Este trabajo es apoyado por el NIH R01 GM47607, CA163296 y NSF 1.334.493 de CET. Damos las gracias a los miembros del laboratorio Turner para la lectura crítica del manuscrito. Todos los datos que se muestran en este informe fueron reproducidas con permiso de Wormer et al. 2014 11.

1. Preparación de Durotaxis Cámaras <ol><li> Para preparar un solo 6 pocillos placa de cultivo de tejidos, tarar la balanza con un tubo cónico de 50 ml. Pesar aproximadamente 10 g de la solución de base de PDMS en el tubo de 50 ml (la solución es bastante viscoso). </li><li> Para un 90: 1 sustrato (Cumplimiento de ~ 1 kPa), se divide el peso medido de la solución de base de PDMS en el tubo por 90 para determinar la cantidad correcta de solución de agente de reticulación necesario. Añadir la cantidad calculada de la solución reticulante PDMS al mismo tubo. Ejemplo: 10 g / 90 = 0,11 g. Añadir 0,11 g de la solución de agente de reticulación PDMS a la solución base de PDMS. NOTA: La base y reticulantes soluciones son a la vez proporcionan en el kit de PDMS. </li><li> Mezclar vigorosamente la mezcla base / reticulante PDMS durante 5 min a RT usando una espátula pequeña. En esta etapa la mezcla contendrá un gran número de burbujas de aire. </li><li> Centrifugar el sustrato PDMS en una centrífuga de mesa durante 5 min a 50 xg a temperatura ambiente para eliminar el bubbles. Aún si hay burbujas después del 5 min, se centrifuga de nuevo. </li><li> Pipeta 1 ml de 90: 1 PDMS sustrato en cada pocillo del cultivo de tejidos tratados placa de 6 pocillos. Cualquier burbuja de aire restantes presentes en los PDMS pueden ser eliminados en esta etapa haciendo estallar ellos usando una aguja de 21 G. Permitir que el PDMS se extiendan durante 30 minutos en el pozo. </li><li> Hervir 12 mm de vidrio # 1 cubreobjetos en agua destilada durante 5 min. Repetir dos veces y almacenar los cubreobjetos en agua destilada. </li><li> Coloque uno, cubreobjetos se secó en cada pocillo de la placa de cultivo de tejidos tocando suavemente un lado del cubreobjetos en la solución de PDMS a continuación, colocar el cubreobjetos sobre los PDMS. A medida que el cubreobjetos se asienta, los PDMS comenzarán a invadir lo largo de los bordes del cubreobjetos, pero no cubren completamente. Esto generará una interfaz entre el PDMS y el vidrio después del curado (ver la Figura 1A, B). </li><li> Incubar la placa a 70 ° C en un horno durante 16 horas para curar (se endurecen) el PDMS. Coloque el plate en una campana de cultivo celular y UV esterilizar durante 10 minutos. NOTA: Lo mejor es hacer las placas en un par de días de uso. Sin embargo, las placas pueden ser envueltos en Parafilm sin ningún tipo de tampón y se almacenaron a 4 ° C durante un máximo de 2 semanas sin ninguna disminución notable en la calidad. </li></ol> 2. Celular Chapado NOTA: Si el estudio del efecto de la caída siRNA mediada, realice la caída con las instrucciones del fabricante o el protocolo optimizado para el tipo de célula de elección. <ol><li> Escudo durotaxis cada cámara con 1 ml de 10 mg / ml de fibronectina en PBS sin calcio y magnesio durante 1 hora a 37 ° C. Alternativamente, la fibronectina se puede aplicar el día antes del análisis mediante la incubación de los PDMS con 10 mg / ml de fibronectina en PBS durante 16 horas a 4 ° C. Asegúrese de que toda la superficie se sumerge en el fibronectina en solución PBS. </li><li> Preparar desnaturalizado por calor 1% de BSA. Pesar 0,5 g de BSA y se disuelven en 50 ml de PBS.Filtrar esterilizar la solución a través de un filtro y el calor de 0,22 micras a 80 ° C durante 12 min. Nota: Prepare esta solución el día antes de la célula de recubrimiento y se almacena a 4 ° C. </li><li> Aspirar la solución de fibronectina y lavar 3 veces con PBS. Añadir 1 ml de BSA desnaturalizado calor en PBS a cada pocillo e incubar durante 30 min a TA. </li><li> Trypsinize y contar las células de elección, mientras que las cámaras de durotaxis están bloqueando con la BSA desnaturalizado por calor. </li><li> Plate 1 x 10 5 células en un volumen de 2 ml en cada pocillo de la cámara durotaxis, utilizando los medios necesarios para el tipo de célula particular de elección. Permitir que las células se adhieran y se extendieron sobre el sustrato durante 4 horas en un incubador humidificado a 37 ° C con 5% de CO 2. NOTA: las células U2OS se mantienen rutinariamente en DMEM con 10% FBS, complementado con 2 mM L-glutamina, piruvato de sodio 1 mM, y 10 IU / ml de penicilina, 10 mg / ml de estreptomicina. NOTA: El número de células utilizado en este ejemplo está optimizado paraCélulas U2OS. Se puede requerir optimización para otros tipos de células. Esta densidad de las células da suficiente espacio para migrar sin interacciones significativas con otras células. </li></ol> Imaging 3. Células vivas <ol><li> Realizar imágenes de células vivas en un microscopio invertido, con contraste de fase con un objetivo de 10X. El microscopio debe estar equipado con una, cámara húmeda del medio ambiente cerrado, lo que permite el control de la temperatura a 37 ° C y 5% de CO 2 durante la exploración a largo plazo. </li><li> Después de que las células se han extendido durante aproximadamente 3,5 horas, montar la placa en la cámara microscopio. Permitir que la muestra (s) se equilibre en la cámara durante 30 min. </li><li> Establecer automatizado, multi-punto de visita en el microscopio si está disponible. Centrarse en la interfaz entre las 90: 1 PDMS y el cubreobjetos de vidrio y elegir los puntos a la imagen en todo el interfaz con un promedio de 40 puntos por durotaxis cámara. Imagen de las células cada 10 min durante un máximo de 16 horas. NOTA: La reregión de interés aparecerá como dos líneas, con la línea externa correspondiente al borde del cubreobjetos y la línea interior correspondiente a la interfaz real entre el PDMS y el cubreobjetos. Ver Figura 1B. </li></ol> Análisis 4. Datos <ol><li> Generar una hoja de cálculo como en la Tabla 1. </li><li> Cuente el número de eventos de cruce de los PDMS a la superficie de vidrio y viceversa de cada película generada. Anote el número de eventos de cruce en la columna correspondiente en la hoja de cálculo Excel. NOTA: Un evento de cruce se define como el núcleo de la célula que pasa sobre el límite entre el PDMS y productos de vidrio en cualquier dirección. </li><li> Para cuantificar múltiples cruces, contar el número de veces que el celular cruzó la interfaz. Ese número se debe registrar en la columna de excel correspondiente al sustrato sobre el que la célula se encuentra en el extremo de la película. Repita el análisis de todas las células que atraviesa la interfaz en tpelícula que él. Excluir las células que migran fuera del campo de visión durante la exploración. NOTA: También es importante verificar, por recuento de células al inicio del experimento, que las células son capaces de adherirse igual a los PDMS recubiertas de fibronectina y superficies cubreobjetos de vidrio. </li><li> Calcular el porcentaje de células que migró de PDMS a la superficie de vidrio (es decir, se sometió a durotaxis). Añadir el número de cruzar eventos de suave a duro y los múltiples eventos de cruce que terminaron en el disco y se dividen por el número total de eventos de cruce. </li><li> Calcular el porcentaje de múltiples cruces dividiendo el número de cruces de múltiples por el número total de cruces. </li></ol>

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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling.">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139 (5), 891-906 (2009).</li><li>Lo, C. M., Wang, H. B., Dembo, M., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+movement+is+guided+by+the+rigidity+of+the+substrate.">Cell movement is guided by the rigidity of the substrate.</a> Biophysical Journal. 79 (1), 144-152 (2000).</li><li>Plotnikov, S. V., Waterman, C. 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Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+and+focal+adhesion+assembly:+a+close+relationship+studied+using+elastic+micropatterned+substrates.">Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates.</a> Nature Cell Biology. 3 (5), 466-472 (2001).</li><li>Provenzano, P. P., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+signaling+through+the+cytoskeleton+regulates+cell+proliferation+by+coordinated+focal+adhesion+and+Rho+GTPase+signaling.">Mechanical signaling through the cytoskeleton regulates cell proliferation by coordinated focal adhesion and Rho GTPase signaling.</a> Journal of Cell Science. 124 (8), 1195-1205 (2011).</li><li>Wang, Y. L., Pelham, R. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+focal+adhesion-localized+CdGAP+regulates+matrix+rigidity+sensing+and+durotaxis.">The focal adhesion-localized CdGAP regulates matrix rigidity sensing and durotaxis.</a> PloS ONE. 9 (3), e91815 (2014).</li><li>Trichet, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+a+large-scale+mechanosensing+mechanism+for+cellular+adaptation+to+substrate+stiffness.">Evidence of a large-scale mechanosensing mechanism for cellular adaptation to substrate stiffness.</a> Proceedings of the National Academy of Sciences. 109 (18), 6933-6938 (2012).</li><li>Eyckmans, J., Boudou, T., Yu, X., Chen, C. 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The authors have no conflicts to disclose.

Aquí se describe un ensayo simple para estudiar durotaxis en la migración de las células. Una de las principales de este ensayo es la facilidad de preparación de las cámaras durotaxis utilizando PDMS. La rigidez de los sustratos puede ser manipulado fácilmente cambiando la relación de la solución de base de PDMS a reticulante para permitir el estudio de diversas rigideces en el ensayo. Sin embargo, una limitación potencial del sistema es que las células sólo están expuestos a un solo cambio en la rigidez del...

La matriz extracelular (ECM) se compone de una serie compleja de proteínas estructurales y de reticulación incluyendo el colágeno, fibronectina y laminina. Aunque está bien establecido que el ECM proporciona importante apoyo estructural a los tejidos celulares, cada vez hay más pruebas que indican que las células responden activamente a los cambios físicos en su entorno de ECM para regular diversos procesos celulares, incluyendo la supervivencia celular, la diferenciación y migración celular. Por ejemplo, las diferencias en la rigidez de la ECM pueden conducir las células madre mesenquimales hacia diferentes linajes, con sustratos blandos (~ 1 kPa) que promueven linajes neurogénicos mientras sustratos rígidos (~ 25 kPa) promueven la diferenciación osteogénica 1. Del mismo modo, un aumento en la rigidez de la matriz estromal ha sido demostrado para promover la tumorigénesis mamaria de las células epiteliales y la invasión en el tejido circundante 2,3. Un aspecto particularmente interesante de este mechanosignresultados de la actividad aling en un proceso conocido como durotaxis, en el que las células migran preferentemente hacia un sustrato más rígido 4,5. Las células constantemente sentido, las características físicas de su entorno extracelular a través de receptor de integrina de unión a la ECM. Esto, a su vez, promueve la acumulación de numerosas proteínas estructurales y de señalización a sus dominios citoplásmicos para impulsar la formación de estructuras adhesivas conocidas como adhesiones focales o contactos focales 6,7. Desde integrinas no tienen actividad enzimática intrínseca, las señales se transmiten desde el ECM a través de estas proteínas accesorias para coordinar la respuesta de la célula a su entorno cambiante 8. En consecuencia, la identificación y caracterización de las proteínas clave que intervienen en la regulación de mechanosignaling y durotaxis es un área importante de investigación. Varios sistemas de modelos se han desarrollado para estudiar durotaxis in vitro, pero la mayoría tienenutilizados sustratos revestidos con colágeno de poliacrilamida 4. Sin embargo, la preparación de los sustratos de poliacrilamida puede ser técnicamente difícil y el colágeno utilizado en estos ensayos debe ser reticulado químicamente al sustrato 9. Sustratos de polidimetilsiloxano (PDMS) se ha demostrado que exhiben propiedades mecánicas comparables a los sustratos de poliacrilamida al 10. Sin embargo, los sustratos de PDMS se preparan por simple mezcla de una proporción de la base a agente reticulante y estos sustratos pueden estar recubiertos con proteínas ECM sin la necesidad de reticulación química, haciendo así más fácil PDMS una herramienta para estudiar los efectos de rigidez en el comportamiento celular. En este documento, se describe cómo preparar una cámara durotaxis simple en el que un sustrato suave PDMS se integra con un cubreobjetos de vidrio rígido. El ensayo, como se indica a continuación, ofrece un método rápido y sencillo para estudiar durotaxis. Para este estudio se utilizaron células de osteosarcoma U2OS humanas combinadas con mediada por siRNAdesmontables de cdGAP para estudiar el papel de esta proteína de adhesión focal en durotaxis 11. Es importante destacar que este protocolo se puede adaptar fácilmente a las necesidades individuales. Otros tipos de células pueden ser sustituidos por las células U2OS y cualquier proteína pueden ser derribados o sobreexpresa para determinar los efectos sobre el comportamiento celular durante durotaxis. Además, este protocolo puede ser adaptado para incorporar proteínas etiquetadas con fluorescencia para analizar su dinámica y el comportamiento utilizando FRAP o FRET enfoques.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
		</tr>
		<tr>
			<td>#1 Cover glass 12mm</td>
			<td>Fisher Scientific</td>
			<td>12-545-82</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Celltreat</td>
			<td>229106</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro</td>
			<td>15-017-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Cellgro</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Fisher Scientific</td>
			<td>BP356-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Cellgro</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>BD Biosciences</td>
			<td>610077</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>21600-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes</td>
			<td>Celltreat</td>
			<td>229456</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>U2OS cells</td>
			<td>ATCC</td>
			<td>HTB-96</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simplified system for evaluating cell mechanosensing and durotaxis in vitro

Las propiedades de la composición y mecánicas de la matriz extracelular son muy variables entre los tipos de tejidos. Esta diversidad estroma de tejido conectivo en gran medida el comportamiento celular impactos para regular los procesos normal y patológico incluyendo la proliferación celular, la diferenciación, la señalización de adhesión y migración direccional. En este sentido, la capacidad innata de ciertos tipos de células para migrar hacia un sustrato de matriz más rígida, o menos compatible que se conoce como durotaxis. Este fenómeno juega un papel importante durante el desarrollo embrionario, la reparación de heridas y la invasión de células de cáncer. A continuación, describimos un ensayo sencillo para estudiar durotaxis, in vitro, usando sustratos (PDMS) de polidimetilsiloxano. Preparación del descrito durotaxis cámaras crea una interfaz de rigidez entre el gel PDMS relativamente blando y un cubreobjetos de vidrio rígida. En el ejemplo, hemos utilizado estos durotaxis cámaras para demostrar un papel para el cdc42 / Rac1 GTPasa prote activaciónen, cdGAP, en mechanosensing y durotaxis regulación en células de osteosarcoma U2OS humanas. Este ensayo es fácilmente adaptable a otros tipos de células y / o desmontables de otras proteínas de interés, para explorar sus respectivas funciones en mechanosignaling y durotaxis.

Un diagrama esquemático de la cámara de durotaxis se muestra en la Figura 1A. Sustrato PDMS Soft (a 90: mezcla 1 de la base de PDMS a reticulante soluciones) se extiende en una placa de 6 pocillos y un cubreobjetos de vidrio se coloca en la parte superior de los PDMS, que luego cubre parcialmente la superficie superior del cubreobjetos, creando de este modo una interfaz entre los dos sustratos de diferente cumplimiento. La rigidez del sustrato PDMS blando es ~ 1 kPa, que es comparable con el cumplimie...

Watch this Scientific Journal Video about A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro at JoVE.com

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

Un sistema simplificado para la evaluación de la célula Mechanosensing y Durotaxis<em&gt; In Vitro</em

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

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Research

JoVE Journal

Biology

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

8.1K vistas.  SUNY Upstate Medical University. Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

Video: A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non coding sequences. One prominent function carried out by the non coding fraction of the genome is to regulate gene transcription; however, there are no effective methods to broadly predict cis-regulatory elements from primary DNA sequence. We have developed an efficient protocol to functionally evaluate potential cis-regulatory elements through zebrafish transgenesis. Our approach offers significant advantages over cell-culture based techniques for developmentally important genes, since it provides information on spatial and temporal gene regulation. Conversely, it is faster and less expensive than similar experiments in transgenic mice, and we routinely apply it to sequences isolated from the human genome. Here we demonstrate our approach to selecting elements for testing based on sequence conservation and our protocol for cloning sequences and microinjecting them into zebrafish embryos.

We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.

Mosaico transgénesis pez cebra para la evaluación de secuencias potenciadoras

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

Investigación

Biología

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Derivación eficiente de los Derechos Humanos precursores cardiacos y cardiomiocitos de pluripotentes células madre embrionarias humanas mediante la inducción de moléculas pequeñas

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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 ...

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

Ensayo Proteasomal degradación en un sistema libre de células en las plantas

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

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

La cuantificación de la adhesividad de la célula endotelial<em&gt; In Vitro</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

Un método de transposición eficiente In Vitro por un sistema de belleza durmiente transcripcionalmente regulada en un Vector lentivirales defectuosa integración

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.

Ensayo Desafiador durotaxis de una sola célula para evaluar el control mecánico del movimiento celular y los eventos de señalización relacionados

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate ...

Evaluación de los efectos fitoquímicos sobre Helicoverpa armigera mediante ensayos de alimentación

Developing a Feeding Assay System for Evaluating the Insecticidal Effect of Phytochemicals on Helicoverpa armigera

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and agricultural productivity. In response, plants produce several phytochemicals that negatively impact the insect's growth and survival. This protocol demonstrates an obligate feeding assay method to evaluate the effect of a phytochemical (quercetin) on insect growth, development, and survival. Under controlled conditions, the neonates were maintained until the second instar on a pre-defined artificial diet. These second-instar larvae were allowed to feed on a control and quercetin-containing artificial diet for 10 days. The insects' body weight, developmental stage, frass weight, and mortality were recorded on alternate days. The change in body weight, the difference in feeding pattern, and developmental phenotypes were evaluated throughout the assay time. The described obligatory feeding assay simulates a natural mode of ingestion and can be scaled up to a large number of insects. It permits one to analyze phytochemicals' effect on the growth dynamics, developmental transition, and overall fitness of H. armigera. Furthermore, this setup can also be utilized to evaluate alterations in nutritional parameters and digestive physiology processes. This article provides a detailed methodology for feeding assay systems, which may have applications in toxicological studies, insecticidal molecule screening, and understanding chemical effects in plant-insect interactions.

Autor destacado: Desarrollo y desafíos de los ensayos de alimentación para el manejo de plagas de insectos 

This protocol describes the obligate feeding assay to evaluate the potentially toxic effect of a phytochemical on the lepidopteran insect larvae. This is a highly scalable insect bioassay, easy to optimize the sublethal and lethal dose, deterrent activity, and physiological effect. This could be used for screening eco-friendly insecticides.

Desarrollo de un sistema de ensayo de alimentación para evaluar el efecto insecticida de los fitoquímicos en Helicoverpa armigera

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and ...