Este trabajo fue financiado por el Programa de Ciencias Humanas de subvención RGP0024 Frontera.

1. Preparación del ADN de plantilla y la transcripción in vitro <ol><li> El gen de interés se clona en cualquier T7 y / o, por ejemplo promotor SP6 basado plásmido. Para obtener RNC de interés, C-terminal de la meta de polipéptido se extiende mediante la adición de secuencia detención inductor de SecM FXXXXWIXXXXGIRAGP 7. Con el fin de asegurar que el fragmento de polipéptido de interés fluirá hacia fuera del túnel ribosómico, la parte C-terminal de la proteína diana tiene que extenderse por lo menos 30 aminoácidos 12-14. Un flexible glicina-serina enlazador rica puede introducirse entre la proteína y la secuencia de detención SecM para evitar cualquier restricciones conformacionales posibles. </li><li> Para la transcripción in vitro, el ADN plantilla debería ser linealizado con la enzima de restricción de corte aguas abajo de la ORF. Uno tiene que verificar linealización completa del ADN de plásmido mediante la ejecución del producto de digestión de restricción en la electroforesis en gel de agarosa. </li><li> El plasma linealizadoIdentificación se utiliza además para reacción de transcripción in vitro. Diferentes concentraciones de ADN molde se puede analizar para determinar las concentraciones de ADN óptima requerida para la transcripción in vitro. Por lo general, con Megascript Kit de alta Ambion de transcripción rendimiento (Ambion / Life Technologies, Grand Island, NY), 1 mg de ADN linearizado produce ARNm 40-60 g. En la transcripción in vitro se realiza siguiendo las instrucciones del fabricante (Ambion / Life Technologies, Grand Island, NY) . </li><li> Tras la transcripción in vitro, el ARNm se purifica por precipitación de cloruro de litio de acuerdo con las instrucciones del fabricante (Megascript Kit de alta Ambion de transcripción rendimiento, Ambion / Life Technologies, Grand Island, NY). </li><li> integridad ARNm se ve verificada por electroforesis utilizando acrilamida o geles de agarosa. </li></ol> 2. Traducción in vitro Para la traducción in vitro utilizando la estrategia en tiempo real de E. coli 100 HY Kit (5 Prime, Gaithersburg, MD), siga los siguientes pasos: <ol><li> Preparar 100 l de instrucción de la traducción in vitro fabricante de la reacción siguiente. En pocas palabras, en agua libre de nucleasa añadir 24 l mezcla de aminoácidos menos metionina (1 mM), 10 unidades de inhibidor de la ribonucleasa (Invitrogen / Life Technologies, Grand Island, NY), 20 Ci de los desechos radiactivos [35 S] MP Biomedicals-metionina (, Solon, OH), 20 l mezcla de reacción, 24 tampón Reconstitución l y 24 l de E. E. lisado. </li><li> Incubar la reacción a 30 ° C durante 5 min para pre tibia la reacción de traducción. Añadir 1-2 g de ARNm para la reacción de traducción y se incuba a 30 ° C durante 10-15 minutos. </li><li> Se detiene la reacción mediante la colocación de la reacción de traducción en hielo. </li></ol> 3. Aislamiento de polipéptido naciente de reacción de traducción in vitro <ol><li> Para aislar RNC, la reacción de traducción se coloca sobre la parte superior de 4,5 ml de gradiente 5-30% de sacarosa en 20 mM de HEPES-KOH pH 70,5, 15 mM de MgCl 2, 100 mM de acetato de potasio, DTT 1 mM y se centrifugó utilizando Beckman Coulter SW55-Ti rotor a 41.000 rpm, 4 ° C durante 2 horas. </li><li> Tras la centrifugación, el gradiente de sacarosa se fracciona para aislar diferentes poblaciones ribosomal y asociados cadenas nacientes. La separación se controla mediante el sistema de la CIUO programable, con gradiente de densidad de grabación continua a 254 nm utilizando un detector de la CIUO UA-6 absorbancia. </li><li> La fracción (s) que contiene 70 ribosomas se recogen y analizan más dependiendo de la finalidad del experimento. </li></ol> 4. La observación de proteína unida a ribosoma con Tris-tricina SDS-PAGE Para comprobar, si la proteína extendida SecM permanece unida al ribosoma 70S, las fracciones del gradiente se recogieron y se trataron como sigue: <ol><li> La proteína en cada fracción se precipita por adición de ácido tricloroacético (TCA) a una concentración final de 10% y se incubaron a 4 ° C overniGHT. </li><li> Después de la incubación durante la noche, las muestras se centrifugaron a 14.000 X g durante 15 minutos para sedimentar la proteína. Después de la centrifugación el sobrenadante se eliminó y el sedimento se lavó dos veces con acetona solución que contiene: 1 mM Tris-HCl pH 7,6 (04:01). Pellet era aire más seca y se disuelve en tampón de carga de SDS-PAGE. </li><li> La muestra tratada se resolvió y se analizaron por Tris-tricina SDS-PAGE. </li><li> Después de la electroforesis, los geles se fijaron, se secó al vacío utilizando gel de tintorero y se sometió a autorradiografía. La distribución de los polipéptidos nacientes se observaron utilizando phosphoimaging. </li></ol> 5. Los resultados representativos Aquí presentamos un experimento que describe el aislamiento de la longitud completa de bovino gamma-B Cristalina establemente unido a ribosoma 70S. La Figura 1 muestra los pasos implicados en el aislamiento de las especies bovina B gamma-RNC cristalinas. El C-terminal de la Cristalina gamma-B se extiende en general por 32 aminoácidos to asegurar que las extrusiones proteína de longitud completa fuera del túnel ribosomal, lo que también incluye la secuencia SecM estancamiento colocado en la misma C-terminal del polipéptido de fusión. Después de la traducción in vitro, los ribosomas 70S se aislaron por centrifugación en gradiente de sacarosa (Figura 2.1). Con el fin de asegurar que la proteína se mantiene establemente unido al ribosoma, 70S que contienen fracciones se agruparon, tampón desalado, intercambio y se sometió a una ronda adicional de centrifugación en gradiente de sacarosa (Figura 2.2). El resultado presentado en la figura 2 indica claramente que SecM de manera eficiente puede inducir la detención de traslación de las RNC B Gamma-cristalina. <img alt="Figura 1" src="/files/ftp_upload/4027/4027fig1.jpg" /> Figura 1. Explicación esquemática de los pasos implicados en el aislamiento de RNC. C-terminal de bovino gamma-B Cristalina se extendió por 32 secuencia de aminoácidos. Esto se extendíaC-terminal de las regiones también incluye la secuencia de detención SecM. Gamma-B Cristalina con la secuencia se clonó en SecM Pivex 2,3 (T7 basado) plásmido. Para la transcripción in vitro del ADN molde se linealizó por digestión con XbaI. Plantilla linealizado fue utilizado además para la transcripción in vitro. El T7 en la plantilla de ADN es reconocida por la RNA polimerasa T7 que transcribe las aguas abajo gamma-B de genes localizados Cristalina del promotor T7. ARNm se purificó utilizando cloruro de litio método de precipitación. El mRNA purificado se utiliza entonces para la traducción in vitro. Después de la incubación, la reacción de traducción se estratificó en la parte superior del gradiente de 5-30% de sacarosa y se centrifuga en Beckman Coulter SW55-Ti rotor a 41.000 rpm, 4 ° C durante 2 horas. <spuna clase = &quot;pdflinebreak&quot;&gt; <img alt="Figura 2" src="/files/ftp_upload/4027/4027fig2.jpg" /> Figura 2. El aislamiento de las especies bovina Gamma-B Cristalina RNC establemente unido al ribosoma 70S. 1 mRNA g se mezcló en la reacción de 100 l E. E. Sistema de extracto S30 tal como se menciona en la sección de protocolo con 20 Ci [35 S]-metionina y se incubaron a 30 ° C durante 15 min. Después de la incubación, la reacción de traducción se estratificó en la parte superior del gradiente de 5-30% de sacarosa en 20 mM de HEPES-KOH, pH 7,5, 15 mM de MgCl 2, 100 mM de acetato de potasio, DTT 1 mM y se centrifugó en Beckman Coulter SW55-Ti rotor a 41.000 rpm, 4 ° C durante 2 horas. Tras la sedimentación velocidad, el gradiente se descargó y el ribosoma Le Pro se obtuvo. Los datos fueron registrados por el programa PeakTrak (gradiente CIUO gradiente de densidad sistema de fraccionamiento). Se recogieron las fracciones, la proteína fue TCA precipitó y se resolvieron en T 16,5%, 6% de C Tris-Tricina PAGE gel 15. El gel se seca y se expone a autorradiografía. Tras la exposición del gel fue escaneada con Typhoon 9410 escáner de imágenes. La figura 2.1 muestra que la longitud completa de Gamma-B Cristalina está presente en las fracciones 70S del ribosoma. Por lo tanto, la secuencia SecM estancamiento permite el aislamiento de las especies bovina estables B Gamma-RNC cristalinas. Los datos de la Figura 2.2 indican claramente que los RNC aislados son de hecho estable. En el presente experimento las fracciones 70S después de la primera ronda de centrifugación en gradiente de sacarosa se agruparon y la sacarosa se eliminó utilizando Amicon Ultra-4 Unidad centrífuga filtro (Millipore), seguido por tampón de cambio en solución que contenía 20 mM de HEPES-KOH, pH 7,5, 15 mM MgCl 2, 100 mM de acetato de potasio, 1 mM DTT. Esta muestra fue sometida además a una ronda adicional de centrifugación a través de gradiente 5-30% de sacarosa y se fraccionó. Cada fracción fue tratada y analizada como en la Figura 2.1.

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No hay conflictos de interés declarado.

Para obtener resultados reproducibles, la calidad y la concentración de los componentes utilizados para la transcripción y traducción in vitro son críticos. Hemos utilizado comercialmente disponible la transcripción in vitro y los extractos de traducción y que dan resultados eficaces y reproducibles, si se maneja con cuidado. Calidad de ARNm pueden afectar a la traducción, por lo que es de suma importancia para probar la integridad del ARNm antes de usarla para traducción<...

<table border="1"><tbody><tr><td> Nombre del reactivo / Kit </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Megascript T7 Kit de Transcripción de alta rentabilidad </td><td> Ambion </td><td> AM1333 </td></tr><tr><td> RTS 100 Kit de E. coli HY </td><td> 5 Prime </td><td> 2401100 </td></tr><tr><td> Inhibidor de ribonucleasa </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 Unidad de filtro centrífugo </td><td> Millipore </td><td> UFC801008 </td></tr><tr><td> De almacenamiento de fósforo autorradiografía </td><td> GE Healthcare </td><td> El tifón 9410 Imager modo variable </td></tr><tr><td> Los sistemas de gradiente de densidad de fraccionamiento </td><td> Teledyne Isco, Inc. </td><td> CIUO Densidad programable sistema de gradiente </td></tr><tr><td> Gradiente de sacarosa Centrifugation </td><td> Beckman Coulter </td><td> Optima L-90 K Preparativo Ultracentrífuga </td></tr></tbody></table>

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

Una amplia investigación ha proporcionado evidencias que sugieren que la proteína de amplios plegado en la célula es un proceso de co-traduccional 1-5. Sin embargo, el camino exacto que sigue la cadena polipeptídica durante la co-translacional de plegado para alcanzar su forma funcional es todavía un enigma. A fin de comprender este proceso y para determinar la conformación exacta de los compuestos intermedios plegables co-traduccionales, es esencial para desarrollar técnicas que permiten el aislamiento de RNC que transportan las cadenas nacientes de tamaños predeterminados para permitir su análisis estructural adicional. SecM (monitor de secreción) es una E. 170 aminoácidos E. proteína que regula la expresión de la corriente abajo Seca (secreción de conducción) ATPasa en el operón SECM-seca 6. Nakatogawa e Ito originalmente encontrado que una secuencia de 17 aminoácidos de largo (150-FSTPVWISQA QGIRA G P-166) en la región C-terminal de la proteína SecM es suficiente y necesaria paraoriginar la detención de la elongación en el SecM Gly165, produciendo de esta manera peptidil-tRNA-glicil estable vinculado a la P-ribosomal sitio de 7-9. Más importante aún, se encontró que esta secuencia de 17 amino ácido larga se pueden fusionar a la C-terminal de virtualmente cualquier proteína de longitud completa y / o truncado permitiendo así la producción de RNC llevan cadenas nacientes de tamaños predeterminados 7. Así, cuando se fusiona o se inserta en la proteína diana, la secuencia SecM estancamiento produce detención de la elongación de la cadena polipeptídica y genera RNC estables tanto in vivo en E. células de E. e in vitro en un sistema libre de células. Centrifugación en gradiente de sacarosa se utiliza además para aislar RNC. Los RNC aisladas pueden ser utilizados para analizar las características estructurales y funcionales de los compuestos intermedios plegables co-traduccionales. Recientemente, esta técnica se ha utilizado con éxito para obtener información sobre la estructura de los ribosomas varias cadenas unidas nacientes 10,11. Aquí se describe el aislamiento de las especies bovina Gamma-B Cristalina RNC fusionado a SecM y generada en un sistema de traducción 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

Se describe aquí una técnica que ahora se utilizan habitualmente para aislar de forma estable consolidados complejos ribosoma cadena naciente (RNC). Esta técnica aprovecha el descubrimiento de que un joven de 17 aminoácidos de longitud SecM &quot;secuencia de la detención&quot; puede detener la elongación de la traducción en un procariota (<em&gt; E. E.</em&gt;) Del sistema, cuando se inserta en (o fusionado al extremo C-terminal) de virtualmente cualquier proteína.

Usar secuencias de detención SecM como una herramienta para aislar Ribosome polipéptidos enlazados

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 vistas.  Cleveland State University. Se describe aquí una técnica que ahora se utilizan habitualmente para aislar de forma estable consolidados complejos ribosoma cadena naciente (RNC). Esta técnica aprovecha el descubrimiento de que un joven de 17 aminoácidos de longitud SecM "secuencia de la detención" puede detener la elongación de la traducción en un procariota ( E. E.) Del sistema, cuando se inserta en (o fusionado al extremo C-terminal) de virtualmente cualquier proteín...

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.

Microfabricated Post-Array-detectores (mPADs): un enfoque para aislar las fuerzas mecánicas

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

Investigación

Biología

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.

Luz correlativa y Microscopía Electrónica (CLEM) como una herramienta para visualizar moléculas microinyección y sus objetivos eucariotas subcelulares


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.

Bioensayo electroantenográfico como herramienta de detección de compuestos volátiles de las plantas hospederas

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.

El aislamiento de los ribosomas polipéptidos nacientes Bound<em&gt; In vitro</em&gt; Para la identificación de sitios de traslación pausa a lo largo del 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 como modelo para identificar Traducción Deterioro

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.

El examen de las proteínas unidas al ADN naciente en células de mamífero utilizando BrdU-chip-Slot-occidental Técnica

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.

Asistida por láser microdisección (LAM) como herramienta para la transcripción de perfiles de distintos tipos de células

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.

Célula activada por el magnético clasificación estrategias para aislar y purificar sinovial líquido mesenquimales células madre derivadas de un modelo de conejo

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 método de alta salida para aislar los pericytes cerebrales del ratón

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