Questo lavoro è stato sostenuto da una borsa Marie Curie di reinserimento Grant (IRG249084). Ringraziamo tutti i membri del laboratorio Kurant.

Per seguire le dinamiche di gioco cellulare per apoptosi in vivo due popolazioni di cellule devono essere etichettati: fagociti e cellule apoptotiche. Per contrassegnare le popolazioni di cellule fagocitarie che usiamo una linea Drosophila contenente il marcatore simulare cytGFP, che le etichette esclusivamente le cellule fagocitarie nell&#39;embrione: macrofagi, cellule gliali e ectoderma 8. Si possono utilizzare diversi marcatori per i fagociti, comprese le linee che contengono un driver Gal4 hemocyte specifici (CRQ-Gal4) o glia-specifica Gal4 conducente (repo-Gal4) e un reporter fluorescente geneticamente codificato sotto controllo UAS (UAS-GFP). Per monitorare le cellule apoptotiche durante liquidazione cellulare per apoptosi usiamo diversi apoptosi / marcatori fagocitosi. Annessina V (Molecular Probes) serve come marker precoce di cellule apoptotiche 10; Phiphilux (OncoImmunin) è un fluorogenico caspasi-3 substrato, che è utilizzato come un secondo apoptosis marcatore, e LysoTracker (Molecular Probes) funziona come un marcatore phagosome. Si inietta questi reagenti che utilizzano il sistema di microiniezione PicoPump PV 820. 1. Preparativi <ol><li> Colla eptano: Srotolare il nastro biadesivo e mettere il più possibile in una fiala di scintillazione, riempire con eptano, chiude la fiala con parafilm, e rock per 24 ore. Aggiungi eptano se la colla è troppo spessa. </li><li> Permettiamo le mosche a deporre su un succo d&#39;uva agar piastra preriscaldata + lievito di pasta per 2 ore, e poi trasferire la piastra a 25 ° C per 10-12 ore (a seconda del livello a scelta) prima di microiniezione e l&#39;ora delle registrazioni lapse di embrioni iniettati. Raccogliamo embrioni da piastre di agar mantenuta a 25 ° C, fornendo così da embrioni stadio 15 o 16 di sviluppo. </li><li> Preparazione Needle: Per preparare gli aghi per l&#39;iniezione, si usa un &#39;strumento Sutter&#39; ago di estrattore e sottili filamenti di vetro a pareti (tubo capillare FHC). La punta del capillare in pool è rotton per un diametro di 0,5-2 micron. </li><li> Coverslip con colla: Usando una pipetta disperdere una goccia di colla eptano in una linea nel mezzo del vetrino. Lasciare asciugare per alcuni secondi. Preparare un paio di queste lamelle. </li></ol> 2. Embrione Preparazione <ol><li> Raccogliere gli embrioni da piastre di agar e trasferirli in un colino cellula (SPL Life Sciences) utilizzando un pennello pulito e acqua corrente. Il filtro è rinviato un becher di raccolta delle acque reflue. </li><li> Lavare embrioni nel setaccio cella utilizzando acqua finché tutto il lievito in pasta viene rimosso. </li><li> Asciugare l&#39;acqua in eccesso pulendo la parte esterna del filtro utilizzando Kimwipes. </li><li> Posizionare il filtro cella in una scatola di Petri pulita e aggiungere abbastanza il 50% di candeggina per coprire embrioni nel colino cellula. Dechorionate gli embrioni per 2 min occasionalmente (2-3 volte) mescolando. </li><li> Risciacquare con acqua molto fino a quando gli embrioni sciolto l&#39;odore di candeggina. </li><li> Posizionare il filtro cella in un ambiente pulitoCapsula di Petri e coprire gli embrioni con acqua per evitare prosciugamento. </li><li> Prima di iniziare l&#39;impostazione embrioni, caricare 1 ml di reagente desiderato in due aghi (a volte un ago può essere intasato). Ci vogliono circa 5 min per il liquido di raggiungere la punta dell&#39;ago. </li><li> Tagliare un pezzo rettangolare di succo d&#39;uva agar e metterlo su un vetrino da microscopio. Trasferire embrioni dechorionated dalla piastra di Petri al pezzo agar utilizzando un pennello pulito. </li><li> Sotto un microscopio a fluorescenza dissezione seleziona adeguatamente allestita embrioni che esprimono il marcatore GFP usando un pennello bagnato e posizionare ogni embrione vicino al bordo del pezzo agar in fila uno dopo l&#39;altro (Figura 1A). Gli embrioni devono essere posizionati con il loro lato ventrale per fagocitosi imaging glia nel Sistema Nervoso Centrale (SNC). </li><li> Istituito circa 10 embrioni in una riga. </li><li> Fissare gli embrioni di un coprioggetto rivestito in mezzo con una striscia di colla eptano (Figura 1B, C </strong&gt;). </li><li> Porre il vetrino nella camera di disidratazione per circa 5 minuti (questo dipende dalla umidità della stanza e la quantità da iniettare). </li><li> Dopo l&#39;essiccazione fino coprire gli embrioni con olio halocarbon 700 (Sigma) per evitare ulteriore disidratazione. </li><li> Porre il vetrino su un vetrino da microscopio con gli embrioni rivolti verso l&#39;alto. Si può mettere una goccia d&#39;acqua sul vetrino per impedire lo spostamento del vetrino. Gli embrioni sono ora pronti per l&#39;iniezione. Ubicazione iniezione è tipicamente sulla parte laterale nel mezzo dell&#39;embrione. </li></ol> 3. Embrione di iniezione <ol><li> Applicare l&#39;ago di un micromanipolatore. </li><li> Per rompere la punta dell&#39;ago poggiare il bordo del vetrino nel campo di vista e regolare l&#39;ago stesso piano focale. Molto spostare con attenzione il vetrino fino a raggiungere la punta dell&#39;ago e la rompe. </li><li> Concentrandosi sugli embrioni, inserire l&#39;ago nel petrolio e assicuratevi di avere una goccia di liquido dall&#39;ago. Significa che l&#39;ago è statorotto bene. </li><li> Senza toccare il supporto dell&#39;ago spostare l&#39;embrione in l&#39;ago e iniettare una goccia nel embrione. Spostare l&#39;embrione via e procedere al successivo fino a che tutti gli embrioni vengono iniettati. </li></ol> 4. Imaging Noi immagine gli embrioni su un microscopio invertito confocale con un obiettivo 40X o 100X. Cerchiamo un embrione ben posizionato con il sistema nervoso centrale in mezzo, che mostra una forte espressione GFP e una buona etichettatura delle cellule apoptotiche dopo l&#39;iniezione. Per le registrazioni in tempo lasso di embrioni vivi scegliamo solitamente 5 o 6 fette confocale (2 micron di spessore ciascuno). Successivamente, facciamo una proiezione di 3 fette (spessore 6 micron) per osservare cellule intere. Usiamo marker per le cellule apoptotiche che sono stabili e non candeggiare facilmente. Per evitare di GFP sbiancamento facciamo le registrazioni ad intervalli di 60 sec. In alcuni casi gli embrioni possono iniziare a rotazionedurante il tempo di registrazione video. Quindi, diamo uno sguardo di tanto in tanto alla registrazione al fine di fermare e ricominciare, se necessario.

<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>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Gioco cellulare per apoptosi è un ultima fase critica di apoptosi, che è altamente dinamico. Pertanto studi in tempo reale del processo sono di massima importanza. Qui si descrive un protocollo che consente il monitoraggio della clearance cellulare per apoptosi nello sviluppo di embrioni di Drosophila vivere. In questa procedura, due popolazioni di cellule devono essere marcati usando specifici marcatori: cellule apoptotiche e fagociti. Il giornalista simulare cytGFP è adatto per il monitoraggio dei...

La corretta eliminazione delle cellule indesiderate o aberranti attraverso apoptosi e successiva fagocitosi è cruciale per lo sviluppo embrionale e per omeostasi tissutale nell&#39;adulto. Fagocitosi delle cellule apoptotiche o di liquidazione per apoptosi è un processo altamente dinamico che si svolge in quattro fasi: (1) il reclutamento dei fagociti al cellulare per apoptosi (&#39;find-me&#39;), (2) il riconoscimento della cellula come un obiettivo per fagocitosi ( &#39;mangia-me&#39;) e (3) engulfment, seguito da (4) maturazione phagosome e la degradazione della particella apoptotico 1-5. Ci sono due tipi di fagociti: macrofagi &#39;professionali&#39; e le cellule dendritiche immature e la &#39;non professionale&#39; cellule vicine tessuto residenti, che sono cruciali per la liquidazione cellulare per apoptosi durante lo sviluppo dei metazoi 6,7. Sviluppo in Drosophila, l&#39;eliminazione delle cellule superflue attraverso apoptosi si verifica in tre mfasi Ain, prima nella metà alla fine embrione, poi a metà pupa, e di nuovo nei primi anni adulto. Durante l&#39;embriogenesi particelle apoptotici vengono rimosse dai fagociti &#39;professionali&#39;, macrofagi e da ectoderma il &#39;non professionale&#39; e glia 8,9. Nel nostro precedente lavoro abbiamo identificato un recettore dei fagociti, Six-micron-under (SIMU), che si esprime esclusivamente in fagociti durante l&#39;embriogenesi. Utilizzando il promotore simulazione abbiamo generato un marcatore specifico per le popolazioni di cellule fagocitarie (simu-cytGFP) che ci permette di monitorare i macrofagi, ectoderma e glia contemporaneamente in un live embrione in via di sviluppo 8. Inoltre, utilizzando marcatori specifici per le cellule in apoptosi in diversi stadi di apoptosi, siamo in grado di seguire le dinamiche di gioco cellulare per apoptosi 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 corretta eliminazione delle cellule indesiderate o aberrante attraverso l&#39;apoptosi e la successiva fagocitosi (clearance cellulare per apoptosi) è cruciale per lo sviluppo normale in tutti gli organismi metazoi. Gioco cellulare per apoptosi è un processo altamente dinamico intimamente associato con la morte delle cellule, le cellule apoptotiche unengulfed sono a malapena visibili in vivo in condizioni normali. Al fine di comprendere le diverse fasi del gioco cellulare per apoptosi e per confrontare fagociti &#39;professionali&#39; - macrofagi e cellule dendritiche a &#39;non professionale&#39; - le cellule vicine tessuto residenti, imaging in vivo vivo del processo è estremamente utile. Qui si descrive un protocollo per lo studio di liquidazione cellulare per apoptosi in vivo embrioni di Drosophila. Per seguire le dinamiche delle diverse fasi della fagocitosi che utilizziamo marcatori specifici per le cellule apoptotiche e fagociti. Inoltre, siamo in grado di monitorare i due sistemi fagocita in parallelo: macrofagi &#39;professionali&#39; e &#39;semi-profeglia ssional &#39;nel sistema nervoso centrale di sviluppo (SNC). Il metodo descritto qui impiega l&#39;embrione di Drosophila come un ottimo modello per studi in tempo reale di gioco cellulare per apoptosi.

Fotogrammi rappresentativi di un film in cui le cellule apoptotiche sono marcate con annessina V, e glia, ectoderma e macrofagi sono etichettati con simu-cytGFP sono illustrati nella Figura 2. Ogni frame è una proiezione di 3 fette 2 micron ciascuna. L&#39;embrione di scena 15 è posizionato correttamente mostrando il SNC embrionali in mezzo con glia ben marcato (g). I macrofagi (m), che sono per lo più al di fuori del sistema nervoso centrale, mostrano una forte ...

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

Qui si descrive un metodo efficace per lo studio delle dinamiche di gioco cellulare per apoptosi<em&gt; In vivo</em&gt;. Questo metodo impiega vivere<em&gt; Drosophila</em&gt; Embrioni come un potente modello per il monitoraggio fagocitosi delle cellule apoptotiche mediante etichettatura specifica delle cellule apoptotiche e fagociti.

Vivere Imaging di apoptotica delle cellule libera nel corso<em&gt; Drosophila</em&gt; Embriogenesi

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 Views.  Technion-Israel Institute of Technology. Qui si descrive un metodo efficace per lo studio delle dinamiche di gioco cellulare per apoptosi In vivo. Questo metodo impiega vivere Drosophila Embrioni come un potente modello per il monitoraggio fagocitosi delle cellule apoptotiche mediante etichettatura specifica delle cellule apoptotiche e fagociti.

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.

Vivere Imaging della migrazione cellule gliali nella Eye Disco Drosophila immaginale

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

Ricerca

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.

Immagini dal vivo di Drosophila melanogaster Migrazioni Hemocyte embrionali

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.

Preparazione di Sviluppo e degli adulti Drosophila Brains e retina per l&#39;imaging dal 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 approccio inverso di test genetici per ridondanza funzionale durante l&#39;embriogenesi

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.

Preparazione individuale Drosophila Egg Chambers per l&#39;imaging dal 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.

Vivo Imaging delle proteine ​​GFP-etichettati in<em&gt; Drosophila</em&gt; Gli oociti

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 Imaging di aggregazione proteica in cellule vive

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.

Il pomodoro / GFP-FLP / FRT Metodo per Live Imaging del Mosaico adulti<em&gt; Drosophila</em&gt; cellule fotorecettrici

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;

L&#39;analisi citologica della spermatogenesi: Preparati Live e fisse di<em&gt; Drosophila</em&gt; Testicoli

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 biosensore tracce non apoptotica caspasi attività 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 ...