Ce travail a été financé par Human Frontier Science Program de subvention RGP0024.

1. Préparation Patron de l&#39;ADN et la transcription in vitro <ol><li> Le gène d&#39;intérêt est cloné dans un T7 et / ou, par exemple sur la base promoteur SP6 plasmide. Pour obtenir RNC d&#39;intérêt, C-terminale du polypeptide cible est étendu par adjonction séquence inducteur d&#39;arrêt de SecM FXXXXWIXXXXGIRAGP 7. Afin d&#39;assurer que le fragment polypeptide d&#39;intérêt se extruder hors du tunnel ribosomique, la partie C-terminale de la protéine cible doit être augmentée d&#39;au moins 30 acides aminés 12-14. Un flexible glycine-sérine lieur riche peut être introduite entre la protéine et la séquence d&#39;arrêt SecM à éviter toutes contraintes possibles de conformation. </li><li> Pour la transcription in vitro, l&#39;ADN modèle doit être linéarisé par l&#39;enzyme de restriction coupe en aval de l&#39;ORF. Il faut vérifier linéarisation complète de l&#39;ADN plasmidique en exécutant le produit de digestion de restriction à l&#39;électrophorèse sur gel d&#39;agarose. </li><li> Le plasma linéariséIdentifiant est en outre utilisée dans la réaction de transcription in vitro. Concentration différente de la matrice ADN peuvent être testés pour déterminer la concentration d&#39;ADN optimale requise pour la transcription in vitro. En général, avec Ambion Trousse MEGAscript à haut rendement de transcription (Ambion / Life Technologies, Grand Island, NY), 1 pg d&#39;ADN linéarisé donne 40-60 ARNm pg. La transcription in vitro se fait suivant les instructions du fabricant (Ambion / Life Technologies, Grand Island, NY) . </li><li> Après transcription in vitro, l&#39;ARNm est purifié par précipitation au chlorure de lithium selon les instructions du fabricant (Ambion de MEGAscript Kit Haut de transcription rendement, Ambion / Life Technologies, Grand Island, NY). </li><li> l&#39;intégrité ARNm est encore vérifiée par électrophorèse utilisant de l&#39;acrylamide ou des gels d&#39;agarose. </li></ol> 2. La traduction in vitro Pour la traduction in vitro en utilisant le RTS 100 E. coli HY Kit (5 Premier, Gaithersburg, MD), suivez les étapes ci-dessous: <ol><li> Préparer 100 ul d&#39;instruction dans le fabricant de réaction de traduction in vitro suivante. En bref, dans la nucléase l&#39;eau libre ajouter 24 ul mélange d&#39;acides aminés moins méthionine (1 mM), 10 unités d&#39;inhibiteur de ribonucléase (Invitrogen / Life Technologies, Grand Island, NY), 20 pCi de matières radioactives [35 S]-méthionine (MP Biomedicals, Solon, OH), 20 pl mélange réactionnel, 24 pl de tampon de reconstitution et 24 pi de E. coli lysat. </li><li> Incuber la réaction à 30 ° C pendant 5 min à l&#39;avance au chaud la réaction de traduction. Ajouter 1-2 ug d&#39;ARNm de la réaction de traduction et incuber à 30 ° C pendant 10-15 min. </li><li> Arrêter la réaction en plaçant la réaction de traduction sur la glace. </li></ol> 3. Isolement de polypeptide naissant de réaction de traduction in vitro <ol><li> Pour isoler RNC, la réaction de traduction est posée sur le dessus de 4,5 ml de gradient de saccharose 5-30% dans 20 mM HEPES-KOH pH 70,5, 15 mM MgCl 2, 100 mM d&#39;acétate de potassium, DTT 1 mM et centrifugé à l&#39;aide Beckman Coulter SW55-Ti rotor à 41000 rpm, 4 ° C pendant 2 heures. </li><li> Après centrifugation, gradient de saccharose est fractionné pour isoler les différentes populations ribosomique et associés des chaînes naissantes. La séparation est contrôlée à l&#39;aide de la CITP système programmable gradient de densité avec un enregistrement en continu à 254 nm en utilisant un détecteur de CITP UA-6 absorbance. </li><li> La fraction (s) contenant des ribosomes 70S sont recueillies et analysées en outre en fonction du but de l&#39;expérience. </li></ol> 4. Observation de la protéine liée à ribosome avec Tris-tricine SDS-PAGE Pour vérifier si la protéine SecM prolongée reste attaché aux 70S du ribosome, fractions du gradient ont été collectées et traitées comme suit: <ol><li> La protéine dans chaque fraction a été précipité par addition d&#39;acide trichloracétique (TCA) à une concentration finale de 10% et incubées à 4 ° C overniGHT. </li><li> Après une nuit d&#39;incubation, les échantillons ont été centrifugés à 14.000 X g pendant 15 min à la pastille de la protéine. Surnageant de centrifugation suivant a été éliminé et le culot lavé deux fois avec de l&#39;acétone solution contenant: 1 mM de Tris-HCl pH 7,6 (4:1). Pellet était de l&#39;air encore séché et dissous dans un tampon de chargement SDS-PAGE. </li><li> L&#39;échantillon traité a été résolu et analysé par Tris-tricine SDS-PAGE. </li><li> Après l&#39;électrophorèse, les gels ont été fixés, séché à l&#39;aide de gel sous vide teinturier et soumis à une autoradiographie. La distribution des polypeptides naissants ont été observées à l&#39;aide phosphoimaging. </li></ol> 5. Les résultats représentatifs Nous présentons ici une expérience décrivant l&#39;isolement de la pleine longueur bovine Gamma-B cristallin stable lié aux ribosomes 70S. La figure 1 illustre les étapes impliquées dans l&#39;isolement de l&#39;espèce bovine RNC cristallines Gamma-B. Le C-terminale de la Gamma-cristallin B a été prolongé d&#39;ensemble de 32 acides aminés to s&#39;assurer que les extrusions de protéines pleine longueur la sortie du tunnel du ribosome, ce qui inclut également la séquence SecM décrochage mis à la très extrémité C-terminale du polypeptide de fusion. Après la traduction in vitro, les ribosomes 70S ont été isolées par centrifugation sur gradient de saccharose (Figure 2.1). Afin de veiller à ce que la protéine reste stable lié au ribosome, les 70S contenant fractions ont été réunies, dessalée, un échange de tampon et soumis à une série supplémentaire de centrifugation en gradient de saccharose (Figure 2.2). Le résultat présenté dans la figure 2 montre clairement que SecM peut induire efficacement arrestation de translation des RNC cristallines Gamma-B. <img alt="Figure 1" src="/files/ftp_upload/4027/4027fig1.jpg" /> Figure 1. Explication schématique des étapes impliquées dans l&#39;isolement de RNC. C-terminale de bovins Gamma-B cristallin a été prolongée de 32 séquence d&#39;acides aminés. Cette étendueC-terminales des régions inclut également la séquence d&#39;arrêt SecM. Gamma-B cristallin avec une séquence SecM a été cloné dans PIVEX 2.3 (basé sur T7) plasmide. Pour la transcription in vitro de l&#39;ADN modèle a été linéarisé par XbaI. Modèle linéarisé a en outre été utilisé pour la transcription in vitro. Le T7 dans la matrice d&#39;ADN est reconnu par l&#39;ARN polymérase T7 qui transcrit en aval gamma-B cristallin gène situées du promoteur T7. ARNm a été purifié en utilisant la méthode de précipitation au chlorure de lithium. L&#39;ARNm purifié a ensuite été utilisé pour la traduction in vitro. Après l&#39;incubation, la réaction de traduction a été superposé au-dessus de gradient de saccharose 5-30% et centrifugé dans Beckman Coulter SW55-Ti rotor à 41000 rpm, 4 ° C pendant 2 heures. <spune classe = &quot;pdflinebreak&quot;&gt; <img alt="Figure 2" src="/files/ftp_upload/4027/4027fig2.jpg" /> Figure 2. Isolement de bovins Gamma-B cristallin stable RNC liés au ribosome 70S. 1 ug d&#39;ARNm a été mélangé dans 100 ul de réaction E. Système coli Extrait S30 tel que mentionné dans la section du protocole avec 20 pCi de [35 S]-méthionine et incubées à 30 ° C pendant 15 min. Après l&#39;incubation, la réaction de traduction a été superposé au-dessus de gradient de saccharose 5-30% dans 20 mM HEPES-KOH pH 7,5, 15 mM MgCl 2, 100 mM d&#39;acétate de potassium, DTT 1 mM et centrifugé dans Beckman Coulter SW55-Ti rotor à 41000 rpm, 4 ° C pendant 2 heures. Après la sédimentation de vitesse, le gradient a été déchargé et le ribosome pro le a été obtenu. Les données ont été enregistrées par le programme PeakTrak (gradient de densité CITP fractionnement dégradé système). Les fractions ont été recueillies, la protéine a été précipité TCA et résolu sur T 16,5%, 6% C Tris-Tricine PAGE gel à 15. Le gel a été séché et exposé pour autoradiographie. Après l&#39;exposition du gel a été numérisé à l&#39;aide du scanner Typhoon imagerie 9410. Figure 2.1 montre que sur toute la longueur Gamma-B cristallin est présent dans 70S du ribosome fractions. Ainsi, la séquence SecM décrochage permet l&#39;isolement de l&#39;écurie de l&#39;espèce bovine RNC cristallines Gamma-B. Les données de la figure 2.2 indiquent clairement que les RNC isolés sont en effet stables. Dans l&#39;expérience en cours des fractions 70S après première série de centrifugation en gradient de saccharose ont été regroupés et le saccharose a été éliminé à l&#39;aide Amicon ultra-4 Unité de filtre centrifuge (Millipore), suivie par un tampon d&#39;échange en solution contenant 20 mM de HEPES KOH pH 7,5, 15 mM MgCl 2, 100 mM acétate de potassium, DTT 1 mM. Cet échantillon a été ensuite soumis à une série supplémentaire de centrifugation à travers gradient de saccharose 5-30% et fractionnés. Chaque fraction a été traité et analysé comme dans la Figure 2.1.

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+a+peptide+tagging+system+in+degradation+of+proteins+synthesized+from+damaged+messenger+RNA.">Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA.</a> Science. 271, 990-993 (1996).</li><li>Evans, M. S., Ugrinov, K. G., Frese, M. A., Clark, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogeneous+stalled+ribosome+nascent+chain+complexes+produced+in+vivo+or+in+vitro.">Homogeneous stalled ribosome nascent chain complexes produced in vivo or in vitro.</a> Nat. Methods. 2, 757-762 (2005).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

Pour obtenir des résultats reproductibles, la qualité et la concentration des composants utilisés pour la transcription in vitro et la traduction sont essentielles. Nous avons utilisé commercialement disponibles dans la transcription in vitro et des extraits de la traduction et elles donnent des résultats efficaces et reproductibles, s&#39;il est manipulé avec soin. Qualité de l&#39;ARNm peut affecter la traduction, de sorte qu&#39;il est d&#39;une importance capitale pour test...

<table border="1"><tbody><tr><td> Nom de réactif / Kit </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> MEGAscript T7 haute Kit de transcription rendement </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> Inhibiteur de la ribonucléase </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 Unité de filtre centrifuge </td><td> Millipore </td><td> UFC801008 </td></tr><tr><td> Luminophore de mémorisation autoradiographie </td><td> GE Healthcare </td><td> Typhoon 9410 imageur mode variable </td></tr><tr><td> Systèmes gradient de densité de fractionnement </td><td> Teledyne Isco, Inc </td><td> CITP système programmable gradient de densité </td></tr><tr><td> Le saccharose dégradé Centrifugation </td><td> Beckman Coulter </td><td> Optima L-90 K préparative Ultracentrifugeuse </td></tr></tbody></table>

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

Des recherches approfondies ont fourni des preuves suffisantes indiquant que repliement des protéines dans la cellule est un processus de co-traductionnelle 1-5. Toutefois, la voie exacte que la chaîne polypeptide suit au cours de co-traductionnelle de pliage pour réaliser sa forme fonctionnelle est encore une énigme. Afin de comprendre ce processus et de déterminer la conformation exacte des intermédiaires co-traductionnelles de pliage, il est essentiel de développer des techniques qui permettent l&#39;isolement des porteurs de chaînes naissantes RNC de tailles prédéterminées afin de permettre leur analyse ultérieure structurelle. SecM (moniteur sécrétion) est un acide aminé 170 E. protéines coli qui régule l&#39;expression de l&#39;aval Seca (conduite sécrétion) ATPase dans l&#39;opéron SECM-secA 6. Nakatogawa et Ito initialement que une séquence de 17 acides aminés de longueur (G 150-FSTPVWISQA QGIRA P-166) dans la région C-terminale de la protéine SecM est nécessaire et suffisant pourprovoquer de décrochage de l&#39;allongement SecM à Gly165, produisant ainsi peptidyl-glycyl-ARNt stable lié à la ribosomal P place 7-9. Plus important encore, il a été constaté que cette séquence de 17 acides aminés de long peut être fusionné à l&#39;extrémité C-terminale de pratiquement n&#39;importe quelle protéine de pleine longueur et / ou tronquée permettant ainsi la production de RNC porteurs de chaînes naissantes de tailles prédéterminées 7. Ainsi, lorsqu&#39;il est fondu ou est insérée dans la protéine cible, la séquence de décrochage SecM produit arrêt de l&#39;allongement de la chaîne polypeptidique et génère RNC stables in vivo dans E. coli et les cellules in vitro dans un système acellulaire. Centrifugation en gradient de saccharose est en outre utilisé pour isoler RNC. Les RNC isolés peuvent être utilisés pour analyser les caractéristiques structurales et fonctionnelles des intermédiaires co-traductionnelles de pliage. Récemment, cette technique a été utilisée avec succès pour mieux comprendre la structure de plusieurs chaînes de ribosomes liés naissantes 10,11. Ici on décrit l&#39;isolement de bovin gamma-B cristallin fusionné à RNC SecM et généré dans un système de traduction 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

Nous décrivons ici une technique qui est maintenant couramment utilisé pour isoler de manière stable liés ribosome complexes de la chaîne naissante (RNC). Cette technique tire parti de la découverte que de 17 acides aminés de long &quot;séquence d&#39;arrêt&quot; SecM peut arrêter élongation de la traduction dans un procaryote (<em&gt; E. coli</em&gt;) Du système, lorsqu&#39;il est inséré dans (ou fusionné à l&#39;extrémité C-terminale) de pratiquement toute la protéine.

Utilisation de la séquence d&#39;arrêt SecM comme un outil pour isoler les polypeptides Ribosome 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

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JoVE Journal

Biology

12.3K vues.  Cleveland State University. Nous décrivons ici une technique qui est maintenant couramment utilisé pour isoler de manière stable liés ribosome complexes de la chaîne naissante (RNC). Cette technique tire parti de la découverte que de 17 acides aminés de long "séquence d'arrêt" SecM peut arrêter élongation de la traduction dans un procaryote ( E. coli) Du système, lorsqu'il est inséré dans (ou fusionné à l'extrémité C-terminale) de pratiquement t...

Vidéo: 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.

Microfabriqués post-Array-détecteurs (mPADs): une approche pour isoler les forces mécaniques

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

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Biologie

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.

Lumière corrélative et microscopie électronique (CLEM) comme un outil pour visualiser des molécules et de leurs microinjecté eucaryotes Sous-cellulaires cibles


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.

Essais biologiques électroantennographique comme outil de dépistage pour les substances volatiles de la plante hôte

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.

Isolement du ribosome Bound polypeptides naissants<em&gt; In vitro</em&gt; Pour identifier les sites de pause de translation le long d&#39;ARNm

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 comme un modèle pour identifier traduction Dépréciation

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.

Examen des protéines liées à Nascent ADN dans les cellules de mammifères utilisant la technique BrdU-Chip-Slot-Ouest

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.

assistée par laser microdissection (LAM) comme outil de profilage transcriptionnel des types cellulaires individuels

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.

Cellule magnétique activé tri des stratégies visant à isoler et purifier Synovial fluide mésenchymateuses cellules souches dérivées d’un modèle de lapin

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.

Une méthode de sortie élevée pour isoler les péricartétes cérébraux de la souris

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