Este trabalho foi apoiado por uma Marie Curie Reintegração Grant (IRG249084). Agradecemos a todos os membros do laboratório Kurant.

Para seguir as dinâmicas de depuração de células apoptóticas in vivo duas populações de células deve ser rotulada: células fagocitárias e células apoptóticas. Para marcar populações de células fagocíticas usamos uma linha de Drosophila que contém o marcador simular cytGFP, que rotula as células fagocíticas exclusivamente no embrião: macrófagos, células gliais e ectoderma 8. Pode-se usar diferentes marcadores de células fagocíticas, incluindo linhas contendo um driver Gal4 específico hemocyte (CRQ-Gal4) ou glia específico Gal4 condutor (repo-Gal4) e um repórter fluorescente geneticamente codificado sob controle UAS (uas-GFP). Para monitorar as células apoptóticas durante a remoção de células apoptóticas usamos diferente apoptose / marcadores de fagocitose. A anexina V (Molecular Probes), que serve como um marcador precoce de células apoptóticas 10; Phiphilux (OncoImmunin) é um f luorogénico de substrato caspase-3, o qual é utilizado como um apopto depoissis marcador e Lyso Tracker (Molecular Probes) funciona como um marcador phagosome. Nós injetar estes reagentes usando o sistema de microinjeção PicoPump PV 820. 1. Preparando-se <ol><li> Heptano cola: Desenrole a fita dupla face e colocar tanto quanto você pode em um frasco de cintilação, encha com heptano, vedar o frasco com Parafilm e rock por 24 horas. Adicionar heptano se a cola é muito espesso. </li><li> Nós permitimos que as moscas para colocar em uma placa de ágar suco de uva pré-aquecido + levedura colar de 2 horas, e depois transferir a placa a 25 ° C por 10-12 horas (depende do estágio desejado) antes da microinjeção e gravações lapso de tempo de embriões injetados. Nós recolher embriões a partir das placas de agar mantido a 25 ° C, proporcionando, assim, a partir de embriões de estágio de 15 ou 16 anos de desenvolvimento. </li><li> Preparação da agulha: Para preparar agulhas para injeção, nós usamos um &quot;instrumento Sutter &#39;extrator de agulhas e finos filamentos de vidro de paredes (tubo capilar FHC). A ponta do capilar agrupada está quebradon a um diâmetro de 0,5-2 mM. </li><li> Lamela com cola: Utilizando uma ponta de pipeta de dispersar uma gota de cola de heptano numa linha no meio da lamela. Deixe secar por alguns segundos. Prepare algumas dessas lamelas. </li></ol> 2. Preparação embrião <ol><li> Coletar embriões a partir de placas de ágar e transferi-los para um filtro de células (SPL Life Sciences), utilizando um pincel limpo e água corrente. O filtro é realizada ao longo de um copo para recolher águas residuais. </li><li> Lavar os embriões no filtro de células usando água até que toda a pasta de levedura é removida. </li><li> Seca-se o excesso de água, limpando o exterior do filtro utilizando Kimwipes. </li><li> Colocar o filtro de células numa placa de Petri limpo e adicionar 50% de branqueador suficiente para cobrir os embriões no filtro celular. Dechorionate os embriões para 2 min ocasionalmente (2-3 vezes), misturando-os. </li><li> Enxágüe com água extensivamente até embriões soltos o odor lixívia. </li><li> Coloque o filtro de células em um ambiente limpoPlaca de Petri e cobrir os embriões com água para evitar ressecamento. </li><li> Antes de iniciar a configuração embriões, carregar 1 ul do reagente desejado para as duas agulhas (por vezes, uma agulha pode ser obstruído). Demora cerca de 5 min para o líquido atinja a ponta da agulha. </li><li> Corte um pedaço retangular de ágar suco de uva e coloque-o sobre uma lâmina de microscópio. Transferência de embriões dechorionated da placa de Petri para a peça de agar usando um pincel limpo. </li><li> Sob um microscópio de dissecção fluorescente seleccionar adequadamente encenado embriões que expressam o marcador GFP usando um pincel molhado e colocar cada embrião perto da borda da peça de ágar numa linha um após o outro (Figura 1A). Os embriões devem ser colocadas com o lado ventral para cima para a fagocitose imagiologia por glia no Sistema Nervoso Central (SNC). </li><li> Configure cerca de 10 embriões em uma linha. </li><li> Anexar os embriões para uma lamela revestida no meio, com uma tira de cola heptano (Figura 1B, C </strong&gt;). </li><li> Coloque a lamela na câmara de desidratação por aproximadamente 5 minutos (isso depende da umidade da sala ea quantidade a ser injetada). </li><li> Após a secagem até cobrir os embriões com óleo halocarbono 700 (Sigma) para evitar a desidratação. </li><li> Coloque a lamela numa lâmina de microscópio com os embriões voltados para cima. Você pode colocar uma gota de água sobre a lâmina para evitar que se deslocam da lamela. Os embriões estão prontos para injeção. Local de injecção é tipicamente na face lateral no meio do embrião. </li></ol> 3. Injeção de embrião <ol><li> Coloque a agulha de um micromanipulador. </li><li> Para quebrar a ponta da agulha de colocar a extremidade da lamela no campo de visão e ajustar a agulha para o mesmo plano focal. Muito se mover cuidadosamente a lamela até atingir a ponta da agulha e quebra. </li><li> Incidindo sobre os embriões, coloque a agulha no óleo e certifique-se de obter uma gota de líquido da agulha. Isso significa que a ponta da agulha foiassim quebrada. </li><li> Sem tocar o suporte de agulha move o embrião dentro da agulha e injectar uma gota para o embrião. Mova o embrião longe e avançar para a próxima até que todos os embriões são injetados. </li></ol> 4. Imagem Nós imagem os embriões em um microscópio confocal invertido com objetiva de 40X ou 100X. Procuramos um embrião bem posicionado com o sistema nervoso central, no meio, o que demonstra uma forte expressão da GFP e uma boa marcação das células apoptóticas após a injecção. Para o período de gravações lapso de embriões vivos costumamos escolher 5 ou 6 fatias confocal (2 mm de espessura cada). Depois, fazemos uma projecção de 3 fatias (6 m de espessura), a fim de observar as células inteiras. Nós usamos marcadores de células em apoptose, que são estáveis ​​e não lixívia facilmente. Para evitar GFP branqueamento fazemos gravações em intervalos de 60 seg. Em alguns casos, os embriões podem começar a rolardurante o tempo de gravação de vídeo. Portanto, vamos dar uma olhada de vez em quando na gravação, a fim de parar e começar de novo, se necessário.

<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>

Os autores declaram que não têm interesses financeiros concorrentes.

Desembaraço celular por apoptose é um último estágio crítico de apoptose, que é altamente dinâmico. Portanto, estudos sobre o processo em tempo real são de extrema importância. Aqui nós descrevemos um protocolo que permite o monitoramento de folga celular por apoptose no desenvolvimento de embriões de Drosophila vivendo. Neste processo, duas populações de células deve ser marcado com marcadores específicos: células apoptóticas e fagócitos. O repórter simular cytGFP é adequado para m...

A eliminação adequada de células indesejadas ou aberrante através de apoptose e fagocitose subsequente é crucial para o desenvolvimento embrionário, bem como para a homeostase dos tecidos no adulto. A fagocitose de células apoptóticas ou na depuração de células apoptóticas é um processo altamente dinâmico, que prossegue em quatro passos: (1) o recrutamento de fagócitos para a célula apoptótica (&#39;encontrar-me&#39;), (2) reconhecimento da célula como um alvo para a fagocitose ( &#39;comer-me&#39;) e (3) imersão, seguido de (4) e da maturação do fagossoma e degradação da partícula apoptótica 1-5. Existem dois tipos de fagócitos: macrófagos &quot;profissionais&quot; e as células dendríticas imaturas, e os &quot;não-profissional&quot; células vizinhas tecido residentes, que são cruciais para a liberação celular por apoptose durante metazoan desenvolvimento 6,7. No desenvolvimento de Drosophila, a eliminação de células supérfluas por apoptose ocorre em três metrosfases Ain, pela primeira vez em meados da década de embrião e, em seguida, em meados pupa, e novamente na adulta. Durante a embriogênese partículas apoptóticos são removidos por fagócitos &quot;profissionais&quot;, macrófagos e pelo &quot;não-profissional&quot; ectoderma e glia 8,9. No nosso trabalho anterior, identificaram um receptor fagocitária, de seis mícrons, sob (SIMU), que é expresso exclusivamente em células fagocíticas durante a embriogénese. Usando o promotor simulação que gerou um marcador específico para as populações de células fagocíticas (simular cytGFP) que nos permite monitorar macrófagos, ectoderma e glia simultaneamente em um embrião em desenvolvimento ao vivo 8. Além disso, por utilização de marcadores específicos para células apoptóticas em diferentes fases da apoptose, que é capaz de seguir as dinâmicas de depuração de células apoptóticas 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

A eliminação adequada de células indesejáveis ​​ou aberrante através de apoptose e fagocitose subseqüente (clearance celular por apoptose) é crucial para o desenvolvimento normal em todos os organismos metazoários. Recarga celular por apoptose é um processo altamente dinâmico intimamente relacionado com a morte das células, as células apoptóticas unengulfed são mal vistas in vivo sob condições normais. A fim de compreender as diferentes etapas de depuração de células apoptóticas e comparar os fagócitos &quot;profissionais&quot; - macrófagos e células dendríticas em &#39;não profissional&#39; - células vizinhas tecido residente, in vivo de imagens em directo do processo é extremamente valiosa. Aqui nós descrevemos um protocolo para estudar a liberação celular por apoptose em embriões de Drosophila ao vivo. Para acompanhar a dinâmica das diferentes etapas da fagocitose usamos marcadores específicos para células em apoptose e fagócitos. Além disso, podemos monitorar dois sistemas em paralelo fagócitos: macrófagos &#39;profissionais&#39; e &#39;semi-profeglia ssional &#39;no sistema nervoso central em desenvolvimento (SNC). O método aqui descrito emprega o embrião de Drosophila como um modelo excelente para estudos de depuração de células apoptóticas em tempo real.

Representativos de fotogramas de um filme em que as células apoptóticas são rotulados com anexina V, e as células gliais, ectoderme e macrófagos são rotulados com simular cytGFP são mostrados na Figura 2. Cada quadro é uma projeção de três fatias de 2 m cada. O embrião de fase 15 está adequadamente posicionada mostrando o SNC embrionárias em meio com células gliais bem marcado (g). Macrófagos (m), que são na sua maioria fora do SNC, mostram forte ex...

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

Descrevemos aqui um método eficaz para o estudo da dinâmica de depuração de células apoptóticas<em&gt; In vivo</em&gt;. Este método emprega viver<em&gt; Drosophila</em&gt; Embriões como um poderoso modelo de monitorização da fagocitose de células apoptóticas através de marcação específica de células apoptóticas e fagócitos.

Imagens ao vivo de Apuramento celular por apoptose durante<em&gt; Drosophila</em&gt; Embriogênese

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 visualizações.  Technion-Israel Institute of Technology. Descrevemos aqui um método eficaz para o estudo da dinâmica de depuração de células apoptóticas In vivo. Este método emprega viver Drosophila Embriões como um poderoso modelo de monitorização da fagocitose de células apoptóticas através de marcação específica de células apoptóticas e fagócitos.

Vídeo: 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.

Imagens ao vivo da migração das células da glia no Disco Drosophila Eye Imaginal

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

Biologia

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.

Imagens ao vivo de Drosophila melanogaster Embrionárias Migrações hemócitos

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.

Preparação de Desenvolvimento e Adultos Drosophila Brains e retina de imagens ao 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.

A abordagem inversa Genética para Teste de Redundância funcional durante a embriogênese

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.

Preparação Individual Drosophila Ovos Câmaras de imagens ao vivo

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.

Imagens ao vivo de Proteínas GFP-rotulados em<em&gt; Drosophila</em&gt; Oócitos

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.

Formação de imagens 4D de agregação de proteínas em 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.

O tomate / GFP-FLP / FRT Method for Imaging ao vivo de Mosaico Adulto<em&gt; Drosophila</em&gt; As 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;

Citológica Análise da espermatogênese: Preparados ao vivo e fixo<em&gt; Drosophila</em&gt; 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 ...

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.

In Vivo Biosensor Tracks apoptótica não-Caspase Atividade em Drosophila

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