Este trabalho foi financiado pelo Programa Ciência das Fronteiras Humanas concessão RGP0024.

1. Preparação de ADN de modelo e transcrição in vitro <ol><li> O gene de interesse é clonado em qualquer e T7 / ou, por exemplo SP6 promotor baseado plasmídeo. Para obter RNC de interesse, C-terminal do polipéptido alvo é estendido por adição de sequência de captura indutor do SECM FXXXXWIXXXXGIRAGP 7. A fim de assegurar que o fragmento de polipéptido de interesse será extrudir para fora do túnel ribossomal, a parte C-terminal da proteína alvo deve ser ampliada de pelo menos 30 aminoácidos 12-14. Um linker flexível Glicina-Serina rica pode ser introduzido entre a proteína ea sequência de captura SECM para evitar quaisquer constrangimentos conformacionais possíveis. </li><li> Para transcrição in vitro, o DNA modelo deve ser linearizado com a enzima de restrição de corte a jusante da ORF. Uma das necessidades para verificar a linearização completa do DNA de plasmídeo, executando o produto da digestão de restrição em electroforese em gel de agarose. </li><li> O plasma linearizadaid é ainda utilizado para reacção de transcrição in vitro. Concentração diferente de ADN molde pode ser testado para identificar a concentração de DNA óptima necessária para transcrição in vitro. Geralmente com Kit Ambion da transcrição de alto rendimento MEGAscript (Ambion / Life Technologies, Grand Island, NY), 1 ug de DNA linearizado produz 40-60 ug de mRNA. Transcrição in vitro é efectuado de acordo com as instruções do fabricante (Ambion / Life Technologies, Grand Island, NY) . </li><li> Após transcrição in vitro, o mRNA é purificado por precipitação com cloreto de lítio de acordo com as instruções do fabricante (Kit Ambion da transcrição MEGAscript de alto rendimento Ambion, / Life Technologies, Grand Island, NY). </li><li> integridade do mRNA é ainda verificado por electroforese em acrilamida ou utilizando géis de agarose. </li></ol> 2. Tradução in vitro Para a tradução in vitro utilizando o RTS 100 E. coli HY Kit (5 Prime, Gaithersburg, MD) siga os passos abaixo: <ol><li> Prepare-se 100 ul de instrução in vitro fabricante reação tradução seguinte. Resumidamente, em água livre de nuclease adicionar 24 ul mistura de ácido amino menos metionina (1 mM), 10 unidades de inibidor da ribonuclease (Invitrogen / Life Technologies, Grand Island, NY), 20 uCi de radioactivos [35 S] Biomedicals-metionina (MP, Solon, OH), 20 ul da mistura da reacção, 24 ul de tampão Reconstituição e 24 ul de E. coli lisado. </li><li> Incubar a reacção a 30 ° C durante 5 min de pré reacção a quente de tradução. Adicionar 1-2 ug de mRNA para a reacção de tradução e incubar a 30 ° C durante 10-15 min. </li><li> Parar a reacção através da colocação da reacção de tradução em gelo. </li></ol> 3. Isolamento de polipéptido nascente in vitro a partir de reacção de tradução <ol><li> Para isolar RNC, a reacção de tradução é colocado em camadas sobre 4,5 ml de gradiente 5-30% de sacarose em 20 mM pH HEPES-KOH 70,5, 15 mM de MgCl2, 100 mM de acetato de potássio, 1 mM de DTT e centrifugou-se utilizando Beckman SW55 Coulter-rotor Ti a 41.000 rpm, 4 ° C durante 2 horas. </li><li> Após a centrifugação, o gradiente de sacarose é fracionado para isolar diferentes populações ribossômico e associados cadeias nascentes. A separação é monitorizado utilizando o sistema de gradiente ISCO programável Densidade com gravação contínua a 254 nm usando um detector de ISCO UA-6 absorvância. </li><li> A fração (s) contendo ribossomos 70S são coletadas e analisadas mais, dependendo do objetivo do experimento. </li></ol> 4. Observação de proteína ligada ao ribossomo com SDS-PAGE Tris-tricina Para verificar, se a proteína SECM estendida permanece ligado aos ribossomas 70S, as fracções de gradiente foram recolhidas e tratadas como se segue: <ol><li> Proteína em cada fracção foi precipitado por adição de ácido tricloroacético (TCA) para uma concentração final de 10% e incubadas a 4 ° C overnight. </li><li> Após incubação durante a noite, as amostras foram centrifugadas a 14.000 X g durante 15 min a pelete a proteína. Após centrifugação o sobrenadante foi removido eo sedimento lavado duas vezes com acetona solução contendo: 1 mM de Tris-HCl pH 7,6 (04:01). Sedimento foi ainda mais ar seco e dissolvido em SDS-PAGE de tampão de carregamento. </li><li> A amostra tratada foi resolvido e analisadas por Tris-tricina SDS-PAGE. </li><li> Após a electroforese, os géis foram fixados, secou-se utilizando vácuo gel Dyer e submetido a autorradiografia. A distribuição de polipéptidos nascentes foram observadas utilizando phosphoimaging. </li></ol> 5. Os resultados representativos Apresentamos aqui uma experiência que descreve o isolamento do full-length bovina Gamma-B cristalina estavelmente ligado aos ribossomas 70S. A Figura 1 ilustra os passos envolvidos no isolamento de bovinos RNC Gamma-B cristalina. O C-terminal da crystallin Gamma-B foi estendida geral por 32 t de aminoácidosó assegurar que a extrusão de comprimento total de proteína para fora do túnel ribossomal, o que também inclui a sequência SECM stalling colocado no muito C-terminal do polipéptido de fusão. Após a tradução in vitro, os ribossomas 70S foram isolados por centrifugação de gradiente de sacarose (Figura 2.1). A fim de assegurar que a proteína permanece estavelmente ligado ao ribossoma, as fracções contendo 70S foram reunidas, tampão, dessalinizado trocados e submetido a uma rodada adicional de centrifugação em gradiente de sacarose (Figura 2.2). O resultado apresentado na Figura 2 sugere claramente que SECM pode eficientemente induzir a apreensão de translação das RNC Gamma-B cristalina. <img alt="A Figura 1" src="/files/ftp_upload/4027/4027fig1.jpg" /> Figura 1. Explicação esquemático dos passos envolvidos no isolamento de RNC. Terminal C de bovino Gamma-B cristalina foi prolongada por 32 sequência de aminoácidos. Este estendidaC-terminais regiões também inclui sequência de captura SECM. Gamma-B cristalina com seqüência SECM foi clonado em pIVEX 2,3 (T7 based) plasmídeo. Para transcrição in vitro do DNA molde foi linearizado por Xbal. Modelo linearizado foi ainda utilizado para transcrição in vitro. A T7 no molde de ADN é reconhecido por T7 RNA polimerase que transcreve a jusante Gamma-B do gene crystallin localizados do promotor T7. mRNA foi purificado utilizando lítio método de precipitação de cloreto. O mRNA purificado foi então usado para tradução in vitro. Após a incubação, a reacção de tradução foi derramado por cima de gradiente 5-30% de sacarose e centrifugou-se em Beckman SW55 Coulter-rotor Ti a 41.000 rpm, 4 ° C durante 2 horas. <spuma class = &quot;pdflinebreak&quot;&gt; <img alt="A Figura 2" src="/files/ftp_upload/4027/4027fig2.jpg" /> Figura 2. Isolamento de bovinos Gamma-B cristalina RNCS estável ligado ao ribossomo 70S. MRNA mg 1 foi misturado em 100 l reação E. coli Sistema Extracto S30 como mencionado na secção protocolo com 20 uCi [35 S]-metionina e incubadas a 30 ° C durante 15 min. Após a incubação, a reacção de tradução foi derramado por cima de gradiente 5-30% de sacarose em 20 mM de HEPES-KOH, pH 7,5, 15 mM de MgCl2, 100 mM de acetato de potássio, 1 mM de DTT e centrifugou-se em Beckman SW55 Coulter-rotor Ti a 41.000 rpm, 4 ° C durante 2 horas. Após sedimentação velocidade, o gradiente foi descarregado eo ribossoma pró le foi obtido. Os dados foram registrados pelo programa PeakTrak (gradiente de densidade ISCO sistema de fracionamento gradiente). As fracções foram recolhidas, a proteína foi precipitada TCA e resolvidos em t 16,5%, 6% C Tris-Tricine gel PAGE 15. O gel foi seco e exposto para autorradiografia. Após a exposição do gel foi digitalizado utilizando imagens do scanner Typhoon 9410. Figura 2.1 mostra que o full-length Gamma-B cristalina está presente em frações 70S dos ribossomas. Assim, SECM sequência stalling permite o isolamento dos estáveis ​​bovina RNC Gamma-B cristalina. Os dados da Figura 2.2 indicam claramente que os isolados RNC são realmente estável. No experimento actual das fracções 70S após a primeira ronda de centrifugação em gradiente de sacarose foram reunidas e da sacarose foi removido utilizando Amicon Ultra-4 unitário centrífuga de filtro (Millipore), seguido por tampão de troca em solução contendo 20 mM de HEPES-KOH, pH 7,5, 15 mM MgCl2, 100 mM de acetato de potássio, 1 mM de DTT. Esta amostra foi submetido a uma rodada adicional de centrifugação através de gradiente 5-30% de sacarose e fraccionado. Cada fracção foi tratado e analisados ​​como na Figura 2.1.

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Para obter resultados reprodutíveis, a qualidade ea concentração dos componentes utilizados para transcrição in vitro e tradução são críticos. Usámos comercialmente disponível in vitro transcrição e extractos de tradução e dão resultados reprodutíveis e eficientes, se manuseados com cuidado. Qualidade de ARNm pode afectar a tradução, por isso, é de extrema importância para testar a integridade do mRNA antes de o utilizar para tradução in vitro. Tempo de...

<table border="1"><tbody><tr><td> Nome de reagente / Kit </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> MEGAscript T7 Kit de Transcrição de alto 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> Inibidor da ribonuclease </td><td> Invitrogen </td><td> 15518012 </td></tr><tr><td> Trans [35 S]-Label </td><td> Biomedicals MP </td><td> 0151006 </td></tr><tr><td> Amicon Ultra-4 unitário Centrífuga filtro </td><td> Millipore </td><td> UFC801008 </td></tr><tr><td> Armazenamento de fósforo autoradiografia </td><td> GE Healthcare </td><td> Typhoon 9410 imager modo variável </td></tr><tr><td> Densidade Sistemas de Fracionamento Gradiente </td><td> Teledyne Isco, Inc. </td><td> ISCO Sistema Gradiente programável Densidade </td></tr><tr><td> Sacarose Gradiente Centrifugation </td><td> Beckman Coulter </td><td> Optima L-90 K ultracentrífuga Preparativo </td></tr></tbody></table>

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

A pesquisa extensiva tem forneceram evidências que sugerem que a proteína amplos dobrando na célula é um processo de co-translacional 1-5. No entanto, o caminho exato que cadeia polipeptídica segue durante a co-traducional dobrar para atingir sua forma funcional ainda é um enigma. A fim de compreender este processo e para determinar a conformação exacta dos intermediários de co-translacionais de dobragem, é essencial ao desenvolvimento de técnicas que permitem o isolamento de RNC que transportam cadeias nascentes de tamanhos pré-determinados para permitir a sua posterior análise estrutural. SECM (monitor secreção) é um aminoácido 170 E. coli proteína que regula a expressão do jusante SecA ATPase (condução secreção) no operão SECM-seca 6. Nakatogawa e Ito originalmente descobriu que uma sequência de aminoácidos 17 de comprimento (150-FSTPVWISQA QGIRA G P-166) na região C-terminal da proteína SECM são necessários e suficientes paracausar bloqueamento dos alongamento SECM em Gly165, produzindo assim peptidil-glicil-tRNA estavelmente ligado ao ribossoma P-site 7-9. Mais importante, verificou-se que esta sequência de aminoácidos 17 de comprimento pode ser fundido com o terminal C de virtualmente qualquer proteína de comprimento completo e / ou truncado, permitindo assim a produção de RNC que transportam cadeias nascentes de tamanhos pré-determinados 7. Assim, quando fundidos ou inserida na proteína alvo, SECM sequência stalling produz prisão do alongamento da cadeia polipeptídica e gera RNC estáveis ​​tanto in vivo em E. células de Escherichia e in vitro num sistema isento de células. Centrifugação em gradiente de sacarose é ainda utilizado para isolar RNC. Os RNC isolados podem ser utilizados para analisar as características estruturais e funcionais dos intermediários de co-translacionais dobráveis. Recentemente, esta técnica tem sido usada com sucesso para obter insights sobre a estrutura de vários ribossomas ligados a cadeias nascentes 10,11. Aqui nós descrevemos o isolamento de bovinos Gamma-B cristalina RNCS fundido a SECM e gerado em um sistema de tradução 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

Descrevemos aqui uma técnica que é rotineiramente usado para isolar estavelmente ligados ribossomo complexos da cadeia nascente (RNC). Esta técnica aproveita a descoberta de que um jovem de 17 aminoácidos longo SECM &quot;seqüência de prisão&quot; pode parar de alongamento de tradução em uma procariótica (<em&gt; E. coli</em&gt;) Do sistema, quando inseridos em (ou fundido com o terminal C) de virtualmente qualquer proteína.

Usando Seqüência de Detenção SECM como uma ferramenta para isolar Ribossomos Polipeptídeos encadernados

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 visualizações.  Cleveland State University. Descrevemos aqui uma técnica que é rotineiramente usado para isolar estavelmente ligados ribossomo complexos da cadeia nascente (RNC). Esta técnica aproveita a descoberta de que um jovem de 17 aminoácidos longo SECM "seqüência de prisão" pode parar de alongamento de tradução em uma procariótica ( E. coli) Do sistema, quando inseridos em (ou fundido com o terminal C) de virtualmente qualquer proteína.

Vídeo: 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 Pós-Array-detectores (mPADs): uma abordagem para isolar Forças Mecânica

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

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.

Microscópio óptico e Eletrônica (CLEM) como uma ferramenta para visualizar moléculas microinjetados e seus eucarióticas Sub-celulares Metas


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.

Bioensaio eletroantenográfica como uma ferramenta de triagem para voláteis da planta hospedeira

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 de Ribossomos ligados a polipeptídeos nascentes<em&gt; In vitro</em&gt; Identificar Sites Pausa translacionais Junto 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 como um modelo para identificar Tradução 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.

Exame de proteínas ligadas ao DNA em células de mamíferos Nascente usar a técnica de-BrdU-chip-Slot Ocidental

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.

assistida por laser Microdissecção (LAM) como ferramenta para transcricional Profiling de tipos de células individuais

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.

Magnético-ativado da pilha classificação estratégias para isolar e purificar sinovial fluidos derivados mesenquimais células-tronco de um modelo de coelho

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.

Um método de alta saída para isolar pericites cerebrais do rato

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