Este trabajo fue apoyado por el NIH subvención NS042197 a MD. Azad Jammu y Cachemira fue apoyado por becas y T32GM008719 F30NS068006.

1. Producción de agujas de microinyección <ol><li> Prefabricados agujas de microinyección están disponibles comercialmente (por ejemplo. Femtotips de Eppendorf) y son útiles si uno no se está realizando un gran número de micro-inyecciones. Sin embargo, para aquellos que desean establecer a largo plazo las capacidades de la microinyección, una alternativa es la producción de agujas de microinyección en el laboratorio con paredes delgadas de vidrio de borosilicato capilares y un extractor de aguja comercial. Esto también permite que la forma de agujas de ser variada, que pueden ser útiles para diferentes tipos de células. </li><li> Con el extractor de la aguja Narishige PC-10 microinyección, conecte los cuatro pesos y el uso de un programa de un solo paso de tracción (Paso 1 de ajuste) con el ajuste de calor con respecto a 58,0 (calentador N º 2). Asegúrese de colocar el elemento de calefacción en el centro de cada capilar de modo que las dos agujas resultantes son de una longitud similar. </li><li> Tire de varios capilares (alrededor de 2 capilares para cada proteína de interés). </li><li> Agujas almacenar en un recipiente, mientras que estar seguro de no dañar la punta de la aguja. Materiales como la espuma o el Blu-Tack se pueden utilizar en recipientes para agujas de microinyección. </li></ol> 2. La preparación de mezclas de proteínas para la inyección <ol><li> Preparar un buffer de microinyección 10 veces con 1 M de cloruro de potasio y fosfato de potasio 0,1 M (KPI) buffer (mezcla equimolar de K 2 HPO 4 y KH 2 PO 4) a un pH de 7,4. Este búfer puede almacenar a largo plazo a temperatura ambiente. </li><li> Para visualizar la inyección de un colorante fluorescente como rodamina dextrano hay que añadir. Diluir buffer de microinyección the10x en agua y disolver rodamina dextrano en polvo para preparar una solución buffer de microinyección de 5x que contiene 20-40 mg / ml de rodamina dextrano. </li><li> Almacenar esta solución en la oscuridad a 4 ° C. Debería ser suficiente para un máximo de 100 preparaciones individuales de proteínas solución. Otros colorantes, como isotiocianato de fluoresceína-dextrano puede sustituir. </li><li> Prepare citocromo c mediante la disolución de las poblaciones de purificar el citocromo c en el agua a una concentración de 20 mg / ml. Tienda de citocromo c a -80 ° C para almacenamiento a largo plazo y evitar los ciclos de congelación-descongelación mediante el almacenamiento de citocromo c en el pequeño (10 l) alícuotas. </li><li> Prepare una mezcla de 10 l para la inyección de proteínas mediante la combinación de dos buffer de microinyección l 5x contienen rodamina dextrano con 3 l de agua y 5 citocromo c l para una concentración final de 10 c mg / mL en el citocromo 1x tampón (100 mM KCl, 10 mM KP i, 4-8 mg / ml de rodamina dextrano). </li></ol> 3. Microinyección citoplasmática del citocromo c <ol><li> Justo antes de la microinyección, centrifugar la mezcla de citocromo C y buffer de microinyección a 16.000 g durante 10 minutos a 4 ° C para separar cualquier partícula que pueda obstruir las agujas de microinyección. </li><li> Durante la centrifugación, a su vez en el microinyector para permitir que la presión del aire para construir. </li><li> Coloque un plato de células en el centro de la platina del microscopio y establecer el foco en las células. Para reducir al mínimo la cantidad de tiempo que las células se mantienen fuera de la incubadora, las células normalmente se devuelven a la incubadora dentro de los 30 min. </li><li> Pipeta de 0,5 a 1 l de la superficie superior de la mezcla de la proteína en el extremo romo de los capilares. Tenga cuidado de no pipeta de las partículas que han sido centrifugada para la parte inferior del tubo. En un minuto, la mezcla de proteína va a distribuir a la punta de la aguja a través de la acción capilar. </li><li> Coloque la aguja con firmeza al titular de la capilaridad de la micromanipulador y la posición de la aguja de modo que la punta pasa a través de la luz transmitida del microscopio aproximadamente a un ángulo de 45 °. </li><li> Ajustar la posición de la punta de la aguja de modo que está situado en el centro del campo de visión. Al centro de la aguja, usar el micromanipulador para mover la aguja mientras mira por el ocular del microscopio. La sombra de la aguja debe ser visible. Ajustar la aguja de modo que su sombra se ve sólo en la mitad del campo de vista, lo que indica que la punta de la aguja se centra sobre las células. </li><li> Establecer el microinyector al modo de flujo continuo y ajustar la presión de trabajo de 20 a 100 hPa. Cada aguja puede requerir una presión de trabajo diferentes, y la presión de trabajo es probable que el ajuste necesario en el procedimiento de microinyección para mantener un flujo constante. </li><li> Baje la aguja con el mando de grueso a una posición justo por encima de las células. Para ello, elevar el plano focal del microscopio a una posición justo por encima de las células. Luego baje la aguja hacia las células con el mando hasta que la gruesa punta de la aguja está en foco. </li><li> Volver a centrar la punta de la aguja dentro del campo de visión y aumentar la ampliación al cambiar el objetivo del microscopio. </li><li> Baje lentamente la aguja con el unti manipulador de mando bienl que la aguja está ligeramente por encima del plano focal de las células. </li><li> Compruebe el flujo de la mezcla de proteínas mirando a la fluorescencia de color rojo de la rodamina. La mezcla de proteínas debe ser salir de la aguja como un chorro fino y constante. Las agujas deben ser reemplazados y re-cargada si la aguja está en peligro o si el flujo es demasiado fuerte. A veces, la punta del capilar de vidrio se cierra y no se ve el flujo de la aguja. Si esto ocurre, vuelva a colocar la aguja con cuidado o inferior a la parte inferior de la placa de cultivo a la ruptura suavemente la punta de la aguja. </li><li> La posición de la punta de la aguja de modo que apunte hacia una celda de aproximadamente un ángulo de 45 °. Luego, con un movimiento suave, bajar la aguja mientras se mueve hacia la célula. Con un movimiento suave y en segundo lugar, inmediatamente invertir la dirección de la aguja para extraer de la célula. </li><li> Una célula con éxito a menudo se inyecta un poco oleaje y se puede confirmar mediante la visualización de la rodamina fluorescente de color rojo dentro de la célula. En ocasiones, una célula se inyecta accidentalmente en el núcleo, que será visible. </li><li> Continuar inyectando células mediante el ajuste de la platina del microscopio hasta unos 50-100 células se inyectan. Cuando se mueve la platina del microscopio, asegúrese de levantar la aguja de microinyección de manera que se aclara la parte superior de las células. </li></ol> 4. Los resultados representativos: La microinyección citoplasmática del citocromo c imita su liberación de la mitocondria durante la apoptosis. Por lo tanto, como era de esperar, los fibroblastos rápidamente la apoptosis en la microinyección citosólica de citocromo c de la especie bovina (Fig. 1A). Para asegurarse de que el procedimiento de inyección por sí sola no es responsable de la muerte celular, la inyección de levadura de citocromo c sirve como un control importante, ya que la levadura del citocromo c es incapaz de activar las caspasas 12. Curiosamente, post-mitóticas neuronas simpáticas son muy resistentes a la citosólica de citocromo c (Fig. 1B) 8,13. Nuestro laboratorio ha identificado que la XIAP caspasa inhibidor endógeno es un inhibidor de la clave de activación de la caspasa en las neuronas 14. Por lo tanto, para que las neuronas mueren después de la inyección del citocromo c, XIAP primero debe convertirse en inactiva. Por ejemplo, la microinyección de citocromo c en XIAP - / - las neuronas simpáticas es suficiente para permitir la activación de las caspasas y apoptosis en estas células (Fig. 2). <img src="/files/ftp_upload/2773/2773fig1.jpg" alt="Figura 1"/> Figura 1. Microinyección citoplasmática del citocromo c provoca la rápida muerte de los fibroblastos, pero no las neuronas. A) De tipo salvaje MEFs o (B) después del día 5 de tipo salvaje neuronas simpáticas se microinyección con bovinos citocromo c (10 mg / ml), junto con rodamina-dextrano para marcar las células inyectadas. Las imágenes muestran el mismo campo de las células inmediatamente después de la inyección (0 h), o en el momento indicado. Las flechas indican las células inyectadas. Barra de escala, 20 micras. <img src="/files/ftp_upload/2773/2773fig2.jpg" alt="Figura 2"/> Figura 2. XIAP neuronas deficientes son susceptibles a la microinyección citoplasmática del citocromo c. Postnatal día 5 neuronas simpáticas de los ratones knock-out XIAP se microinyección con bovinos citocromo c (10 mg / ml), junto con rodamina-dextrano para marcar las células inyectadas. Las imágenes muestran el mismo campo de las células inmediatamente después de la inyección (0 h), o 5 horas después de la citocromo c microinyección (5 horas). Barra de escala, 20 micras.

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	<li>Danial, N. N., Korsmeyer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death:+critical+control+points.">Cell death: critical control points.</a> Cell. 116, 205-219 (2004).</li><li>Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expanding+role+of+mitochondria+in+apoptosis.">The expanding role of mitochondria in apoptosis.</a> Genes Dev. 15, 2922-2933 (2001).</li><li>Hengartner, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biochemistry+of+apoptosis.">The biochemistry of apoptosis.</a> Nature. 407, 770-776 (2000).</li><li>Fuentes-Prior, P., Salvesen, G. 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O., Orrenius, S., Zhivotovsky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+induced+by+microinjection+of+cytochrome+c+is+caspase-dependent+and+is+inhibited+by+Bcl-2.">Apoptosis induced by microinjection of cytochrome c is caspase-dependent and is inhibited by Bcl-2.</a> Cell Death Differ. 5, 660-668 (1998).</li><li>Li, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+induction+of+apoptosis+by+microinjection+of+cytochrome+c.+Bcl-XL+has+activity+independent+of+cytochrome+c+release.">Cell-specific induction of apoptosis by microinjection of cytochrome c. Bcl-XL has activity independent of cytochrome c release.</a> J. Biol. Chem. 272, 30299-30305 (1997).</li><li>Deshmukh, M., Johnson, E. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+cell-free+system+of+neuronal+apoptosis+-+comparison+of+premitochondrial,+mitochondrial,+and+postmitochondrial+phases.">Establishment of a cell-free system of neuronal apoptosis - comparison of premitochondrial, mitochondrial, and postmitochondrial phases.</a> J. Neurosci. 17, 6165-6178 (1997).</li><li>Neame, S. J., Rubin, L. L., Philpott, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+cytochrome+c+activity+within+intact+neurons+inhibits+apoptosis.">Blocking cytochrome c activity within intact neurons inhibits apoptosis.</a> J. Cell Biol. 142, 1583-1593 (1998).</li><li>Potts, P. R., Singh, S., Knezek, M., Thompson, C. 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Todos los procedimientos experimentales con animales fueron aprobados por el Cuidado de Animales institucional y el empleo en la Universidad de Carolina del Norte.

La microinyección de citocromo c directamente en el citoplasma de las células es una herramienta única y poderosa que permite estudios de la regulación post-mitocondrial de la apoptosis. Es importante destacar que esta técnica permite la activación directa de la apoptosis aguas abajo de la mitocondria, sin el uso de agentes que causan daño celular o mitocondrial. Aunque este protocolo se ha centrado en la microinyección de citocromo c para los estudios sobre la apop...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> DM IRE2 microscopio invertido </td><td> Leica </td><td></td><td></td></tr><tr><td> PC-10 Microinyección Extractor de agujas </td><td> Narishige </td><td></td><td></td></tr><tr><td> MWO-202 Micromanipulador </td><td> Narishige </td><td></td><td></td></tr><tr><td> FemtoJet microinyector </td><td> Eppendorf </td><td></td><td></td></tr><tr><td> De pared delgada Boroscilicate capilar de vidrio con microfilamentos </td><td> AM Sistemas </td><td> 615000 </td><td> 4 pulgadas de largo, 1,00 mm de diámetro exterior de 0,75 mm de diámetro interno </td></tr><tr><td> Rodamina B isotiocianato de dextrano </td><td> Sigma-Aldrich </td><td> R9379 </td><td> Peso molecular medio ~ 70.000 Da </td></tr><tr><td> Bovina proteína citocromo c </td><td> Sigma-Aldrich </td><td> C3131 </td><td></td></tr></tbody></table>

activation of apoptosis by cytoplasmic microinjection of cytochrome c

La apoptosis, o muerte celular programada, es una vía conservada y muy regulado por el cual las células mueren 1. La apoptosis puede ser activado cuando las células encuentran una amplia gama de tensiones citotóxicos. Estos insultos iniciar cascadas de señalización que pueden llegar a causar la liberación de citocromo c desde el espacio intermembrana mitocondrial al citoplasma 2. La liberación de citocromo c de la mitocondria es un evento clave que desencadena la activación rápida de las caspasas, proteasas celulares claves que finalmente ejecutar la muerte celular 3-4. La vía de la apoptosis está regulada en los puntos aguas arriba y aguas abajo de la liberación del citocromo c de la mitocondria 5. Con el fin de estudiar la regulación post-mitocondrial de activación de las caspasas, muchos investigadores han recurrido a la microinyección directa de citoplasma holocytochrome c (hemo-adjunto) de proteínas en las células 6-9. Citocromo c es normalmente localizada en la mitocondria, donde la unión de un grupo hemo es necesario para poder activar la apoptosis 10-11. Por lo tanto, para activar directamente las caspasas, es necesario inyectar la proteína c holocytochrome en lugar de su cDNA, porque si bien la expresión de citocromo c de las construcciones de cDNA se traducirá en la orientación mitocondrial y el apego del hemo, será secuestrado de caspasas citosólica. Por lo tanto, la microinyección directa citosólica de la proteína purificada hemo-adjunto del citocromo c es una herramienta útil para simular la liberación del citocromo c mitocondrial y la apoptosis, sin el uso de insultos tóxicos que causan daño celular y mitocondrial. En este artículo se describe un método para la microinyección de citocromo c de proteínas en las células, a través del ratón fibroblastos embrionarios (MEFs) y primaria neuronas simpáticas como ejemplos. Aunque este protocolo se centra en la inyección de citocromo c de las investigaciones de la apoptosis, las técnicas mostradas aquí también se puede adaptar fácilmente para la microinyección de otras proteínas de interés.

Watch this Scientific Journal Video about Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c at JoVE.com

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

En este protocolo, se describe la microinyección directa citoplasma del citocromo<em&gt; C</em&gt; Las proteínas en los fibroblastos y las principales neuronas simpáticas. Esta técnica permite la introducción de la citocromo<em&gt; C</em&gt; Proteínas en el citoplasma de las células e imita la liberación de citocromo<em&gt; C</em&gt; De las mitocondrias, que se produce durante la apoptosis.

La activación de la apoptosis mediante microinyección citoplasmática del citocromo C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

activation-of-apoptosis-by-cytoplasmic-microinjection-of-cytochrome-c

Research

JoVE Journal

Biology

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

17.3K vistas.  University of North Carolina. En este protocolo, se describe la microinyección directa citoplasma del citocromo C Las proteínas en los fibroblastos y las principales neuronas simpáticas. Esta técnica permite la introducción de la citocromo C Proteínas en el citoplasma de las células e imita la liberación de citocromo C De las mitocondrias, que se produce durante la apoptosis.

Video: Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Generación de transgénicos estable C. elegans con microinyección

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Investigación

Biología

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinyección de oocitos de Xenopus laevis

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

Microinyección de embriones de pez cebra para analizar la función génica

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

Generación de transgénicos C. elegans Por la transformación biolística

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

Análisis de la cinética de exportación nuclear de ARNm en células de mamífero por microinyección

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

El análisis de la función de las pequeñas GTPasas de la microinyección de plásmidos en células epiteliales polarizadas

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

La visualización de la función respiratoria mitocondrial utilizando citocromo C Oxidasa / Succinato Deshidrogenasa (COX / SDH) de doble etiquetado Histoquímica

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are evident. A means to detect apoptosis in its earliest, reversible stages would afford a pre-clinical 'window' during which preventive or therapeutic measures could be taken to protect the heart from permanent damage. We present herein a simple and robust method to conjugate human Annexin V (ANX), which avidly binds to cells in the earliest, reversible stages of apoptosis, to superparamagnetic iron oxide (SPIO) nanoparticles, which serve as an MRI-detectable contrast agent. The conjugation method begins with an oxidation of the SPIO nanoparticles, which oxidizes carboxyl groups on the polysaccharide shell of SPIO. Purified ANX protein is then added in the setting of a sodium borate solution to facilitate covalent interaction of ANX with SPIO in a reducing buffer. A final reduction step with sodium borohydride is performed to complete the reduction, and then the reaction is quenched. Unconjugated ANX is removed from the mix by microcentrifuge filtration. The size and purity of the ANX-SPIO product is verified by dynamic light scattering (DLS). This method does not require addition to, or modification of, the polysaccharide SPIO shell, as opposed to cross-linked iron oxide particle conjugation methods or biotin-labeled nanoparticles. As a result, this method represents a simple, robust approach that may be extended to conjugation of other proteins of interest.

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Early detection of apoptosis may identify at-risk cell populations in a variety of diseases. Here we demonstrate a method to link an early apoptosis-detection protein (Annexin V) to a MRI-detectable iron oxide nanoparticle (SPIO). This method may be extended to other proteins of interest to generate MRI-detectable molecular imaging probes.

Síntesis de un<em&gt; En vivo</em&gt; RM-detectable apoptosis de la sonda

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

Una sola molécula de imágenes del Reglamento Gene<em&gt; En vivo</em&gt; Utilización de la activación cotraduccional por escisión (COTRAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Análisis de la apoptosis en embriones de pez cebra en Todo el montaje de inmunofluorescencia para detectar la caspasa 3 activada

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

La activación de la apoptosis mediante microinyección citoplasmática del citocromo C

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