Questo lavoro è stato finanziato dal programma Human Frontier Science concessione RGP0024.

1. Preparazione del DNA template e trascrizione in vitro <ol><li> Il gene di interesse viene clonato in qualsiasi T7 e / o ad esempio promotore SP6 base plasmide. Per ottenere RNC di interesse, C-terminale del polipeptide bersaglio viene esteso aggiungendo inducendo sequenza di arresto SECM FXXXXWIXXXXGIRAGP 7. Al fine di garantire che il frammento del polipeptide di interesse sarà estrudere fuori del tunnel ribosomiale, il C-terminale della proteina bersaglio deve essere ampliato di almeno 30 amminoacidi 12-14. Una glicina-serina linker flessibile ricco può essere introdotto tra la proteina e la sequenza arresto SECM evitare vincoli possibili conformazionali. </li><li> Per la trascrizione in vitro, DNA stampo deve essere linearizzato con l&#39;enzima di restrizione taglio a valle della ORF. Si deve verificare linearizzazione completa del DNA plasmide eseguendo il prodotto della digestione di restrizione elettroforesi su gel di agarosio. </li><li> Il plasma linearizzatoid è ulteriormente utilizzato per la reazione di trascrizione in vitro. Diversa concentrazione di DNA stampo può essere testato per identificare concentrazione ottimale di DNA necessarie per la trascrizione in vitro. Generalmente con Kit MEGAscript Ambion Trascrizione di alto rendimento (Ambion / Life Technologies, Grand Island, NY), 1 mg DNA linearizzato produce mRNA 40-60 mg. Trascrizione in vitro viene effettuata seguendo le istruzioni del fabbricante (Ambion / Life Technologies, Grand Island, NY) . </li><li> Dopo la trascrizione in vitro, mRNA viene purificato mediante precipitazione litio cloruro secondo le istruzioni del produttore (Ambion di MEGAscript alta Kit Trascrizione resa, Ambion / Life Technologies, Grand Island, NY). </li><li> integrità mRNA è ulteriormente verificato mediante elettroforesi utilizzando acrilammide o gel di agarosio. </li></ol> 2. Traduzione in vitro Per la traduzione in vitro utilizzando l&#39;RTS 100 E. coli HY Kit (5 Prime, Gaithersburg, MD), attenersi alla seguente procedura: <ol><li> Preparare 100 pl di istruzione in produttore traduzione in vitro la reazione successivo. In breve, in acqua priva di nucleasi aggiungere 24 microlitri miscela di aminoacidi meno metionina (1 mM), 10 unità di inibitore della ribonucleasi (Invitrogen / Life Technologies, Grand Island, NY), 20 uCi di sostanze radioattive [35 S] Biomedicals-metionina (MP, Solon, OH), 20 pl miscela di reazione, 24 microlitri tampone di ricostituzione e 24 pl di E. coli lisato. </li><li> Incubare la reazione a 30 ° C per 5 min a caldo pre la reazione di traduzione. Aggiungere 1-2 pg di mRNA alla reazione di traduzione e incubare a 30 ° C per 10-15 min. </li><li> Arrestare la reazione ponendo la reazione di traduzione in ghiaccio. </li></ol> 3. Isolamento del polipeptide nascente dalla reazione di traduzione in vitro <ol><li> Per isolare RNC, la reazione di traduzione è sovrapposto di 4,5 ml di saccarosio gradiente 5-30% in 20 mM HEPES pH 7-KOH0,5, 15 mM MgCl 2, 100 mM acetato di potassio, 1 mM DTT e centrifugati utilizzando Beckman Coulter SW55-Ti rotore a 41.000 rpm, 4 ° C per 2 ore. </li><li> Dopo centrifugazione, gradiente di saccarosio è frazionato per isolare diverse popolazioni ribosomali e le relative catene nascenti. La separazione viene monitorata utilizzando il sistema programmabile ISCO gradiente di densità con registrazione continua a 254 nm, utilizzando un rivelatore ISCO UA-6 assorbanza. </li><li> La frazione (s) contenente ribosomi 70S vengono raccolti e analizzati ulteriormente a seconda dello scopo dell&#39;esperimento. </li></ol> 4. Osservazione delle proteine ​​al ribosoma con Tris-tricina SDS PAGE Per verificare, se la proteina SECM estesa rimane attaccato agli anni &#39;70 ribosoma, frazioni del gradiente sono stati raccolti e trattati come segue: <ol><li> Proteine ​​in ciascuna frazione è stata precipitato per aggiunta di acido tricloroacetico (TCA) ad una concentrazione finale del 10% e incubate a 4 ° C overniGHT. </li><li> Dopo incubazione per una notte, i campioni sono stati centrifugati a 14.000 g per 15 minuti per precipitare la proteina. Dopo la centrifugazione supernatante è stato rimosso e il pellet lavata due volte con acetone soluzione contenente: 1 mM Tris-HCl pH 7,6 (4:1). Pellet è stato ulteriormente essiccati all&#39;aria e disciolto in SDS-PAGE tampone di caricamento. </li><li> Il campione trattato è stato risolto e analizzato mediante Tris-tricina SDS-PAGE. </li><li> Dopo l&#39;elettroforesi, gel sono stati fissati, essiccati sotto vuoto usando gel Dyer e sottoposto ad autoradiografia. La distribuzione dei polipeptidi nascenti stati osservati usando phosphoimaging. </li></ol> 5. Risultati rappresentativi Qui vi presentiamo un esperimento che descrive l&#39;isolamento del full-length bovina Gamma-B sonoro superiore cristallino stabilmente legata alle 70S dei ribosomi. La figura 1 illustra passaggi necessari per l&#39;isolamento di bovini Gamma-B cristallina RNC. Il C-terminale della Gamma-B sonoro superiore cristallino è stata estesa complessiva da 32 t aminoacidiO Assicurarsi che full-length estrude proteina fuori del tunnel ribosomale, questo include anche la sequenza SECM stallo posto al molto C-terminale del polipeptide di fusione. Seguendo traduzione in vitro, i ribosomi 70S sono stati isolati mediante centrifugazione gradiente di saccarosio (Figura 2.1). Al fine di garantire che la proteina rimane stabilmente legato al ribosoma, i 70S contenenti frazioni sono state riunite, dissalato, tampone scambiate e sottoposto ad una serie supplementare di centrifugazione gradiente di saccarosio (Figura 2.2). Il risultato illustrato nella figura 2 indica chiaramente che SECM può efficacemente indurre arresto traslazionale dei Gamma-B RNC cristallina. <img alt="Figura 1" src="/files/ftp_upload/4027/4027fig1.jpg" /> Figura 1. Spiegazione schematica i passi necessari per l&#39;isolamento di RNC. C-terminale di bovini Gamma-B sonoro superiore cristallino è stata prorogata dal 32 sequenza aminoacidica. Questa estesaC-terminale regioni comprende anche la sequenza SECM arresto. Gamma-B sonoro superiore cristallino con la sequenza SECM è stato clonato nel Pivex 2.3 (T7 based) plasmide. Per la trascrizione in vitro del DNA stampo è stato linearizzato con XbaI. Modello linearizzato è stato ulteriormente utilizzato per la trascrizione in vitro. Il T7 nel modello DNA è riconosciuto da RNA polimerasi T7 che trascrive le Gamma-B gene sonoro superiore cristallino a valle del promotore T7. mRNA è stato purificato usando cloruro di litio metodo di precipitazione. Il mRNA purificato è stato poi usato per traduzione in vitro. Dopo l&#39;incubazione, la reazione di traduzione è stato stratificato sopra del gradiente 5-30% di saccarosio e centrifugato in Beckman Coulter SW55-Ti rotore a 41.000 rpm, 4 ° C per 2 ore. <spuno class = &quot;pdflinebreak&quot;&gt; <img alt="Figura 2" src="/files/ftp_upload/4027/4027fig2.jpg" /> Figura 2. Isolamento dei bovini Gamma-B sonoro superiore cristallino RNC stabilmente legato al ribosoma 70S. 1 mRNA pg è stato miscelato in 100 microlitri di reazione E. coli sistema S30 estratto come descritto nella sezione protocollo con 20 uCi [35 S]-metionina e incubate a 30 ° C per 15 min. Dopo l&#39;incubazione, la reazione di traduzione è stato stratificato sopra del gradiente di saccarosio 5-30% in 20 mM HEPES-KOH pH 7,5, 15 mM MgCl 2, 100 mM di acetato di potassio, 1 mM DTT e centrifugato in Beckman Coulter SW55-Ti rotore a 41.000 rpm, 4 ° C per 2 ore. A seguito di velocità di sedimentazione, il gradiente è stato scaricato e il ribosoma Pro Tools LE è stato ottenuto. I dati sono stati registrati dal programma PeakTrak (gradiente di densità ISCO sistema gradiente di frazionamento). Le frazioni sono state raccolte, la proteina è stata TCA precipitata e ha deliberato 16,5% T, C 6% Tris-Tricine PAGE gel 15. Il gel è stato essiccato ed esposto per autoradiografia. Dopo l&#39;esposizione il gel è stato analizzato utilizzando Typhoon 9410 scanner imaging. La figura 2.1 mostra che il full-length Gamma-B sonoro superiore cristallino è presente in frazioni 70S dei ribosomi. Così, la sequenza SECM stallo consente l&#39;isolamento della stalla bovina Gamma-B cristallina RNC. I dati in Figura 2,2 indicano chiaramente che i RNC isolate sono infatti stabili. Nell&#39;esperimento corrente delle frazioni 70S dopo primo ciclo di centrifugazione gradiente di saccarosio sono state riunite e il saccarosio è stato rimosso usando Amicon Ultra-4 Unità di filtro centrifugo (Millipore), seguita da tampone scambio in soluzione contenente 20 mM HEPES-KOH pH 7,5, 15 mM MgCl 2, acetato di potassio 100 mM, DTT 1 mM. Questo campione è stato inoltre sottoposto ad una serie supplementare di centrifugazione attraverso gradiente 5-30% di saccarosio e frazionato. Ogni frazione è stata trattati ed analizzati come in Figura 2.1.

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Per risultati riproducibili, la qualità e la concentrazione dei componenti utilizzati per la trascrizione e traduzione in vitro sono critiche. Abbiamo usato commercialmente disponibile la trascrizione in vitro ed estratti di traduzione e danno risultati efficaci e riproducibili, se maneggiato con cura. Qualità di mRNA può influenzare la traduzione, quindi è della massima importanza per testare l&#39;integrità di mRNA prima di utilizzarlo per traduzione in vitro. Il tempo...

<table border="1"><tbody><tr><td> Nome del reagente / Kit </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> MEGAscript T7 alta Kit Trascrizione rendimento </td><td> Ambion </td><td> AM1333 </td></tr><tr><td> RTS 100 E.coli HY Kit </td><td> 5 Prime </td><td> 2401100 </td></tr><tr><td> Ribonucleasi Inhibitor </td><td> Invitrogen </td><td> 15518012 </td></tr><tr><td> Trans [35 S]-Label </td><td> MP Biomedicals </td><td> 0151006 </td></tr><tr><td> Amicon Ultra-4 Unità centrifuga Filtro </td><td> Millipore </td><td> UFC801008 </td></tr><tr><td> Stoccaggio fosforo autoradiografia </td><td> GE Healthcare </td><td> Typhoon 9410 imager modalità variabile </td></tr><tr><td> Gradiente di densità di frazionamento Sistemi </td><td> Teledyne Isco, Inc. </td><td> ISCO programmabile densità del sistema Gradient </td></tr><tr><td> Gradiente di saccarosio Centrifugation </td><td> Beckman Coulter </td><td> Optima L-90 K Preparative ultracentrifuga </td></tr></tbody></table>

using secm arrest sequence as a tool to isolate ribosome bound polypeptides

Numerose ricerche hanno fornito ampie evidenze che suggeriscono che le proteine ​​ripiegare nella cellula è un processo di co-traslazionale 1-5. Tuttavia, il percorso esatto che segue catena polipeptidica durante la co-traslazionale pieghevole per raggiungere la sua forma funzionale è ancora un enigma. Per comprendere questo processo e di determinare l&#39;esatta conformazione degli intermedi pieghevoli co-traslazionale, è necessario sviluppare tecniche che consentono l&#39;isolamento di RNC trasportano nascenti catene di dimensioni predeterminate per consentire loro ulteriori analisi strutturale. SECM (monitor secrezione) è un aminoacido 170 E. coli proteina che regola l&#39;espressione della valle Seca (guida secrezione) ATPasi nel SECM-seca operone 6. Nakatogawa e Ito originariamente scoperto che una sequenza amminoacidica 17 lungo (150-FSTPVWISQA QGIRA G P-166) nella regione C-terminale della proteina SECM è sufficiente e necessariocausare lo stallo di allungamento SECM a Gly165, producendo così peptidil-glicil-tRNA stabilmente legata alla ribosomiale P-site 7-9. Ancora più importante, è stato trovato che questa sequenza amminoacidica 17 lungo può essere fusa al C-terminale di qualsiasi proteina a lunghezza intera e / o troncato permettendo la produzione di RNC trasportano nascenti catene di dimensioni predeterminate 7. Così, quando fuse o inserito nella proteina bersaglio, la sequenza SECM stallo produce arresto del l&#39;allungamento della catena polipeptidica e genera RNC stabili in vivo sia in E. coli e in vitro in un sistema acellulare. Centrifugazione in gradiente di saccarosio è inoltre utilizzata per isolare RNC. I RNC isolati possono essere utilizzati per analizzare le caratteristiche strutturali e funzionali degli intermedi pieghevoli co-traslazionale. Recentemente, questa tecnica è stata utilizzata con successo per acquisire conoscenze nella struttura di alcune catene di ribosoma legati nascenti 10,11. Qui si descrive l&#39;isolamento di bovino Gamma-B sonoro superiore cristallino RNC fusa SECM e generato in un sistema di traduzione in vitro.

Watch this Scientific Journal Video about Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides at JoVE.com

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Descriviamo qui una tecnica che viene ora utilizzato di routine per isolare complessi ribosoma stabilmente legati a catena nascente (RNC). Questa tecnica sfrutta la scoperta che un 17 aminoacidi lungo &quot;sequenza di arresto&quot; SECM può fermare l&#39;allungamento traduzione in un procariota (<em&gt; E. coli</em&gt;) Del sistema, quando inserito (o fusa al C-terminale) di qualsiasi proteina.

Utilizzando Sequence SECM arresto come uno strumento per isolare ribosoma Polipeptidi Bound

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

using-secm-arrest-sequence-as-tool-to-isolate-ribosome-bound

Research

JoVE Journal

Biology

12.3K Views.  Cleveland State University. Descriviamo qui una tecnica che viene ora utilizzato di routine per isolare complessi ribosoma stabilmente legati a catena nascente (RNC). Questa tecnica sfrutta la scoperta che un 17 aminoacidi lungo "sequenza di arresto" SECM può fermare l'allungamento traduzione in un procariota ( E. coli) Del sistema, quando inserito (o fusa al C-terminale) di qualsiasi proteina.

Video: Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

Microfabbricazione post-Array-Detector (mPADs): un approccio per isolare le forze meccaniche

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

Ricerca

Biologia

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.

Luce Correlativo e Microscopia Elettronica (CLEM) come strumento per visualizzare molecole microiniezione e dei loro sub-cellulari eucariotiche Obiettivi


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

Bioassay Electroantennographic come strumento di screening per Volatili piante ospiti

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolamento dei ribosomi Polipeptidi Bound nascenti<em&gt; In vitro</em&gt; Per identificare i siti di pausa traslazione lungo mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

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 ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus laevis come un modello per individuare Traduzione Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

Esame di proteine ​​legate al DNA Nascente in cellule di mammifero utilizzando la tecnica BrdU-chip-Slot-occidentale

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

Laser-assistita Microdissection (LAM) come strumento per trascrizionale Profiling di tipi di cellule individuali

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Magnetico-Activated Cell Sorting strategie per isolare e purificare Synovial cellule staminali mesenchimali liquido derivato da un modello di coniglio

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

Un metodo ad alta uscita per isolare i periciti cerebrali dal mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...