby-tinto membrane di nitrocellulosa. </li><li> Blocco Bloccare la membrana con 5% di latte scremato in TBST (10 mM Tris-HCl, 140 mM NaCl, 0,05% Tween 20, pH 7,4) per 1 ora a temperatura ambiente. </li&gt; <li> Anticorpo primario Diluire anticorpo anti-epitopo primario in 1% di latte scremato in TBST alla concentrazione raccomandato dal produttore e incubare con la membrana bloccata per 1 ora a temperatura ambiente o per una notte a 4 ° C con agitazione. </li><li> Risciacquo Sciacquare la membrana viene sciacquato con 20 ml TBST per 15 minuti una volta e due volte per 5 minuti a temperatura ambiente con agitazione. </li><li> Anticorpo secondario Diluire anticorpo secondario (ad esempio IgG anti-coniglio) coniugato con perossidasi di rafano (HRP) in 1% di latte scremato in TBST come raccomandato dal fabbricante e incubare con la membrana per 1 ora a temperatura ambiente con agitazione. </li><li> Rivelazione Sciacquare nuovamente la membrana come descritto al punto 3.4. Dopo il risciacquo finale in TBST, visualizzare le proteine ​​di interesse, con un substrato chemiluminescente HRP, più comunemente utilizzando un kit ECL. </li></ol>

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Questo test si basa sulla espressione delle proteine ​​testati in tessuti vegetali, quindi, il potenziale processo di degradazione proteasoma avviene ovviamente già all&#39;interno dei tessuti viventi. Abbiamo protein assay destabilizzazione, tuttavia, solo negli estratti, con il tempo di campionamento di zero che serve come punto di riferimento iniziale. Quindi, definiamo come un saggio privo di cellule. Un aspetto importante per il successo di questo test è la corretta scelta del vet...

Il proteasoma percorso ubiquitin/26S sta emergendo come un meccanismo diffuso per il controllo di diverse reazioni biologiche, tra cui regolazione trascrizionale, la progressione del ciclo cellulare e trasduzione del segnale, recettori down-regulation o endocitosi, tra gli altri processi 1-4. In questo percorso, la proteina bersaglio viene etichettato da ubiquitina residui che vengono prima collegati tramite un legame thiolester to-ubiquitina attivando l&#39;enzima E1 e poi traslocato a un residuo di acido cisteina amino ubiquitina-coniugazione enzima E2, infine, E2 interagisce con ubiquitina ligasi E3 , con conseguente polyubiquitination del substrato proteico. In definitiva, le proteine ​​polyubiquitinated sono riconosciuti e degradati dal proteasoma 26S. In questo meccanismo, l&#39;enzima E3 specifica il substrato e funge da elemento regolatore chiave del sistema proteasoma ubiquitin/26S (UPS). Ligasi E3 possono agire in modo indipendente, come ligases dominio RING, o come parte di un SCF multisubunit (S) complesso proteico kp1/Cullin/F-box, come ad esempio F-box ligases dominio. SCF-mediate vie di degradazione del proteasoma sono coinvolti nella regolazione della trascrizione, ciclo cellulare, la trasduzione del segnale 5-10 e molte altre importanti funzioni cellulari. Oltre a questi ruoli critici nella regolazione dei processi cellulari, UPS prende il palco centrale in molte interazioni pianta-patogeno. Ad esempio, una crescente evidenza suggerisce che diversi agenti patogeni delle piante, tra cui Agrobacterium tumefaciens, si basano su l&#39;UPS host per per facilitare il processo di infezione 11. Agrobacterium provoca escrescenze neoplastiche sulle piante, che rappresentano i suoi ospiti naturali, e può anche trasformare una vasta gamma di altri eucarioti, da funghi 1,2 a cellule umane 12,13. Durante la sua infezione, Agrobacterium esporta un elemento DNA (T-DNA) e vari virulenza (vir) proteine ​​nella cellula ospite 12-13. Una di queste proteine ​​è VirF, la prima proteina F-box trovatiessere codificati da un genoma procariotico 14. Come parte del complesso SCF ubiquitina ligasi, VirF, e il suo omologo ospitante funzionale VBF 15, facilitare infezione Agrobacterium tramite la degradazione delle proteine ​​UPS-mediata, che facilita presumibilmente uncoating del invadere batterica T-DNA dalle proteine ​​batteriche e ospitanti accompagnamento, VirE2 e VIP1 rispettivamente 16,17. È interessante notare che molte proteine ​​F-box, tra cui VirF, sono intrinsecamente instabili a causa della loro proteolisi, che è mediata da attività autoubiquitination 18,19 o da altri ligasi E3 per i quali le proteine ​​F-box possono fungere da substrati 20-23. Quando si studia attività biochimiche delle proteine ​​F-box, altri ligasi ubiquitina, e / o le loro substrati, sarebbe molto utile un test semplice e affidabile per la degradazione del proteasoma. Qui si descrive un tale protocollo per analizzare la stabilità della proteina in una cellula-Libero Sistema. In questo saggio, la stabilità del substrato UPS viene analizzato in presenza o assenza di uno dei componenti essenziali del percorso di degradazione proteasoma, come una proteina F-box, in un sistema acellulare. In generale, esprimiamo la proteina (s) testato in tessuti vegetali, preparare estratti privi di cellule di questi tessuti e monitorare la quantità di proteina (s) di interesse mediante western blotting. Il meccanismo UPS-dipendente di degradazione proteica è dimostrata dalla inclusione di inibitori del proteasoma specifici e / o tramite coespressione di forma dominante negativa di un componente SCF, Cullin. Considerando che illustriamo questo dosaggio utilizzando proteasoma degradazione della proteina Arabidopsis VIP1 17 dalla proteina F-box VBF 15, può essere impiegato per studiare la stabilità di eventuali altri substrati del proteasoma.

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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Protein assay kit</td>
			<td style="width:129px;">Bio-Rad</td>
			<td style="width:119px;">500-0001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Proteinase inhibitor cocktail&#160;</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td style="width:119px;">S8820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mini-Protean system</td>
			<td style="width:129px;">Bio-Rad</td>
			<td style="width:119px;">165-8000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Semi-dry western blotting SD electrotransfer system</td>
			<td style="width:129px;">Bio-Rad</td>
			<td style="width:119px;">170-3940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Affinity Purified Rabbit Anti-Ha</td>
			<td style="width:129px;">icllab</td>
			<td>RHGT-45A-Z</td>
			<td></td>
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			<td height="42" style="height:42px;width:247px;">Goat anti-Rabbit IgG Peroxidase Conjugate</td>
			<td style="width:129px;">Thermo Scientific</td>
			<td style="width:119px;">31460</td>
			<td></td>
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			<td height="42" style="height:42px;width:247px;">BioTrace, NT nitrocellulose transfer membrane</td>
			<td style="width:129px;">Pall Corportation</td>
			<td style="width:119px;">27377-000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Immobilon western chemiluminescent HRP substrate</td>
			<td style="width:129px;">EMD Millipore</td>
			<td style="width:119px;">WBKL S0 050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">MG132</td>
			<td style="width:129px;">EMD Millipore</td>
			<td>474790-1MG</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Lactacystin</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td>L6785</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Thermo Scientific Pierce Fast Western Blot Kit, ECL Substrate</td>
			<td style="width:129px;">Pierce</td>
			<td style="width:119px;">35055</td>
			<td></td>
		</tr>
	</tbody>
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assaying proteasomal degradation in a cell-free system in plants

Il pathway ubiquitina-proteasoma per la degradazione delle proteine ​​è emerso come uno dei meccanismi più importanti per la regolazione di un ampio spettro di funzioni cellulari in quasi tutti gli organismi eucarioti. In particolare, nelle piante, il sistema proteasoma ubiquitin/26S (UPS) regola la degradazione delle proteine ​​e contribuisce significativamente allo sviluppo di una vasta gamma di processi, compresa la risposta immunitaria, lo sviluppo e la morte cellulare programmata. Inoltre, sempre più evidenze suggeriscono che numerosi agenti patogeni delle piante, quali Agrobacterium, sfruttano l&#39;UPS di accoglienza per l&#39;infezione efficiente, sottolineando l&#39;importanza della continuità nelle interazioni pianta-patogeno. La specificità di substrato della UPS è raggiunto dalla ubiquitina ligasi E3 che agisce di concerto con la E1 e E2 ligasi di riconoscere e marcare molecole proteiche specifici destinati a degradazione da dandole catene di molecole di ubiquitina. Una classe delle ligasi E3 è la SCF (Skp1 / Cproteina Ullin / F-box) complessa, che riconosce specificamente i substrati UPS e obiettivi per l&#39;ubiquitinazione attraverso la sua componente proteica F-box. Per studiare il ruolo potenziale di UPS in un processo biologico di interesse, è importante mettere a punto un saggio semplice e affidabile per degradazione proteica UPS-mediata. Qui, descriviamo un tale saggio usando un sistema acellulare pianta. Questo test può essere adattato per lo studio dei ruoli di degradazione delle proteine ​​regolamentato in diversi processi cellulari, con un focus particolare sulle interazioni proteina-substrato F-box.

Figura 1, adattato da Zaltsman et al. 17, illustra esperimenti rappresentativi per il rilevamento di degradazione del proteasoma in un sistema acellulare. In particolare, dimostriamo destabilizzazione di un impianto destinato alla difesa proteina VIP1 dalla proteina F-box VBF attraverso la via SCF VBF in N. benthamiana. Arabidopsis VBF e VIP1 (HA-VIP1) proteine ​​HA-tagged stati transitoriamente coespressi, e HA-VIP1 proteina contenuto all&#3...

Watch this Scientific Journal Video about Assaying Proteasomal Degradation in a Cell-free System in Plants at JoVE.com

Assaying Proteasomal Degradation in a Cell-free System in Plants

Degradazione delle proteine ​​mirata rappresenta un importante meccanismo di regolazione per la funzione delle cellule. Essa si verifica attraverso una via ubiquitina-proteasoma conservati, che attribuisce catene polyubiquitin alla proteina bersaglio che poi servire &quot;tag&quot; molecolari per il proteasoma 26S. Qui, descriviamo un saggio semplice e affidabile senza cellula per proteasoma degradazione delle proteine.

Saggio del proteasoma degradazione in un sistema di libero-Cell nelle piante

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

assaying-proteasomal-degradation-in-a-cell-free-system-in-plants

Research

JoVE Journal

Biology

14.3K Views.  Stony Brook University, State University of New York.  Degradazione delle proteine ​​mirata rappresenta un importante meccanismo di regolazione per la funzione delle cellule. Essa si verifica attraverso una via ubiquitina-proteasoma conservati, che attribuisce catene polyubiquitin alla proteina bersaglio che poi servire "tag" molecolari per il proteasoma 26S. Qui, descriviamo un saggio semplice e affidabile senza cellula per proteasoma degradazione delle proteine. 

Video: Assaying Proteasomal Degradation in a Cell-free System in Plants

Maintaining Wolbachia in Cell-free Medium

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ascertain the viability of the purified Wolbachia and (3) maintain the now extracellular Wolbachia in cell-free medium.  Purified Wolbachia remain alive in the extracellular phase but do not replicate until re-inoculated into eukaryotic cells.  Extracellular Wolbachia purified in this manner will remain viable for at least a week at room temperature, and possibly longer.  Purified Wolbachia are suitable for micro-injection, DNA extraction and other applications. 


This video describes a method for purifying Wolbachia pipientis from an Anopheles gambiae cell line and then culturing the endosymbiont in cell-free medium. An assay for viability of the bacterium is demonstrated.

Il mantenimento di Wolbachia in Cell-free medio

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ...

Ricerca

Biologia

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Saggiare l&#39;attività chinasi di LRRK2 In vitro</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &amp;#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

Ricostituzione della β-catenina degradazione nel<em&gt; Xenopus</em&gt; Estrai Egg

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

Measuring Diurnal Rhythms in Autophagic and Proteasomal Flux

Cells employ several methods for recycling unwanted proteins and other material, including lysosomal and non-lysosomal pathways. The main lysosome-dependent pathway is called autophagy, while the primary non-lysosomal method for protein catabolism is the ubiquitin-proteasome system. Recent studies in model organisms suggest that the activity of both autophagy and the ubiquitin-proteasome system is not constant across the day but instead varies according to a daily (circadian) rhythm. The ability to measure biological rhythms in protein turnover is important for understanding how cellular quality control is achieved and for understanding the dynamics of specific proteins of interest. Here we present a standardized protocol for quantifying autophagic and proteasomal flux in vivo that captures the circadian component of protein turnover. Our protocol includes details for mouse handling, tissue processing, fractionation, and autophagic flux quantification using mouse liver as the starting material.

We describe our protocol for measuring biological rhythms in protein catabolism via autophagy and the proteasome in mouse liver.

Misurazione dei ritmi diurni nel flusso autofagico e proteasomico

Cells employ several methods for recycling unwanted proteins and other material, including lysosomal and non-lysosomal pathways. The main ...

A Simple Protocol for Mapping the Plant Root System Architecture Traits

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar tolerance to environmental challenges. An experimental protocol is presented for setting up the hydroponic system, plantlet growth, RSA spreading, and imaging. The approach used a magenta box-based hydroponic system containing polypropylene mesh supported by polycarbonate wedges. Experimental settings are exemplified by assessing the RSA of the plantlets under varying nutrient (phosphate [Pi]) supply. The system was established to examine the RSA of Arabidopsis, but it is readily adaptable to study other plants like Medicago sativa (Alfalfa). Arabidopsis thaliana (Col-0) plantlets are used in this investigation as an example to understand the plant RSA. Seeds are surface sterilized by treating ethanol and diluted commercial bleach, and kept at 4 &#176;C for stratification. The seeds are germinated and grown on a liquid half-MS medium on a polypropylene mesh supported by polycarbonate wedges. The plantlets are grown under standard growth conditions for the desired number days, gently picked out from the mesh, and submersed in water-containing agar plates. Each root system of the plantlets is spread gently on the water-filled plate with the help of a round art brush. These Petri plates are photographed or scanned at high resolution to document the RSA traits. The root traits, such as primary root, lateral roots, and branching zone, are measured using the freely available ImageJ software. This study provides techniques for measuring plant root characteristics in controlled environmental settings. We discuss how to (1) grow the plantlets, and collect and spread root samples, (2) obtain pictures of spread RSA samples, (3) capture the images, and (4) use image analysis software to quantify root attributes. The advantage of the present method is the versatile, easy, and efficient measurement of the RSA traits.

We use simple laboratory tools to examine the root system architecture (RSA) of Arabidopsis and Medicago. The plantlets are grown hydroponically over mesh and spread using an art brush to reveal the RSA. Images are taken using scanning or a high-resolution camera, then analyzed with ImageJ to map traits.

Un semplice protocollo per mappare i tratti dell'architettura del sistema di root delle piante

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar ...

Quantificazione dell'infiltrazione adiposa del muscolo scheletrico mediante decellularizzazione

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

Autore in primo piano: Quantificazione basata sulla decellularizzazione dell'infiltrazione adiposa del muscolo scheletrico  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.

Quantificazione basata sulla decellularizzazione dell'infiltrazione grassa del muscolo scheletrico

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic ...

Incorporamento di reazioni CFPS in una varietà di matrici di idrogel

Methods for Embedding Cell-Free Protein Synthesis Reactions in Macro-Scale Hydrogels

Synthetic gene networks provide a platform for scientists and engineers to design and build novel systems with functionality encoded at a genetic level. While the dominant paradigm for the deployment of gene networks is within a cellular chassis, synthetic gene networks may also be deployed in cell-free environments. Promising applications of cell-free gene networks include biosensors, as these devices have been demonstrated against biotic (Ebola, Zika, and SARS-CoV-2 viruses) and abiotic (heavy metals, sulfides, pesticides, and other organic contaminants) targets. Cell-free systems are typically deployed in liquid form within a reaction vessel. Being able to embed such reactions in a physical matrix, however, may facilitate their broader application in a wider set of environments. To this end, methods for embedding cell-free protein synthesis (CFPS) reactions in a variety of hydrogel matrices have been developed. One of the key properties of hydrogels conducive to this work is the high-water reconstitution capacity of hydrogel materials. Additionally, hydrogels possess physical and chemical characteristics that are functionally beneficial. Hydrogels can be freeze-dried for storage and rehydrated for use later. Two step-by-step protocols for the inclusion and assay of CFPS reactions in hydrogels are presented. First, a CFPS system can be incorporated into a hydrogel via rehydration with a cell lysate. The system within the hydrogel can then be induced or expressed constitutively for complete protein expression through the hydrogel. Second, cell lysate can be introduced to a hydrogel at the point of polymerization, and the entire system can be freeze-dried and rehydrated at a later point with an aqueous solution containing the inducer for the expression system encoded within the hydrogel. These methods have the potential to allow for cell-free gene networks that confer sensory capabilities to hydrogel materials, with the potential for deployment beyond the laboratory.

Autore in primo piano: un nuovo approccio per l'incorporazione di reazioni di sintesi proteica cell-free negli idrogel 

Here, we present two protocols for embedding cell-free protein synthesis reactions in macro-scale hydrogel matrices without the need for an external liquid phase.

Metodi per incorporare reazioni di sintesi proteica cellulo-free in idrogel su macroscala

Synthetic gene networks provide a platform for scientists and engineers to design and build novel systems with functionality encoded at a genetic level. ...