Ringraziamo Polan Santos e Darren Obbard per le illustrazioni di Drosophila in Fig. 2a, Marcelo Jacobs-Lorena per l&#39;uso del insectary JHMRI. Questo lavoro è stato sostenuto dalla borsa di studio Fondazione Life Science Research (HLT), Comitato Università assegni di Hong Kong AoE / B-07/99 (MCF), e NIH concede NS096677, NS037402 e NS083373 (JMH). Ho Lam Tang è un Shurl e Kay Curci Foundation Fellow della Fondazione Life Sciences Research.

1. Preparazione di mosche CaspaseTracker <ol><li> Per preparare CaspaseTracker (DQVD) vola per esperimenti, eseguire questa croce: UBI-CaspaseTracker x G-TRACE (UAS-RFP; UAS-FLP; Ubi&gt; Arresto&gt; GFP-NLS), trasferendo 7-10 femmina vergine (o maschio) mosche trasportano il substrato caspasi biosensore mCD8-DIAP1-GAL4 guidato dal promotore dell&#39;ubiquitina 35 insieme con lo stesso numero di maschio (o femmina) linea G-TRACE, che hanno la seconda bilanciatore cromosoma cyo per evitare letalità della combinazione omozigote di G- TRACE (UAS-RFP, UAS-FLP, e Ubi&gt; Arresto&gt; GFP-NLS) 34. Inserire vola in un flaconcino alimento fresco con pasta di lievito fresco come fonte proteica. </li><li> Incubare i file a 18 ° C per 5 a 7 giorni (massimo 2 settimane) e quindi rimuovere il genitore aria dal flacone per evitare il sovraffollamento con la nuova progenie, e per impostare nuove fiale di allevamento. </li><li> Continuare incubazione a 18 ° C fino a quando progenie vola Eclose. Selezionare la non cyo [non-cURLy ala] progenie della corretta genotipo di CaspaseTracker, che sono transgenici per CaspaseTracker e G-35 oligoelementi. </li><li> Allo stesso tempo, generare controllo parentale mosche W118 non transgenici e caspasi-insensitive (DQVA) vola in parallelo per verificare specifiche RFP CaspaseTracker e GFP fluorescenza. </li><li> Eseguire la PCR genotipizzazione per confermare i file con CaspaseTracker utilizzando i primer: 5&#39;-TCCCCCGGGCTGCAGGAATTC, 3&#39;-TGGAATTGGGGTACGTCTAGA, producendo un prodotto di 3.897 bp. Per la genotipizzazione loci G-Trace, utilizzare i seguenti primer per GFP (474 ​​bp), 5&#39;-CAC GAC TTC TTC AAG TCC ATG GCC CCC G, 3&#39;-CTT GTA CAG CTC GTC CAT GCC GAG AGT GAT C; per RFP (258 bp), 5&#39;-GGC TGC TTC ATC TAC AAG GTG AAG TTC ATC GG, 3&#39;-GAT GTC CAG CTT GGA GTC CAC GTA GTA GTA GC; e per Flpase (655 bp), 5&#39;-CCACCTAAGGTGCTTGTTCGTCAGTTTGTGG, 3&#39;-GCC TAC TAA CGC TTG TCT TCT TTG CTG TCA C. Per la genotipizzazione GAL4 nel vettore pUWR (554 bp), 5&#39;-GAA GCA CAC CTT CGC ATC GCT CAGTCA CGC, 3&#39;-TGG TGG AAT GGT ACG TCT AGA. </li></ol> 2. Preparazione del tessuto, colorazione e montaggio <ol><li> Per dissezioni mosca, evitare tessuti dissezionati di attaccarsi alle punte di plastica per pipette e provette da centrifuga, consigli cappotto e tubi con albumina 1% di siero bovino (BSA) disciolti in acqua. </li><li> Dissect vola su un cuscinetto di silicone con pinze. <ol><li> Per evitare di danneggiare le punte delle pinze su una superficie dura quando si esegue la dissezione, eseguire la dissezione su una superficie di silicio. Per la realizzazione di lastre di dissezione silicone, sciogliere il silicio nel kit commerciale secondo le istruzioni del produttore (Materials List). Dopo la fusione il silicone, aggiungere 3 a 4 mL di un tessuto di diametro piatto di coltura 60mm. piatti di silicio possono essere riutilizzati. </li><li> Anestetizzare una mosca con CO 2, e trasferirlo ad una piastra in silicone con 1 ml di PBS freddo. Introdurre la CO 2 ad un flacone contenente il volo usando una cerbottana, e quindi trasferire al volo t anestetizzatoOA Pad che ha una fornitura di CO 2. </li><li> Utilizzare un paio di pinze per tenere la testa mosca, ed una seconda coppia di pinze per tirare torace nella direzione opposta per scollegare la testa mosca dal corpo di rivestimento senza danneggiare la connessione tra la testa e foregut. </li><li> Usare 2 paia di pinze per rimuovere le ali e le gambe dal torace. </li><li> Utilizzare un paio di pinze per tenere il torace, e un altro paio di pinze per tirare l&#39;addome per separarlo dal torace di nuovo senza danneggiare la connessione tra il foregut e midgut. </li><li> Sezionare organi interni compreso il cervello, intestino, tubuli malpighiani e le ovaie, come precedentemente dimostrato. 39-41 </li><li> Rimuovere tutti i pezzi della cuticola e gli organi di grasso con una pinza da questi tessuti producono forti autofluorescenza. La pazienza e la pratica sono tenuti a preparare un sistema di organi completo come mostrato. </li></ol></li><li> Trasferimento tessuti sezionati a 1,5 ml provette da centrifuga, e fissare i tessuti with 0,5 mL di 4% paraformaldeide in PBS (tampone fosfato salino) a temperatura ambiente per 20 a 30 minuti con rotazione al buio per evitare sbiancamento RFP e GFP nei tessuti. Evitare la fissazione prolungata che può compromettere le successive risultati di colorazione. </li><li> Rimuovere la paraformaldeide delicatamente pipettando e lavare 3 volte con 0,5 mL di PBST (PBS + 0,1% Triton X-100) a RT. </li><li> Permeabilize i tessuti con 0,5 ml PBST a 4 ° C per una notte con un leggero scuotimento. incubazione più brevi possono causare permeabilizzazione e il compromesso di colorazione risultati incompleti. <ol><li> Se è necessario ridurre la colorazione di fondo, non incubando tessuti in 0,5 mL PBST con 1% BSA, invece di PBS. </li></ol></li><li> Lavare i tessuti 3 volte con 0,5 mL PBST. </li><li> Per macchiare nuclei e filamentosa actina (F-actina), si applicano 0,5 ml PBST con 10 mg / mL di Hoechst 33342 blu colorante nucleare e 0,3 micron Alexa Fluor 633 falloidina F-actina macchia e incubare simultaneamente con i tessuti per 1 ora a RT with leggera rotazione nel buio. Evitare colorazione prolungata in quanto ciò aumenterà lo sfondo. <ol><li> Per immunocolorazione, incubare fisso e permeabilizzate tessuti con 0,1% Triton X-100 in 0,5 ml di PBS notte a 4 ° C, macchia con diluizione 1: 100 di anticorpi anti-ELAV o con diluizione 1: 200 di anticorpi anti-caspasi-3 notte a 4 ° C (300 mL). Seguire dal corrispondente anticorpo secondario diluito 1: 100 e incubare per 1 a 3 ore a RT. Vedere Materiali Lista per specifiche informazioni di anticorpi. </li></ol></li><li> Lavare i tessuti 3 volte, ognuna per 5 min in 0,5 mL PBST con rotazione dolce. </li><li> Con una pipetta, rimuovere tutti PBST, e poi aggiungere 200 microlitri anti-candeggina agente di montaggio (vedi materiali) per coprire completamente i tessuti per 1-3 ore a temperatura ambiente, o 4 ° C durante la notte. tessuti ottimali che hanno completamente assorbito l&#39;agente di montaggio si depositano sul fondo della provetta. </li><li> Pre-pulire il vetrino con acqua o etanolo al 70% e trasferire i tessuti con l&#39;agente di montaggio per i glass slitta. </li><li> Posizionare con cura un vetrino di vetro sopra i tessuti. Applicare vaselina per il vetrino e la polizza di copertura per evitare di distruggere il tessuto da sovracompressione. Rimuovere l&#39;agente di fissaggio in più con la carta velina. </li><li> Sigillare la polizza di copertura mediante l&#39;applicazione di smalto lungo i bordi del vetrino per evitare la fuoriuscita di agenti di montaggio dai tessuti. </li></ol> 3. Microscopia confocale <ol><li> Utilizzare un confocale epi-microscopio a fluorescenza invertito. Tuttavia, microscopi up-destra possono anche essere utilizzati. Per i tessuti di immagine utilizzando piastrelle, mantenere la temperatura ambiente stabile per evitare la deriva di messa a fuoco e spostamento del piano xy a causa della dilatazione termica e la contrazione dei componenti. sistemi di controllo ambientale e sistemi di compensazione attenzione deriva aiutano ad evitare questo problema a causa di perdita di termo-equilibrio del sistema microscopio. </li><li> Posizionare il vetrino sul palco del microscopio. Prendete un paio di immagini di prova con bassa risoluzione (125 x 125 pixel) unad determinare il numero appropriato di piastrelle necessarie per coprire l&#39;intera regione di interesse (ROI). </li><li> Impostare il sistema di scansione microscopio per catturare immagini con risoluzione di scansione ad esempio 500 x 500 pixel. Selezionare l&#39;obiettivo come un 20X NA 0.8 o un obiettivo Plan-Apochromat 63X NA 1.4 per analizzare i tessuti attraverso vetrino, in modo che ciascuna delle immagini (piastrelle) sovrapposizioni del 20% su tutti i lati. </li><li> Impostare la sequenza di immagini da più lungo al più breve lunghezze d&#39;onda di eccitazione, come la luce di lunghezza d&#39;onda lunga provoca inferiore fotometabolismo. La sequenza dalla più lunga alla più corta lunghezza d&#39;onda di eccitazione è: (i) 633 nm per falloidina F-actina e per gli anticorpi caspasi attivate, (ii) 561 nm per RFP, (iii) 488 nm per GFP, e (iv) 405 nm per Hoechst macchia nucleare. </li><li> Durante l&#39;imaging, minimizzare l&#39;esposizione delle cellule alla eccitazione laser fluorescente durante il processo di imaging per evitare fotometabolismo riducendo l&#39;intensità del laser per ottenere immagini di alta qualità di tessuti. </li><li> Dopo iomaghi sono raccolti, l&#39;unione delle immagini utilizzando il software microscopio. </li></ol>

The authors have nothing to disclose.

Qui illustriamo la costruzione e funzionamento interno di CaspaseTracker che facilitano il rilevamento di una diffusa attività di caspasi basale nei tessuti sani. I passaggi critici per rilevare l&#39;attività della caspasi non apoptotica in vivo sono: 1) la generazione di mosche con il transgene biosensore, 2) verificare la funzione giornalista specifiche caspasi con opportuni controlli, 3) praticando tecniche di dissezione di osservare tutti i sistemi di organi interni di adulti Drosophila, e 4) at...

Caspasi sono proteasi cisteina che mediano la morte cellulare per apoptosi, con l&#39;adesione molte proteine ​​intracellulari dopo residui di aspartato chiave. Ad esempio, caspasi iniziatrici attivano le caspasi effettrici, nucleasi DNA derepress, componenti del citoscheletro Cleave e alterano la composizione lipidica delle membrane cellulari di smantellare rapidamente le cellule e stimolare il loro riconoscimento e engulfment da parte delle cellule che smaltiscono i cadaveri cellula vicina. 1-4 Si stima che miliardi di cellule muoiono al giorno nel corpo umano, e l&#39;apoptosi è un importante meccanismo di morte delle cellule tumorali indotta da chemioterapia. 5 un diverso insieme di caspasi può causare la morte cellulare da processi non apoptotici distinte per stimolare l&#39;immunità innata. 6 Perciò, maggior parte della ricerca sulle caspasi è concentrata sulla loro funzioni pro-morte. È interessante notare che le prove nelle prime fasi del campo ha rivelato che gli stessi caspasi responsabili della promozione della morte cellulare hanno anche non-morte funzioni. Studi pionieristici hanno dimostrato che caspasi sono coinvolte in diverse funzioni cellulari in cellule sane, tra cui la regolazione della proliferazione cellulare e la migrazione durante l&#39;embriogenesi. 7-9 Caspases sono necessari per l&#39;individualizzazione spermatid in Drosophila 10,11, per bloccare un percorso di morte cellulare necroptotic alternativa nei topi 12,13, e per l&#39;elaborazione microRNA in C. elegans. 14,15 In forse la più lunga vissuto cellule, i neuroni, caspasi e altri macchinari apoptotica sono implicati nella regolazione dell&#39;attività neuronale da potatura terminazioni sinaptiche, un processo che si ritiene essere essenziale per rafforzare altri sinapsi per l&#39;apprendimento e la memoria 16. 18 E &#39;possibile che caspasi facilitare la potatura sinaptica da un tipo di mini-apoptosi di minuscole proiezioni neuronali senza morte cellulare intero. 19 Tuttavia, caspasi possono avere funzioni alternative non correlati ad eventi apoptosi-like. 20,21 duplice ruolos nella vita e la morte non sono riservate caspasi; BCL-2 proteine della famiglia e citocromo c hanno ruoli in energetica cellulari in cellule sane, ma sono anche parte del nucleo via apoptotica che viene attivato da molti tipi di stress cellulare. 22-25 Anche se non è provato, sembra logico che l&#39;evoluzione ha legato giorno : i processi di morte-posti di lavoro all&#39;interno delle stesse molecole per garantire la tempestiva eliminazione delle cellule inadatti o indesiderabili. Allo stato attuale, i meccanismi molecolari di attività caspasi non-apoptotica non sono comprese, e la misura dell&#39;attività di caspasi non apoptotica durante lo sviluppo embrionale e nei tessuti adulti non è noto. Una sfida importante è la difficoltà nel distinguere giornaliere di posti di lavoro da death-lavori di caspasi. In contrasto con l&#39;apoptosi e pyroptosis, quando l&#39;attività delle caspasi è amplificato da una cascata proteolitica, si prevede che i giorno-lavori di caspasi che si verifichi a livelli molto più bassi di attività enzimatica, probabilmente al di sotto di rilevamento da molti Techn disponibileologies. Prima del lavoro qui presentato, altri svilupparono una varietà di biosensori caspasi per scopi diversi. I biosensori SCAT (ad esempio, ECFP-DEVD-Venere) individuare rapidamente l&#39;attività della caspasi in tempo reale in cellule in coltura e tessuti animali utilizzando FRET. 26,27 Al caspasi scissione, la frazione GFP nucleare mirato di Apoliner (mCD8-RFP-DQVD- nucGFP) subisce rilocalizzazione subcellulare in pochi minuti quando la sua membrana plasmatica-tether viene scisso dalla caspasi. 28 allo stesso modo, ApoAlert-pCaspase3-Sensor (NES-DEVD-YFP-NLS) rilocalizza dal citoplasma al nucleo su caspasi scissione. 29,30 Altro di recente, il cromoforo in iCasper è stato abilmente progettato per fluorescenza quando spaccati dalla caspasi, permettendo la rilevazione di attività biosensore in tempo reale nei neuroni di embrioni di Drosophila, ma soprattutto in associazione con la morte delle cellule di sviluppo. morte 31 caspasi-dipendente dei neuroni olfattivi durante l&#39;invecchiamento, è stato demonstvotata da immuno-rilevamento della forma caspasi-spaccati di biosensori CPV (ad esempio, mCD8-PARP-Venere). 32,33 È importante sottolineare che la forma attiva della caspasi-3 è stato rilevato in assenza di morte cellulare da immunostain sensibile a spine di neuroni in coltura, e nel soma utilizzando la fluorescenza caspasi-dipendente del colorante CellEvent giornalista nucleare, ma sono stati riscontrati a causa di foto-tossicità difficoltà, anche se la morte cellulare è stata ritardata fino a dopo l&#39;eliminazione della colonna vertebrale. 19 Pertanto, sono necessari nuovi biosensori caspasi per rilevare e tenere traccia delle cellule con l&#39;attività delle caspasi basale in vivo. Per superare queste difficoltà, abbiamo generato un romanzo a due biosensore colore caspasi, designato CaspaseTracker. Questa strategia combina una versione modificata del Drosophila caspasi-sensitive Apoliner biosensore 28 con la Drosophila G-TRACE sistema FRT ricombinasi 34 per etichettare definitivamente e monitorare le cellule in vivo. &lt;sup&gt; 35 Il sistema G-TRACE GAL4-attivato permette di livelli molto bassi di caspasi per attivare CaspaseTracker, con conseguente espressione RFP nel citoplasma e l&#39;espressione GFP nucleare mirata permanente in una cella che ha mai sperimentato l&#39;attività delle caspasi. 35 Questo sistema può etichettare cellule per tutta la vita in animali interi utilizzando Drosophila melanogaster, un sistema modello trattabili e ampiamente utilizzati per lo studio delle caspasi e morte cellulare. 36-38

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			<td height="21" style="height:21px;width:273px;">CONSUMABLES AND REAGENTS</td>
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			<td height="21" style="height:21px;">Vectashield</td>
			<td style="width:229px;">Vector Products</td>
			<td style="width:225px;">H-1000</td>
			<td>Mounting medium</td>
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			<td height="21" style="height:21px;">Forceps</td>
			<td style="width:229px;">Ted Pella</td>
			<td style="width:225px;">#505 (110mm, #5)</td>
			<td>Dumont tweezer biology grade, stainless steel</td>
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			<td height="21" style="height:21px;">Hanging Drop Slides</td>
			<td style="width:229px;">Fisher Scientific</td>
			<td style="width:225px;">12-565B</td>
			<td>Glass slides</td>
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			<td height="21" style="height:21px;">Hoechst 33342</td>
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			<td>DNA stain</td>
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			<td height="21" style="height:21px;">Alexa Fluor 633 Phalloidin</td>
			<td style="width:229px;">Molecular Probes</td>
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			<td>Actin stain</td>
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			<td height="42" style="height:42px;width:273px;">Rat-Elav-7E8A10 anti-elav antibody</td>
			<td style="width:229px;">&#160;Developmental Studies Hybridoma Bank (DSHB)</td>
			<td style="width:225px;">Antibody Registry ID:&#160; AB_528218&#160;</td>
			<td>Stain for Drosophla pan-neuronal ELAV</td>
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			<td height="21" style="height:21px;width:273px;">Cleaved caspase-3 (Asp175) antibody</td>
			<td style="width:229px;">Cell Signaling Technology</td>
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			<td>Stain for active fragment of caspase-3</td>
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			<td height="21" style="height:21px;">ProLong Gold antifade reagent</td>
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			<td>to preserve fluorophores&#160;</td>
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			<td height="21" style="height:21px;width:273px;">ProLong Diamond Antifade Mountant</td>
			<td style="width:229px;">Life Technologies</td>
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			<td>to preserve fluorophores&#160;</td>
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			<td height="21" style="height:21px;width:273px;">SylGard 182 Silicone Elastomer Kit</td>
			<td style="width:229px;">Dow Corning&#160;</td>
			<td style="width:225px;">Product code: 0001023934</td>
			<td>for dissection plates</td>
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			<td height="21" style="height:21px;width:273px;">EQUIPMENT</td>
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			<td></td>
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			<td height="21" style="height:21px;">LSM780 confocal microscope</td>
			<td style="width:229px;">Carl Zeiss</td>
			<td style="width:225px;">N/A</td>
			<td>Imaging</td>
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			<td height="21" style="height:21px;width:273px;">Carl Zeiss Stereomicroscope Stemi 2000&#160;</td>
			<td style="width:229px;">Carl Zeiss</td>
			<td style="width:225px;">N/A</td>
			<td>Drosophila dissection</td>
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			<td height="42" style="height:42px;width:273px;">AmScope Fiber Optic Dual Gooseneck Microscope Illuminator, 150W</td>
			<td style="width:229px;">AmScope</td>
			<td style="width:225px;">WBM99316&#160;</td>
			<td>Light source</td>
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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.

Ci sono due componenti fondamentali che permettono CaspaseTracker per rilevare l&#39;attività delle caspasi nelle cellule sane normali (Figura 1A). Il primo di questi è un 146 aminoacidi polipeptide caspasi-cleavable modello della caspasi biosensore Apoliner (Figura 1b). 28 Questo polipeptide è derivato da DIAP1 (Drosophila inibitore di apoptosi) contenente un singolo sito caspasi naturale che è scisso durante l&#39;apoptosi tipic...

Watch this Scientific Journal Video about In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila at JoVE.com

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

Per rilevare le cellule sane in animali interi che contengono bassi livelli di attività delle caspasi, il biosensore altamente sensibile designato CaspaseTracker è stato generato per Drosophila. attività biosensore caspasi-dipendente viene rilevato in cellule sane lunga durata durante gli organi interni di animali adulti allevati in condizioni ottimizzate in assenza di stimoli morte.

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 ...

in-vivo-biosensor-tracks-non-apoptotic-caspase-activity-in-drosophila

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JoVE Journal

Biology

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

9.0K Views.  Johns Hopkins University Bloomberg School of Public Health.  Per rilevare le cellule sane in animali interi che contengono bassi livelli di attività delle caspasi, il biosensore altamente sensibile designato CaspaseTracker è stato generato per Drosophila. attività biosensore caspasi-dipendente viene rilevato in cellule sane lunga durata durante gli organi interni di animali adulti allevati in condizioni ottimizzate in assenza di stimoli morte. 

Video: In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Mesoscopici fluorescenza per Tomografia In-vivo Imaging di sviluppo Drosophila</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Ricerca

Biologia

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Preparazione di embrioni per la microscopia elettronica del Drosophila Tubo cuore embrionale

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

In vivo centrifugazione di embrioni di Drosophila

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

Imaging in vivo di intatto Larve di Drosophila a livello sub-cellulare Risoluzione

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Patch clamp e Tecniche di perfusione per la valutazione dei canali ioni Espresso in Xenopus Ovociti

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

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.

Imaging Forma Modifica cella in Living Drosophila Embrioni

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 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.

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  ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

Un In vivo La reticolazione Approccio per isolare complessi proteici da Drosophila Embrioni

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Ricostituzione funzionale e attività Canale Misure di purificata di tipo selvatico e mutante CFTR proteine

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Misura della larvali attività nel<em&gt; Drosophila</em&gt; Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...