Este trabajo fue apoyado por el subsidio GM096076 del programa AREA NIH.

1. Preparación de moscas para la disección <ol><li> Anestesiar moscas sanas del genotipo deseado (por ejemplo, expresión de una proteína GFP-etiquetados) que están a menos de una semana de edad. Seleccione 10-15 hembras de gran tamaño con redondeados de color crema abdomen. </li><li> Transfiera las moscas seleccionados y unos pocos machos (3-5) a un vial que contiene alimento nuevo a la ligera yeasted y permitir que las moscas para engordar durante dos días a 25 ° C. Consulte Weil et al. 6 para los detalles de la preparación de Drosophila. Engorde de las moscas se asegura de que una variedad de etapas, especialmente etapas 8-12, estará disponible para la formación de imágenes. Mal cebados o unfattened vuela por lo general contienen sólo etapas muy tempranas (1-6) y maduros ovocitos (fase 14). </li></ol> 2. Preparación de Ovocitos de Imagen Usando un microscopio compuesto Upright <ol><li> Preparar un portaobjetos de vidrio estándar mediante la colocación de dos cubreobjetos al portaobjetos, aproximadamente 1 cm de separación, utilizando de engrase de siliconae. Utilice sólo grasa suficiente para asegurar un buen sellado entre el cubreobjetos y portaobjetos. Presionando firmemente sobre cada cubreobjetos se extienda la grasa de la capa fina y uniforme. Añadir un poco de aceite de halocarbono 27 (Sigma) para la unión entre los cubreobjetos y se deslizan. Servirán como espaciadores para evitar herir a los ovocitos aislados en los pasos siguientes. No. de 1,5 cubreobjetos trabajar bien, con un espesor medio (0,16 a 0,18 mm) que coincide con la de ovocitos en fase tardía. </li><li> Colocar 2-3 gotas de aceite de halocarbono 27 sobre la superficie de un cubreobjetos de 22 mm 2. Para obtener los ovocitos sanos posibles, una hembra se retira del vial alimentos utilizando pipeteo con la boca y suavemente depositado en el aceite de halocarbono sin anestesiar primero. </li><li> Con fuertes pinzas de disección (como Dumont # 5), echar la mosca contra el cubreobjetos y salga de la punta del abdomen. Eliminar los ovarios arrastrándolos a lo largo de la superficie del cubreobjetos, bajo el aceite. Esto promoverá la adhesion de los oocitos para el cubreobjetos. </li><li> Con cuidado separar los ovarios, en busca de ovocitos que son al menos 10B etapa de la ovogénesis. Los ovocitos en esta etapa pueden ser identificados mediante la búsqueda de cámaras de huevo en el que el ovocito ocupa al menos el 50-60% del volumen total de la cámara de huevos. En este punto de la ovogénesis, las células del folículo que recubren el ovocito se han convertido en columnar, y rodean el ovocito en todos los lados excepto por una pequeña región donde las conexiones con las células nodrizas permanecer. Usa los fórceps para extraer ovocitos individuales de la vaina ovariole. </li><li> Proceder, usando a las mujeres 1-3, 5-8 oocitos intactos hasta que se han aislado en el cubreobjetos. Retire cualquier tejido inutilizable. </li><li> Invierta cuidadosamente los cubreobjetos que contiene los ovocitos en el portaobjetos pre-preparado para que los oocitos se coloca entre los dos espaciadores. No ejerza presión sobre el cubreobjetos invertido como que dañará los ovocitos. Si es necesario, agregue una pequeña cantidad de aceite de hidrocarburo halogenado para tél bordes del &quot;sandwich&quot; para asegurar el espacio está lleno. (Opcional:. Dejar reposar diapositiva durante varios minutos para permitir la sedimentación de cubreobjetos) El portaobjetos ahora está listo para formación de imágenes. </li></ol> 3. Preparación de Ovocitos de Imagen Usando un microscopio compuesto invertido <ol><li> Los ovocitos se disecan esencialmente como se describe en la sección 2, excepto que las pequeñas cajas de Petri con una pieza de fondo de cristal (MatTek, 35 mm) se utilizan en lugar de cubreobjetos. No es necesario para cubrir la muestra, y después de la disección, el tejido puede ser captado directamente en los platos, pero se debe tener cuidado para asegurar que los oocitos permanecen cubiertos con aceite y no se permite que se seque. </li></ol> 4. Imágenes en directo <ol><li> Trae un ovocito en el foco o utilizando DIC óptica de contraste de fases y examinar el ovocito para detectar cualquier signo de daño, tales como fugas de citoplasma, o abombamiento de las células del folículo. Sólo imagen ovocitos que parecen sanos y en buen estado. </li><li> Streaming se puede controlar directamente, sin la necesidad de etiquetado GFP, desde vesículas de yema en el citoplasma son autofluorescentes 7. Esta señal puede ser considerado en el rango FITC del alcance pero es probable que requiera más altos valores de ganancia que los utilizados para las proteínas GFP-etiquetados. </li><li> Las mejores imágenes de transmisión se obtienen a partir de secciones ópticas que están justo debajo de la superficie del ovocito que descansa sobre el cubreobjetos de vidrio / Petri de vidrio. Aquí, el ovocito es ligeramente aplanada, para hacer un mejor plano de foco. Las imágenes pueden capturarse a 200X - 1.000 X. Usando la disposición descrita aquí, los objetivos de aire se debe utilizar, ya que el cubreobjetos forma una barrera entre el ovocito y objetivo. Esto es óptimo debido a que minimiza en gran medida la deriva y el movimiento de la muestra. </li><li> Una vez que una región de interés está en el foco, desconecte la óptica brighfield y cambiar a imagen confocal. Con el rápido escaneo / función de vista previa del software, ajustar el enfoque y ganarsegún sea necesario. </li><li> Configuración para la captura variará de microscopio para microscopio, pero los parámetros generales son las siguientes: Utilice una longitud de onda de excitación de ~ 488 nm y una longitud de onda de emisión de ~ 515 nm. Configurar eje Z de la captura de manera que el eje Z permanece constante, con un retraso de 10 segundos entre los marcos. Capturar una secuencia de ensayo de tramas 5-10 para asegurar el ovocito es estacionario y el plano de enfoque es apropiado. </li><li> Una secuencia de lapso de tiempo típico capturar entre 20 a 50 marcos, y puede ser crítica inmediatamente después de la finalización para evaluar la calidad de vídeo. El proceso se puede repetir para otras células en el portaobjetos, o hembras adicionales puede ser diseccionado, según sea necesario. </li></ol> 5. Solución de problemas y común <ol><li> Drift - Cuando se utiliza un microscopio vertical, las células pueden desplazarse a veces debido a la propagación del petróleo. Esto puede minimizarse mediante el uso de sólo aceite suficiente para cubrir el área bajo el cubreobjetos. Si los problemas persisten, use menos g de siliconaREase para separadores colocará en la diapositiva. </li></ol> Nota: Algunos paquetes de software confocal permite el seguimiento de células, pero en general es mejor abandonar formación de imágenes de los ovocitos de la deriva, y utilizar en su lugar otro espécimen. <ol start="2"><li> No hay señales de transmisión streaming - Si no es evidente, es probable que se deba a una de dos causas: o bien el plano focal para la captura de la imagen no es correcto (por lo general demasiado profundamente en el interior del ovocito) o el ovocito está dañado. El tiempo y la práctica puede minimizar tanto los errores. </li></ol>

<ol>
	<li>Becalska, A. N., Gavis, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lighting+up+mRNA+localization+in+Drosophila+oogenesis.">Lighting up mRNA localization in Drosophila oogenesis.</a> Dev. 136, 2493-2503 (2009).</li><li>He, L., Wang, X., Montell, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shining+light+on+Drosophila+oogenesis:+Live+imaging+of+egg+development.">Shining light on Drosophila oogenesis: Live imaging of egg development.</a> Curr. Opin. Gen. Dev. 21, 612-619 (2011).</li><li>King, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ovarian development Drosophila melanogaster. , (1970).</li><li>Spradling, A. C., Bate, M., Martinez-Arias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+genetics+of+oogenesis.">Developmental genetics of oogenesis.</a> The Development of Drosophila melanogaster. 1, 1-70 (1993).</li><li>Serbus, L. R., Cha, B. -. J., Theurkauf, W. E., Saxton, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynein+and+the+actin+cytoskeleton+control+kinesin-driven+cytoplasmic+streaming+in+Drosophila+oocytes.">Dynein and the actin cytoskeleton control kinesin-driven cytoplasmic streaming in Drosophila oocytes.</a> Dev. 132, 3743-3752 (2005).</li><li>Weil, T. T., Parton, R. M., Davis, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+Individual+Drosophila+Egg+Chambers+for+Live+Imaging.">Preparing Individual Drosophila Egg Chambers for Live Imaging.</a> J. Vis. Exp. (60), e3679 (2012).</li><li>Theurkauf, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+microtubule-dependent+cytoplasmic+streaming+in+cappuccino+and+spire+mutant+oocytes.">Premature microtubule-dependent cytoplasmic streaming in cappuccino and spire mutant oocytes.</a> Science. 265, 2093-2096 (1998).</li><li>Morin, X., Daneman, R., Zavortink, M., Chia, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protein+trap+strategy+to+detect+GFP-tagged+proteins+expressed+from+their+endogenous+loci+in+Drosophila.">A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila.</a> PNAS. 98, 15050-15055 (2001).</li><li>R&#246;per, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rtnl1+is+enriched+in+a+specialized+germline+ER+that+associates+with+ribonucleoprotein+granule+components.">Rtnl1 is enriched in a specialized germline ER that associates with ribonucleoprotein granule components.</a> J. Cell Sci. 120, 1081-1092 (2007).</li><li>Pokrywka, N. J., Payne-Tobin, A., Raley-Susman, K. M., Swartzman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubules,+the+ER+and+Exu:+New+associations+revealed+by+analysis+of+mini+spindles+mutations.">Microtubules, the ER and Exu: New associations revealed by analysis of mini spindles mutations.</a> Mech. Dev. 126, 289-300 (2009).</li><li>Petri, W. H., Mindrinos, M. N., Lombard, M. F., Margaritis, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+development+of+the+Drosophila+chorion.ih+a+chemically+defined+organ+culture+medium.">In vitro development of the Drosophila chorion.ih a chemically defined organ culture medium.</a> Wilhelm Roux's Arch., Devl. Biol. 186, 351-362 (1979).</li></ol>

No tengo nada que revelar.

Drosophila ovocitos son un sistema modelo versátil para abordar una variedad de cuestiones relacionadas con el funcionamiento y organización de las células y componentes subcelulares. Una comprensión completa de la función de las proteínas requiere la supervisión de los aspectos espaciales y temporales de la conducta de proteínas. Los avances en ambos sistemas de formación de imágenes y las herramientas genéticas disponibles para Drosophilists hacer posible que incluso pequeños laboratorios para llevar a cabo experimentos de imagen de alta calidad en los ovocitos de vida. Aquí destaco un protocolo para analizar el comportamiento de las proteínas GFP-etiquetados o autofluorescente y estructuras a mediados o finales de la ovogénesis que se puede utilizar en combinación con una variedad de bases genéticas para investigar la función de proteínas. En este protocolo de describir el proceso por el que los oocitos están preparados para la formación de imágenes y los ajustes básicos confocal que permitan la adquisición de vídeo de lapso de tiempo de proteínas fluorescentes y estructuras. Adicional details en la preparación de los ovocitos se describen por Weil et al. 6 Una amplia variedad de cepas de moscas que expresan proteínas que han sido etiquetados endógenamente a través de exón-atrapamiento están disponibles 8, e infinitamente más variantes de la proteína puede ser diseñado y reintroducidos en las moscas. En el trabajo con tejido vivo, la salud de los especímenes es de suma importancia. Cámaras de huevo de la etapa 10B Puede completar la ovogénesis de una manera esencialmente autónomo 11, lo que es ideal para los estudios de imagen a corto plazo. Se debe tener cuidado en varias etapas clave para obtener datos de alta calidad. Durante la disección es importante para manipular ovarios y ovocitos en tan poco como sea posible, y para reducir al mínimo la cantidad de tiempo entre la disección y la terminación de formación de imágenes. Idealmente, una estación pequeña disección debe estar ubicado en la misma sala que alberga el ámbito confocal, y sólo unas pocas cámaras de huevo a la vez debe ser fotografiado. El número recomendado aquí (5-8) Asegura que el tiempo disección se minimiza mientras que maximiza la probabilidad de obtener imágenes de buena calidad. Recomiendo la captura de una corta serie de imágenes para evaluar la calidad de imagen y confirmar que los ajustes de captura están optimizados, antes de capturar una secuencia de lapso de tiempo más largo. La mayoría del software permite que las imágenes individuales para ser salvo, y estas imágenes se pueden analizar en una variedad de maneras usando ImageJ 12 u otro software.

<table border="1"><tbody><tr><td> Nombre </td><td> Empresa </td><td> El número de catálogo </td><td> Comentarios </td></tr><tr><td> Halocarbono 27 </td><td> Sigma-Aldrich </td><td> H8773-100ML </td><td></td></tr><tr><td> # 5SF Pinzas </td><td> Herramientas Artes Ciencias </td><td> 11252-00 </td><td></td></tr><tr><td> Grasa de silicona </td><td> Sigma Aldrich </td><td> Z273554-1EA </td><td></td></tr><tr><td> Con fondo de cristal platos de Petri </td><td> MatTek </td><td> P35G-0-20-C </td><td></td></tr></tbody></table>

live imaging of gfp-labeled proteins in drosophila oocytes

El ovocito Drosophila se ha establecido como un sistema versátil para investigar cuestiones fundamentales tales como la función del citoesqueleto, la organización celular, y la estructura y función de orgánulo. La disponibilidad de varias proteínas etiquetadas con GFP-significa que muchos procesos celulares pueden ser controlados en células vivas en el transcurso de minutos u horas, y el uso de esta técnica, los procesos de transporte, tales como RNP, morfogénesis epitelial, y la remodelación de tejidos se han descrito en gran detalle en Drosophila 1,2 ovocitos. La capacidad para realizar las imágenes de vídeo combinado con un rico repertorio de mutantes permite una enorme variedad de genes y procesos que se examinarán en detalle increíble. Un ejemplo de ello es el proceso de transmisión de ooplasmic, que se inicia en la mitad de la ovogénesis 3,4. Este vigoroso movimiento de las vesículas citoplasmáticas es microtúbulos y 5 kinesin-dependiente y proporciona un sistema útilTEM para la investigación de la función del citoesqueleto en estas etapas. Aquí presento un protocolo para obtener imágenes de lapso de tiempo de vida de los ovocitos mediante prácticamente cualquier configuración de microscopía confocal.

Obtención de imágenes de las proteínas marcadas con GFP puede variar dependiendo de la fuerza con la proteína de la etiqueta se expresa. A mediados de los ovocitos que expresan etapa Reticulon-like 1 (Rtnl-1), el exón etiquetado con GFP 8,9 produce señal fuerte. Rtnl-1 parece co-localizar con los microtúbulos largas presentes durante el flujo ooplasmic 10 (Figura 1A). Rtnl-1 es un buen marcador para la observación de streaming, y es también un indicador de organización de microtúbulos en los ovocitos en fase tardía. Streaming de Ooplasmic en una cámara de huevos de tipo salvaje es evidente tan notable movimiento de vesículas de yema de autofluorescentes cuando se utiliza una velocidad de captura de un cuadro cada 10 seg (Figura 1B). Una proyección máximo de cinco marcos pueden ser generados para marcar el camino de vesículas que se mueven. <img alt="Figura 1" src="/files/ftp_upload/50044/50044fig1.jpg" /> Figura 1. Vivir formación de imágenes de ambos autofluorescentes y GFP-etiquetados estructuras. A) Una sola vez-lapse de imágenes de una etapa de 11 ovocitos que expresan GFP en Rtnl1-400X. Rtnl1-GFP fibras se mueven con un movimiento ondulatorio en los ovocitos de streaming. B) una proyección a cinco máximo de trama de una cámara de la etapa de huevo 10B en la que las vesículas autofluorescentes yema fueron imágenes cada 10 seg para 50 total seg, la ampliación de 400X. Barra de escala = 30 m

Watch this Scientific Journal Video about Live Imaging of GFP-labeled Proteins in Drosophila Oocytes at JoVE.com

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

Un protocolo para la obtención de imágenes en directo de las proteínas GFP-etiquetados o estructuras autofluorescentes en particular<em&gt; Drosophila</em&gt; Ovocitos se describe.

Imágenes en directo de las proteínas GFP-etiquetados en<em&gt; Drosophila</em&gt; Los ovocitos

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

live-imaging-of-gfp-labeled-proteins-in-drosophila-oocytes

Research

JoVE Journal

Biology

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

11.3K vistas.  Vassar College. Un protocolo para la obtención de imágenes en directo de las proteínas GFP-etiquetados o estructuras autofluorescentes en particular Drosophila Ovocitos se describe.

Video: Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Imágenes en directo de la migración de células gliales en el ojo del disco imaginal de Drosophila

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Investigación

Biología

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

La preparación de los Drosophila Los embriones para Live-Imaging utilizando el protocolo de gota

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Imágenes en directo de Drosophila melanogaster Migraciones hemocitos embrionarias

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

Marcaje fluorescente de COS-7 Expresando etiqueta SNAP-proteínas de fusión de imágenes de células vivas

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

Preparación de desarrollo y de adultos Drosophila Los cerebros y retinas de imágenes en vivo

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Imagen de la posición vertical Drosophila Los embriones

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Celular imagen Cambiar forma de vida Drosophila Los embriones

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

Preparación Individual Drosophila Huevos Cámaras de Imágenes en directo

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

De dos y tres dimensiones imágenes de células vivas de las proteínas de respuesta al daño del ADN

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

Imágenes en directo de las proteínas GFP-etiquetados en

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