Patrick Cafferty es apoyado por una beca postdoctoral de la Sociedad de Esclerosis Múltiple de Canadá.

Parte 1: Pre-montaje experimental. <ol><li> Una semana de antelación, compañero de moscas para generar larvas que expresan GFP bajo el control de un promotor glial-específica. Para nuestro experimento visualizar GFP etiquetados con una secuencia de localización nuclear expresado en las células gliales con la polaridad invertida (repo) promotor (3, 4). </li><li> Prepare la cubierta se desliza por lo menos un día antes en remojo 18 mm cubreobjetos redondos de 10 minutos en 1% de poli-L-lisina solución y el aire seco durante la noche. </li><li> El día antes del experimento, una cámara limpia Chamlide cultura magnética en remojo toda la noche los componentes en el 70% de etanol. </li><li> En el día del experimento, preparar un medio de cultivo mediante la adición de suero fetal bovino, la solución de penicilina-estreptomicina, y la insulina a 10 ml de medio de insectos de Schneider utilizando una técnica aséptica. Las concentraciones de trabajo son: 1X de suero fetal bovino, penicilina 100U/ml; 0.1mg/ml estreptomicina, y 0,2 mg / ml de insulina en el medio de los insectos de Schneider (modificado a partir de (5)). </li><li> Permita que la cámara de cultivo se seque al aire en una campana de cultivos celulares. Lavar el etanol residual de la cámara de la cultura de un enjuague con medio de cultivo preparado. </li></ol> Parte 2: La disección de la Drosophila ojo-cerebro complejo. <ol><li> Seleccione una larva de tercer estadio vagando de un lado de un vial de volar y el lugar en una gota de medio de cultivo refrigeradas en una placa de Petri en hielo durante varios minutos. El enfriamiento de la larva se recomienda para disminuir las contracciones peristálticas del cuerpo para facilitar la disección. </li><li> Coloque la larva refrigerados en una gota de medio de cultivo en una placa recubierta Sylgard bajo un microscopio de disección. </li><li> Bajo el microscopio de disección, utilice un par de pinzas Dumont bien para agarrar firmemente la larva aproximadamente un tercio de la distancia entre el extremo posterior. Espere hasta que la larva expulsa sus ganchos de la boca el extremo anterior. Sujete los ganchos de la boca con un segundo par de pinzas en toda su extensión. Para diseccionar la larva, tire lentamente de los dos pares de pinzas en direcciones opuestas. El complejo ojo-cerebro, junto con las glándulas salivales, los órganos de grasa y tejido imaginario, se retirará de la cuerpo de la larva. No tire los ganchos de la boca demasiado rápido de lo contrario se desprenda del complejo ojo-cerebro. </li><li> Recorte las glándulas salivales, cuerpos grasos, y los discos imaginal con ultra-finas tijeras podadoras. Los dos hemisferios del cerebro y los discos de ojos se adjuntará a la cordón nervioso ventral y ganchos de la boca. </li><li> Lugar un deslizamiento mm cubren 18 en un portaobjetos de vidrio. Dejar un borde de la hoja de la cubierta colgando del borde de la diapositiva con el fin de recoger más tarde la hoja de la cubierta. Añadir una gota de medio de cultivo en la hoja de cubierta de 18 mm. Transferir el complejo ojo-cerebro en el medio de cultivo agarrando los ganchos de la boca con unas pinzas. No comprenden el complejo ojo-cerebro directamente con las pinzas o el tejido dañado. </li><li> Con la tijera fina disección, corte la boca ganchos del complejo del cerebro, ojos y desechar. La eliminación de los ganchos de la boca es importante para la imagen en vivo, como los ganchos de la boca seguirá contrayéndose en el medio de cultivo provoca que el tejido se mueva durante la microscopía. </li><li> Para visualizar la migración gliales en el disco imaginal del ojo, corte con cuidado el tallo óptico, el tejido fino que conecta el cerebro y el disco de ojo y alejar o descartar el cerebro (ver figura 1A). Para visualizar las células de dentro del tallo óptico, dejar el cerebro, el tallo óptico, y el disco imaginal del ojo intacto (véase la Figura 1E). </li><li> Levante el cubreobjetos con unas pinzas y dibujar un círculo debajo de todo el tejido de interés con un marcador permanente. Este círculo se ayuda en la localización de los tejidos para microscopia en la parte 4. </li></ol> Parte 3: Montar el disco imaginal del ojo de una cámara de cultivo magnético. <ol><li> Con unas pinzas, agarre la punta colgando del cubreobjetos. Transferir el portaobjetos a la placa inferior de la cámara magnética Chamlide sin alterar el tejido de la cultura. </li><li> Coloque el anillo de silicona en el cuerpo principal de la cámara Chamlide. Instalar el cuerpo principal en la parte superior de la placa inferior. </li><li> Poco a poco agregue caldo de cultivo para la cámara. No agregue el caldo de cultivo muy rápido o el tejido de interés va a ser alterado. No llenar completamente la cámara para permitir el intercambio de gases en la cubierta. </li><li> Con cuidado, coloque la tapa invisible en la parte superior de la cámara de Chamlide. </li></ol> Parte 4: Visualización de la migración de las células gliales en el disco de los ojos. <ol><li> Coloque la cámara de cultivo en el escenario de un microscopio confocal o de fluorescencia. Localizar la muestra con el círculo en el cubreobjetos como referencia el bajo aumento. </li><li> Se centran en la muestra usando una lente de 40x. Permitir que el tejido para resolver sobre el cubreobjetos. Captura de imágenes del disco óptico del ojo o el tallo cada 10-15min en 3-4 horas. El tejido puede contracción y se mueven fuera de foco. Manual de enfocar el tejido antes de la captura de imágenes aliviará ªes un problema. </li></ol> Parte 5: Los resultados representativos: Se realiza correctamente, el protocolo nos ha permitido recoger una serie de imágenes de la GFP-etiquetados células gliales que migraron desde el tallo óptica en el disco imaginal del ojo (Figura 1 BD). Mientras que imágenes en vivo durante un período de 60 minutos fue suficiente para observar los cambios en las posiciones de los núcleos gliales dentro de un tipo de disco imaginal del ojo salvaje, núcleos gliales en un mutante de un gen necesario para la migración de células gliales fracasado por completo para salir del tallo óptico (flechas Figura 1 FH). Hemos cultivado el cerebro los ojos complejos para períodos de hasta 240 minutos antes de la observación de un deterioro de los tejidos cultivados. Después de los 240 minutos de duración punto empezamos a observar las células GFP positivos en el medio de cultivo circundantes cerebrales cultivadas ojos complejos (flecha Figura 2 C). Además de las buenas prácticas agrarias se acumulan en los puntos difusos a lo largo de los tejidos lo que sugiere una ruptura de la integridad del tejido. <img src="/files/ftp_upload/1155/figure1.png" alt="" width="589" height="241" /> Figura 1: Imágenes en vivo de la GFP-etiquetados núcleos gliales en el tipo de desarrollo salvaje y mutante sistemas visuales. A, E) Las fotografías tomadas con contraste de interferencia diferencial (DIC) de microscopía de tipo salvaje y mutante cultivadas imaginal ojo-discos (ED). En la de tipo salvaje, las células gliales son nacidos dentro y migran desde el tallo óptico (OS) en el ojo de disco. El cerebro ha sido removido para facilitar el acoplamiento y la imagen del disco imaginal del ojo. BD) microscopia de fluorescencia del disco imaginal del ojo de tipo salvaje cultivadas en (A) a los 0, 30 y 60 minutos de tiempo pone de manifiesto los puntos GFP-etiquetados núcleos gliales migran dentro del disco del ojo. FH) La microscopia de fluorescencia del mutante ojo-cerebro complejo (E) a los 0, 30 y 60 puntos de minutos de tiempo demuestra un estancamiento de la GFP-etiquetados núcleos gliales en el tallo óptico (flecha). El cerebro (BR), ha sido colocada en el tallo óptico para facilitar la obtención de imágenes de células gliales en el tallo. <img src="/files/ftp_upload/1155/Figure_2.png" alt="" width="600" height="156" /> Figura 2: Imágenes en vivo de las buenas prácticas agrarias con etiquetas de las membranas gliales en un tipo de disco imaginal del ojo salvaje. Hemos logrado cerebrales cultivadas ojos complejos para 240 minutos de períodos. Cultivos de tejido más de 240 minutos comienza a descomponerse. El medio de cultivo que rodean el disco imaginal del ojo (ED), tallo óptico (OS), y el lóbulo del cerebro (BR) a 0 (A) y 150 minutos (B), en comparación con los 300 minutos de duración punto (C), es gratis de las buenas prácticas agrarias de células positivas, indica con una flecha.

<ol>
	<li>Aigouy, B., Lepelletier, L., Giangrande, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+chain+migration+requires+pioneer+cells.">Glial chain migration requires pioneer cells.</a> J. Neurosci. 28, 11635-11641 (2008).</li><li>Silies, M., Yuva, Y., Engelen, D., Aho, A., Stork, T., Klambt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cell+migration+in+the+eye+disc.">Glial cell migration in the eye disc.</a> J. Neurosci. 27, 13130-13139 (2007).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Sepp, K. J., Auld, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+lacZ+enhancer+trap+lines+to+GAL4+lines+using+targeted+transposition+in+Drosophila+melanogaster.">Conversion of lacZ enhancer trap lines to GAL4 lines using targeted transposition in Drosophila melanogaster.</a> Genetics. 151, 1093-1101 (1999).</li><li>Gibson, M. C., Patel, A. B., Nagpal, R., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emergence+of+geometric+order+in+proliferating+metazoan+epitheia.">The emergence of geometric order in proliferating metazoan epitheia.</a> Nature. 442, 1038-1041 (2006).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+(MARCM)+for+Drosophila+neural+development.">Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development.</a> Trends Neurosci. 24, 251-254 (2001).</li></ol>

The authors have nothing to disclose.

En este protocolo se describe la observación de la migración de células gliales en el disco imaginal del ojo mediante microscopía en vivo. En nuestro ejemplo, de tipo salvaje (figura 1 AD), se utilizó un marcador GFP nuclear para observar el movimiento de células gliales en el disco de los ojos en el transcurso de una hora. En un mutante de un gen candidato requerido para la migración de células gliales en la actualidad objeto de estudio en nuestro laboratorio, hemos observado un estancamiento de los n?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Poly-L-lysine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P8920</td>
<td>&nbsp;</td>
<tr>
<td>Schneider's Insect Media</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S0146</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P4458</td>
<td>&nbsp;</td>
<tr>
<td>Insulin solution from bovine pancreas</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I0516</td>
<td>&nbsp;</td>
<tr>
<td>Chamlide Magnetic chamber</td>
<td>Tool</td>
<td>Live cell Instrument</td>
<td>CM-R-10</td>
<td>35 mm dish type chamber for 18 mm coverslip</td>
</tr>
<tr>
<td>Ultra fine clipper scissors</td>
<td>Tool</td>
<td>Fine scientific tools</td>
<td>15200-00</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 forceps</td>
<td>Tool</td>
<td>Fine scientific tools</td>
<td>11251-20</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscope</td>
<td>Microscope</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>Any fluorescent imaging system that has the necessary filters and excitation for GFP can be used. </td>
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live imaging of glial cell migration in the drosophila eye imaginal disc

Las células gliales de vertebrados e invertebrados organismos deben emigrar a las regiones de destino final para ensheath y apoyar las neuronas asociadas. Si bien el progreso reciente ha sido hecho para describir la migración en vivo de las células gliales en el ala en desarrollo pupal (1), los estudios de<em&gt; Drosophila</em&gt; La migración de células gliales que suelen participar el examen de tejido fijado. Análisis en vivo microscópico de las células móviles ofrece la posibilidad de examinar el comportamiento celular en todo el proceso migratorio, incluyendo la determinación de la tasa y los cambios de dirección del crecimiento. Junto con el uso de herramientas genéticas, imágenes en vivo se puede utilizar para determinar las funciones más precisa de genes específicos en el proceso de desarrollo. El trabajo previo de Silies<em&gt; Et al.</em&gt; (2) ha descrito la migración de las células de origen en el tallo óptico, una estructura que conecta el ojo y el cerebro en desarrollo, en el disco imaginal del ojo en el tejido fijado. Aquí planteamos un protocolo para el examen de la migración en vivo de las células gliales en el<em&gt; Drosophila</em&gt; Disco imaginal del ojo. Nos aprovechamos de una línea de Drosophila que expresa GFP en las células de desarrollo para seguir la progresión de las células gliales de tipo salvaje y en los animales mutantes.

Watch this Scientific Journal Video about Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc at JoVE.com

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

A continuación se describe un protocolo para examinar la migración de las células gliales en el desarrollo del ojo de Drosophila mediante el análisis microscópico directo emparejado con GFP etiquetados células gliales.

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 ...

live-imaging-glial-cell-migration-drosophila-eye-imaginal

Research

JoVE Journal

Biology

10.7K vistas.  University of British Columbia - UBC. A continuación se describe un protocolo para examinar la migración de las células gliales en el desarrollo del ojo de Drosophila mediante el análisis microscópico directo emparejado con GFP etiquetados células gliales.

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

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

La disección de los discos imaginales de 3er estadio las larvas de Drosophila

Investigación

Biología

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

Visualizar el vivo Drosophila Gliales-unión neuromuscular con tintes fluorescentes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

Una novela RFP reportero para ayudar en la visualización del disco imaginal del ojo en Drosophila</em

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries ...

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 ...

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 ...

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 ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

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 Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Imágenes de células vivas de las primeras autofagia Eventos: Omegasomes y más allá

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

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, ...

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

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