Este trabajo fue apoyado por una Marie Curie Reintegración Grant (IRG249084). Damos las gracias a todos los miembros del laboratorio Kurant.

Para seguir la dinámica de la liquidación de apoptosis de células in vivo dos poblaciones de células deberá contener: los fagocitos y las células apoptóticas. Con motivo de las poblaciones de células fagocíticas que utilizamos una línea de Drosophila que contiene el marcador simular cytGFP, que las etiquetas de las células fagocíticas exclusivamente en el embrión: macrófagos, células gliales y ectodermo 8. Se pueden utilizar diferentes marcadores de las células fagocíticas, incluidas las líneas que contienen un controlador Gal4 hemocyte específico (CRQ-Gal4) o células gliales específicos Gal4 conductor (repo-Gal4) y un reportero fluorescente codificada genéticamente bajo control UAS (UAS-GFP). Para controlar las células apoptóticas durante el despacho celular apoptótica usamos diferentes apoptosis / marcadores fagocitosis. Anexina V (Molecular Probes) sirve como un marcador temprano de células apoptóticas 10; Phiphilux (OncoImmunin) es un sustrato fluorogénico de caspasa-3, que se utiliza como una apoptosis más tardesis marcador, y LysoTracker (Molecular Probes) funciona como un marcador fagosoma. Se inyecta estos reactivos utilizando el sistema de microinyección PicoPump PV 820. 1. Preparándose <ol><li> Pegamento Heptano: Desenrolle la cinta de doble cara y poner todo lo que puede en un vial de centelleo, se llenan de heptano, cerrar el vial con Parafilm y rock durante 24 horas. Añadir heptano si el pegamento es demasiado grueso. </li><li> Permitimos que las moscas ponen en una uva precalentado jugo de la placa de agar + levadura pegue durante 2 horas y, a continuación, transferir la placa a 25 ° C durante 10-12 horas (depende de la etapa deseada) antes de la microinyección y el tiempo de grabación de lapso de los embriones inyectados. Recopilamos embriones de las placas de agar mantenido a 25 ° C, proporcionando de este modo embriones de la etapa 15 o 16 de desarrollo. </li><li> Preparación de la aguja: Para preparar agujas para inyección, se utiliza un &quot;instrumento Sutter &#39;extractor de agujas y filamentos de vidrio de paredes delgadas (tubo capilar FHC). La punta del capilar combinado está roton para un diámetro de 0,5-2 micras. </li><li> Cubreobjetos con pegamento: Utilizando una punta de pipeta de dispersar una gota del pegamento heptano en una línea en el centro del cubreobjetos. Deje que se seque durante unos segundos. Prepare algunas de estas cubreobjetos. </li></ol> 2. Preparación de embriones <ol><li> Recoger embriones a partir de placas de agar y transferirlos a un filtro de células (SPL Life Sciences) usando un cepillo de pintura limpio y agua corriente. El filtro se coloca sobre un vaso para recoger las aguas residuales. </li><li> Lave los embriones en el filtro de células con agua hasta eliminar toda la pasta de levadura. </li><li> Secar el exceso de agua frotando la parte exterior del filtro usando Kimwipes. </li><li> Coloque el filtro de células en una placa de Petri limpia y agregue suficiente 50% de lejía para cubrir embriones en el filtro celular. Dechorionate los embriones durante 2 min en ocasiones (2-3 veces) agitando. </li><li> Enjuague con agua exhaustivamente hasta embriones sueltas el olor a cloro. </li><li> Coloque el filtro de células en un recipiente limpioPlaca de Petri y cubrir los embriones con agua para evitar que se seque para arriba. </li><li> Antes de iniciar el ajuste de los embriones, la carga 1 l de reactivo deseado en dos agujas (a veces una aguja puede estar obstruida). Se tarda unos 5 minutos para que el líquido para llegar a la punta de la aguja. </li><li> Corte un pedazo rectangular de agar jugo de uva y colocarlo en un portaobjetos de microscopio. Transferencia de embriones dechorionated de la placa de Petri con la pieza agar usando un pincel limpio. </li><li> Bajo un microscopio de disección fluorescente seleccionar adecuadamente por etapas embriones que expresan el marcador GFP usando un cepillo de pintura húmeda y colocar cada embrión cerca del borde de la pieza de agar en una fila uno tras otro (Figura 1A). Los embriones se colocan con su lado ventral hacia arriba para la fagocitosis de imágenes por la glía en el sistema nervioso central (SNC). </li><li> Establecer unos 10 embriones en una fila. </li><li> Fije los embriones a un cubreobjetos recubierto en el centro con una tira de la cola heptano (Figura 1B, C </strong&gt;). </li><li> Coloque el cubreobjetos en la cámara de deshidratación durante aproximadamente 5 min (esto depende de la humedad de la habitación y la cantidad a inyectar). </li><li> Después de secar a cubrir los embriones con aceite de halocarbonos 700 (Sigma) para evitar una mayor deshidratación. </li><li> Coloque el cubreobjetos sobre un portaobjetos de microscopio con los embriones hacia arriba. Usted puede poner una gota de agua en la diapositiva para evitar que se mueva del cubreobjetos. Los embriones están listos para la inyección. Ubicación de inyección es típicamente en el lado lateral en el centro del embrión. </li></ol> 3. Inyección de Embriones <ol><li> Coloque la aguja a un micromanipulador. </li><li> Para romper la punta de la aguja puso el borde del cubreobjetos en el campo de visión y ajustar la aguja en el mismo plano focal. Muy mueva con cuidado el cubreobjetos hasta que llega a la punta de la aguja y la rompe. </li><li> Centrándose en los embriones, colocar la aguja en el aceite y asegúrese de obtener una gota de líquido de la aguja. Esto significa que la punta de la aguja ha sidoroto también. </li><li> Sin tocar el soporte de la aguja mover el embrión en la aguja e inyectar una gota en el embrión. Mueva el embrión de distancia y proceder a la siguiente hasta que se inyectan todos los embriones. </li></ol> 4. Imágenes Nos la imagen de los embriones en un microscopio confocal invertido con un objetivo de 40X o 100X. Buscamos un embrión bien situada, el SNC en el medio, que muestra una fuerte expresión GFP y un buen etiquetado de las células apoptóticas después de la inyección. Por tiempo de grabación de lapso de embriones vivos que solemos elegir 5 o 6 rebanadas confocal (2 micras de espesor cada una). Después, se hace una proyección de 3 rebanadas (6 m de espesor) con el fin de observar las células enteras. Utilizamos marcadores de apoptosis, que son estables y no blanquear fácilmente. Para evitar GFP blanqueo hacemos grabaciones en intervalos de 60 segundos. En algunos casos, los embriones pueden comenzar a rodardurante el tiempo de grabación de vídeo. Por lo tanto, damos un vistazo de vez en cuando en el registro con el fin de parar y empezar de nuevo si es necesario.

<ol>
	<li>Kinchen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+to+die+for:+signaling+to+apoptotic+cell+removal+in+worm,+fly+and+mouse.">A model to die for: signaling to apoptotic cell removal in worm, fly and mouse.</a> Apoptosis. 15, 998-1006 (2010).</li><li>Kinchen, J. M., Ravichandran, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagosome+maturation:+going+through+the+acid+test.">Phagosome maturation: going through the acid test.</a> Nat. Rev. Mol. Cell Biol. 9, 781-795 (2008).</li><li>Kinchen, J. M., Ravichandran, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytic+signaling:+you+can+touch,+but+you+can't+eat.">Phagocytic signaling: you can touch, but you can't eat.</a> Curr. Biol. 18, 521-524 (2008).</li><li>Stuart, L. M., Ezekowitz, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis:+elegant+complexity.">Phagocytosis: elegant complexity.</a> Immunity. 22, 539-550 (2005).</li><li>Lauber, K., Blumenthal, S. G., Waibel, M., Wesselborg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clearance+of+apoptotic+cells:+getting+rid+of+the+corpses.">Clearance of apoptotic cells: getting rid of the corpses.</a> Mol. Cell. 14, 277-287 (2004).</li><li>Henson, P. M., Hume, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+cell+removal+in+development+and+tissue+homeostasis.">Apoptotic cell removal in development and tissue homeostasis.</a> Trends Immunol. 27, 244-250 (2006).</li><li>Kurant, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+the+CNS+clear:+glial+phagocytic+functions+in+Drosophila.">Keeping the CNS clear: glial phagocytic functions in Drosophila.</a> Glia. 59, 1304-1311 (2011).</li><li>Kurant, E., Axelrod, S., Leaman, D., Gaul, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Six-microns-under+acts+upstream+of+Draper+in+the+glial+phagocytosis+of+apoptotic+neurons.">Six-microns-under acts upstream of Draper in the glial phagocytosis of apoptotic neurons.</a> Cell. 133, 498-509 (2008).</li><li>Mergliano, J., Minden, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase-independent+cell+engulfment+mirrors+cell+death+pattern+in+Drosophila+embryos.">Caspase-independent cell engulfment mirrors cell death pattern in Drosophila embryos.</a> Development. 130, 5779-5789 (2003).</li><li>van den Eijnde, S. M., 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=Cell+surface+exposure+of+phosphatidylserine+during+apoptosis+is+phylogenetically+conserved.">Cell surface exposure of phosphatidylserine during apoptosis is phylogenetically conserved.</a> Apoptosis. 3, 9-16 (1998).</li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

Aclaramiento celular apoptótica es una última etapa crítica de la apoptosis, la cual es altamente dinámico. Por lo tanto los estudios en tiempo real del proceso son de suma importancia. Aquí se describe un protocolo que permite la supervisión del despacho de células apoptóticas en el desarrollo de embriones de Drosophila vivir. En este procedimiento, dos poblaciones de células deben marcarse mediante marcadores específicos: las células apoptóticas y los fagocitos. El reportero simular cytGFP</em...

La eliminación adecuada de las células no deseadas o aberrante a través de la apoptosis y la subsiguiente fagocitosis es crucial para el desarrollo embrionario, así como para la homeostasis del tejido en el adulto. La fagocitosis de las células apoptóticas o el aclaramiento celular por apoptosis es un proceso muy dinámico que se desarrolla en cuatro etapas: (1) la contratación de los fagocitos a la célula apoptótica (&quot;find-me»), (2) el reconocimiento de la célula como un objetivo para la fagocitosis ( &quot;comer-me &#39;) y (3) inmersión, seguido de (4) la maduración del fagosoma y la degradación de la partícula apoptosis 1-5. Hay dos tipos de fagocitos: macrófagos &quot;profesionales&quot; y las células dendríticas inmaduras, y el &quot;no profesionales&quot; células de tejidos vecinos residentes, que son cruciales para la remoción de células apoptóticas durante el desarrollo metazoos 6,7. En el desarrollo de Drosophila, la eliminación de células superfluas a través de la apoptosis se produce en tres mfases Ain, por primera vez en la segunda mitad de embriones tarde, entonces a mediados de pupa, y de nuevo a principios del adulto. Durante la embriogénesis partículas apoptóticas son eliminados por los fagocitos &quot;profesionales&quot;, los macrófagos y por ectodermo y glia 8,9 «no profesional». En nuestro trabajo anterior hemos identificado un receptor fagocítico, seis micras-bajo (SIMU), que se expresa exclusivamente en las células fagocíticas durante la embriogénesis. Utilizando el promotor simulación que generó un marcador específico de las poblaciones de células fagocíticas (simu-cytGFP) lo que nos permite monitorear los macrófagos, ectodermo y glia simultáneamente en un concierto embrión en desarrollo 8. Además, mediante el uso de marcadores específicos para células apoptóticas en diferentes etapas de la apoptosis, somos capaces de seguir la dinámica de aclaramiento celular apoptótica in vivo.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Annexin V </td>
 <td>Molecular Probes</td>
 <td>A35108</td>
 <td></td>
 </tr>
 <tr>
 <td>PhiPhiLux G2D2</td>
 <td>OncoImmunin</td>
 <td>A304R2G-5</td>
 <td></td>
 </tr>
 <tr>
 <td>LysoTracker</td>
 <td>Molecular Probes</td>
 <td>L-7528</td>
 <td></td>
 </tr>
 <tr>
 <td>Halocarbon oil 700</td>
 <td>Sigma</td>
 <td>H8898</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Material</td>
 </tr>
 <tr>
 <td>Capillary tubing</td>
 <td>FHC </td>
 <td>30-30-0</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Strainer</td>
 <td>SPL</td>
 <td>93100</td>
 <td></td>
 </tr>
 <tr>
 <td>Paintbrush</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

live imaging of apoptotic cell clearance during drosophila embryogenesis

La eliminación adecuada de las células no deseadas o aberrantes mediante apoptosis y la posterior fagocitosis (aclaramiento de las células apoptóticas) es crucial para el desarrollo normal en todos los organismos metazoos. Aclaramiento celular apoptótica es un proceso muy dinámico íntimamente asociada con la muerte celular, las células apoptóticas unengulfed apenas se observan in vivo en condiciones normales. Con el fin de entender los diferentes pasos de aclaramiento celular apoptótica y de comparar los fagocitos &quot;profesionales&quot; - macrófagos y las células dendríticas a &#39;no profesional&#39; - células vecinas tejido residentes, en vivo de imágenes en vivo del proceso es extremadamente valiosa. Aquí se describe un protocolo para el estudio de aclaramiento celular por apoptosis en embriones de Drosophila vivas. Para seguir la dinámica de los diferentes pasos en la fagocitosis que utilizamos marcadores específicos de células apoptóticas y los fagocitos. Además, podemos controlar dos sistemas de fagocitos en paralelo: los macrófagos &quot;profesionales&quot; y &quot;semi-profeglía ssional &#39;en el sistema nervioso central en desarrollo (SNC). El método descrito aquí utiliza el embrión de Drosophila como un excelente modelo para estudios en tiempo real de despacho de las células apoptóticas.

Marcos representativos de una película en la que las células apoptóticas se etiquetan con Anexina V, y glía, ectodermo y los macrófagos se marcaron con simula-cytGFP se muestran en la Figura 2. Cada cuadro es una proyección de 3 rebanadas de 2 micras cada una. El embrión de la etapa 15 está colocada adecuadamente mostrando el SNC de embriones en el medio con glía bien marcado (g). Los macrófagos (m), en su mayoría fuera del SNC, muestran una fuerte expres...

Watch this Scientific Journal Video about Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis at JoVE.com

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Aquí se describe un método eficaz para estudiar la dinámica de aclaramiento celular apoptótica<em&gt; In vivo</em&gt;. Este método emplea vivir<em&gt; Drosophila</em&gt; Embriones como un poderoso modelo para el monitoreo fagocitosis de las células apoptóticas mediante un etiquetado específico de las células apoptóticas y los fagocitos.

Imágenes de Apoptotic Liquidación de células vivas durante<em&gt; Drosophila</em&gt; Embriogénesis

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

live-imaging-apoptotic-cell-clearance-during-drosophila

Research

JoVE Journal

Biology

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

13.4K vistas.  Technion-Israel Institute of Technology. Aquí se describe un método eficaz para estudiar la dinámica de aclaramiento celular apoptótica In vivo. Este método emplea vivir Drosophila Embriones como un poderoso modelo para el monitoreo fagocitosis de las células apoptóticas mediante un etiquetado específico de las células apoptóticas y los fagocitos.

Video: Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

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

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 ...

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 ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Un enfoque de genética inversa para probar la redundancia funcional durante la embriogénesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

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, ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

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 ...

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.

4D de imágenes de la agregación de proteínas en células vivas

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 ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

El tomate / GFP-FLP / FRT Método para imágenes en vivo del mosaico adultos<em&gt; Drosophila</em&gt; Las células fotorreceptoras

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

Análisis citológico de espermatogénesis: Los preparativos en directo y fijas de<em&gt; Drosophila</em&gt; Testículos

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by available technologies, apoptosis is generally assumed to occur shortly thereafter. Caspases can cleave many functional and structural components to cause rapid and complete cell destruction within a few minutes. However, accumulating evidence indicates that in normal healthy cells the same caspases have other functions, presumably at lower enzymatic levels. Studies of non-apoptotic caspase activity have been hampered by difficulties with detecting low levels of caspase activity and with tracking ultimate cell fate in vivo. Here, we illustrate the use of an ultrasensitive caspase reporter, CaspaseTracker, which permanently labels cells that have experienced caspase activity in whole animals. This in vivo dual color CaspaseTracker biosensor for Drosophila melanogaster transiently expresses red fluorescent protein (RFP) to indicate recent or on-going caspase activity, and permanently expresses green fluorescent protein (GFP) in cells that have experienced caspase activity at any time in the past yet did not die. Importantly, this caspase-dependent in vivo biosensor readily reveals the presence of non-apoptotic caspase activity in the tissues of organ systems throughout the adult fly. This is demonstrated using whole mount dissections of individual flies to detect biosensor activity in healthy cells throughout the brain, gut, malpighian tubules, cardia, ovary ducts and other tissues. CaspaseTracker detects non-apoptotic caspase activity in long-lived cells, as biosensor activity is detected in adult neurons and in other tissues at least 10 days after caspase activation. This biosensor serves as an important tool to uncover the roles and molecular mechanisms of non-apoptotic caspase activity in live animals.

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

To detect healthy cells in whole animals that contain low levels of caspase activity, the highly sensitive biosensor designated CaspaseTracker was generated for Drosophila. Caspase-dependent biosensor activity is detected in long-lived healthy cells throughout the internal organs of adult animals reared under optimized conditions in the absence of death stimuli.

En Vivo biosensor Pistas de apoptosis no caspasa actividad en Drosophila

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by ...