Ringraziamo Jordan Davis, Yanyan Lin, Eric Schadler e Jimiao Zheng per il loro aiuto tecnico con questo studio. Questo lavoro è stato supportato da NSF Career Grant MCB-1054962 a ALA

1. Preparare grande mela Piastre Juice-agar <ol><li> Per fare 4 piastre, aggiungere 375 ml di H 2 O, 11,25 g fly agar e un bar mescolare in un pallone di 1.000 ml. Questo è il Mix A. Autoclave Mix A con il coperchio pallone liberamente ricoperto da un ciclo di 30 min-sterilizzazione per le merci liquide. </li><li> Aggiungere 125 ml di succo di mela, 12,5 g di zucchero da tavola e un ancoretta ad un bicchiere da 500 ml. Questo è Mix B. calore Mix B su una piattaforma riscaldata sotto agitazione e mantenere la temperatura a circa 70 ° C fino alla autoclave di Mix A è terminata. </li><li> Al completamento di Mix A autoclave, trasferire Mix B Mix A. Continuate a mescolare il composto unito delicatamente e lasciate raffreddare per 15 minuti. Evitare di raffreddamento e la solidificazione agar portato prima dell&#39;aggiunta di conservante. </li><li> Prova del 10% (peso / volume) metil 4-idrossibenzoato soluzione in etanolo. Questo agirà come conservante. Aggiungere 3,75 ml della soluzione alla miscela succo-agar mela sopra. Continuate a mescolare per another 5 min. </li><li> Versare 125 ml di succo di mela miscela-agar con conservanti in ciascuna di plastica 300 centimetri 2 o lastra di polistirolo. Lasciate raffreddare a temperatura ambiente e solidificare. Evitare la formazione di bolle d&#39;aria durante il versamento. Nota: Le piastre di agar succo di mela sono pronti per l&#39;uso immediato. Coprire le piastre utilizzate con impacchi chiari e conservare a 4 ° C per un massimo di una settimana. </li></ol> 2. Drosophila Embryo raccolta e il trattamento pre-reticolazione <ol><li> Caricare una gabbia popolazione con circa 40.000 a 50.000 mosche da cui verranno raccolti embrioni. </li><li> Fare due piastre contenenti ciascuno 125 ml di farina di mais-melassa standard, medie 12 e cospargere di circa 4 g di lievito di birra essiccato su ogni piatto. Mettere questi piatti nella gabbia della popolazione in un incubatore a 25 ° C per 48 ore per ingrassare le mosche. </li><li> Warm 2 piastre di succo di mela-agar a 25 ° C. Cospargere circa 1 g di lievito di birra essiccato su ognipiatto. Posizionare le 2 piastre nella gabbia popolazione di iniziare a raccogliere 0-1 embrioni hr-old. </li><li> Estrarre le piastre alla fine dell&#39;insieme 1 hr. Sostituire con 2 nuove piastre se è necessario un altro giro di raccolta. </li><li> Aggiungere acqua sufficiente a coprire le piastre e risospendere gli embrioni delicatamente con un pennello fine. Versare gli embrioni risospeso attraverso un setaccio a doppio strato, fatto di rete metallica in acciaio inox. Nota: Lo strato superiore è realizzato in dimensioni medie dei pori (850 micron), maglia che raccoglie gran parte della mosca mentre lascia passare embrioni. Lo strato inferiore è realizzata in maglia fine (75 micron), che raccoglie gli embrioni pur lasciando passare lievito e acqua. </li><li> Usare il pennello ammenda di trasferire gli embrioni raccolti in un cestino di raccolta realizzato con un tubo da 50 ml e rete di nylon fine. Sciacquare gli embrioni brevemente con acqua, quindi asciugare la rete su carta assorbente per asciugare. </li><li> Immergere gli embrioni nel 50% di candeggina con agitando delicatamente per 3 min. Risciacquare approfonditaly con acqua per rimuovere qualsiasi residuo di candeggina. Asciugare la rete su carta assorbente per asciugare. </li><li> Immergere gli embrioni dechorionated in isopropanolo con leggera agitazione per 15 secondi o fino a quando la ripartizione dei cespi multi-embrione (questo non dovrebbe richiedere più di 30 secondi). Asciugare la rete su carta assorbente per asciugare completamente. </li><li> Immergere gli embrioni in esano per 5 minuti mentre usando una pipetta per lo streaming eptano negli embrioni per tenerli risospeso. Asciugare la rete su carta assorbente per asciugare. </li><li> Lavare gli embrioni con i flussi di Soluzione tampone fosfato, pH 7.4 (PBS) contenente 0,1% Triton X-100 (PBST) per 3 min. </li><li> Smontare il cestello di raccolta e trasferire gli embrioni con la maglia di una provetta da 15 ml contenente 10 ml PBST. Capovolgere delicatamente il tubo per lavare gli embrioni fuori la rete. </li><li> Rimuovere la maglia dal tubo. Inserire il tubo in posizione verticale e lasciare gli embrioni si depositano sul fondo della provetta per gravità. Nota: Circa 100-300microlitri di embrioni è previsto per una raccolta 1 ora da una gabbia. </li><li> Rimuovere il PBST e continuare a funzionare reticolazione immediatamente. </li></ol> 3. Nel vivo reticolazione degli embrioni raccolti <ol><li> Preparare 0,2% di formaldeide in PBST immediatamente prima dell&#39;uso. Aggiungere 10 ml di fissativo formaldeide per gli embrioni e incubare a 25 ° C con agitazione su un agitatore rotante per 10 min. Scartare fissativo inutilizzato alla fine della giornata. </li><li> Placare la reazione di reticolazione <ol><li> Al termine della reticolazione, lasciare che gli embrioni si depositano sul fondo della provetta. </li><li> Rimuovere la soluzione di formaldeide. Immediatamente aggiungere 10 ml di 0,25 M glicina in PBST per placare la reazione di reticolazione. Incubare a 25 ° C con agitazione su un agitatore rotante per 5 min. </li><li> Affittare gli embrioni si depositano sul fondo della provetta. Rimuovere la soluzione di abbattimento. Lavare gli embrioni con 10 ml di PBST tre volte. </li><li> Rimuovere completamente il PSoluzione di lavaggio BST con una pipetta. Conservare embrioni a -80 ° C fino al momento dell&#39;uso. </li></ol></li></ol> 4. Proteine ​​preparazione del campione e Immunoprecipitation <ol><li> Omogeneizzare 500 microlitri embrioni reticolati utilizzando un omogeneizzatore Dounce in 2 ml di tampone di lisi (0.5 M urea, 0,01% SDS, 2% Triton X-100, 2 mM phenylmethanesulfonyl fluoruro [PMSF] e cocktail inibitore di proteasi in PBS). Nota: Eseguire questa e le seguenti operazioni a temperatura ambiente, se non diversamente specificato. </li><li> Trasferire il lisato a 1,5 ml provette da centrifuga e rock delicatamente su una piattaforma rotante per 15 minuti al fine di garantire lisi approfondita. Centrifugare il lisato a 16.000 xg per 5 minuti per far sedimentare i detriti cellulari. Salvare il surnatante in un ambiente pulito tubo da 15 ml. </li><li> Aggiungere 40 microlitri anti-HA agarosio impasto nel surnatante e incubare su piattaforma rotante per 2,5 ore. </li><li> Agglomerare le perline per centrifugazione a 2500 xg per 5 min. Rimuovere il surnatante ma lasciare approximately 250 microlitri nel tubo. Risospendere le sfere nel surnatante rimanente e trasferire in una colonna di spin 1,5 ml con 10 micron di filtro pori. </li><li> Centrifugare la colonna di spin a 12.000 xg per 10 secondi e scartare il flusso attraverso. </li><li> Lavare le perline aggiungendo 200 microlitri di tampone di lavaggio (0,1 M glicina, 1 M NaCl, 1% IGEPAL CA-630, 0,1% Tween-20, in PBS). Centrifugare la colonna a 12.000 xg per 10 secondi e scartare il lavaggio. Ripetere il lavaggio altre due volte. </li><li> Aggiungere 40 microlitri tampone di eluizione (2 mg / ml di HA-peptide in PBS) per le perline e incubare a 37 ° C per 15 minuti con agitazione ogni 5 min. Centrifugare la colonna a 12.000 xg per 10 sec e raccogliere l&#39;eluato. </li><li> Confermare eluizione del complesso mediante analisi western-blot utilizzando anticorpi anti-HA 13. </li></ol>

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La formaldeide è stato comunemente usato come reagente di reticolazione per identificare proteina-proteina e le interazioni proteina-acido nucleico. La sua buona solubilità e permeabilità della membrana cellulare, unitamente alla compatibilità con valle procedure di spettrometria di massa, rendono formaldeide un agente candidato ideale per applicazioni di reticolazione intracellulari 3,15-17. In particolare, è stato usato con successo per identificare mRNA associati Vasa, un critico elicasi RNA di cellul...

Isolamento delle assemblee proteine ​​multisubunit e-DNA o RNA-proteina complessi viene effettuata per identificare complessi proteici, loci genomici riconosciuto da proteine ​​regolatrici che legano il DNA o RNA target di proteine ​​leganti RNA. Diversi metodi permettono l&#39;identificazione genoma dei siti del DNA riconosciuti da fattori di trascrizione o proteine ​​della cromatina (ChIP-Seq) 1 e gli obiettivi di RNA associate a un determinato RNA-binding protein (CLIP-ss) 2. Le librerie di cDNA RNA derivati ​​da o bersagli di DNA sono poi profondamente sequenziati. Questi metodi utilizzano chimica o indotto da UV reticolazione a regolarizzare i complessi seguita da immunoprecipitazione (IP) con un anticorpo contro un componente proteica del complesso studiato. Durante lo sviluppo di un organismo, molti complessi proteici formano transitoriamente. Pertanto, è fondamentale per analizzare la composizione e la funzione di questi complessi in vivo per comprendere i meccanismi molecolari che controllano development. Tale analisi in vivo sarebbe superiore al metodo in vitro dato che è praticamente impossibile riprodurre concentrazioni nativi dei componenti interagenti e ambiente biochimico cellulare in vitro. Qui mostriamo un approccio in vivo che usiamo con successo per isolare grandi complessi proteici da embrioni di Drosophila. In questo metodo, complessi proteici negli embrioni viventi sono reticolati con una bassa concentrazione di formaldeide e successivamente complessi proteici di interesse sono isolati da IP con un anticorpo contro un componente noto dei complessi seguita da purificazione gel dei complessi e analisi di spettrometria di massa a identificare i componenti complessi sconosciuti. Poiché formaldeide è in grado di permeare la membrana cellulare e ha un raggio di reticolazione 2.3-2.7 Å 3, complessi proteici possono essere reticolati in vivo e le componenti complessi sono suscettibili di essere vicini l&#39;uno all&#39;altro. In questo articolodescrivere questo metodo usando l&#39;isolamento di Tudor (Tud) complesso proteico come esempio. Tud è una proteina germinale che è essenziale per lo sviluppo della linea germinale 4-7. Questa proteina contiene 11 domini Tud noti per interagire con arginines denaturato o lysines di altri polipeptidi 8-10. In precedenza, abbiamo generato una linea di Drosophila transgenica che esprime HA-tagged funzionale Tud 5 e, quindi, specifici anticorpi anti-HA viene utilizzato per abbattere complesso Tud dopo reticolazione. Oltre a legami crociati tra proteine, formaldeide può generare acido nucleico reticola-proteina e viene usato in esperimenti di ChIP-seq. Inoltre, in Drosophila, in vivo reticolazione con formaldeide ha permesso l&#39;identificazione di un target di RNA di Vasa RNA elicasi proteine ​​11. Mentre in questo articolo descriviamo un metodo per reticolazione in vivo e purria del complessi proteici da embrioni di Drosophila, questo metodo può essere adattato per altri organismi e tessuti.

<table border="0" cellpadding="0" cellspacing="0" width="858">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Drosophila agar</td>
			<td style="width:235px;">Lab Scientific (http://www.labscientific.com/)</td>
			<td style="width:119px;">FLY-8020-1</td>
			<td style="width:289px;">Do not autoclave the water and agar mix for prolonged period of time as it will cause the apple juice plates to become fragile</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Methyl 4-hydroxybenzoate</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">H5501</td>
			<td style="width:289px;">Other name: TEGOSEPT</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Population cage</td>
			<td style="width:235px;">Flystuff (http://www.flystuff.com/)</td>
			<td style="width:119px;">59-104</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Fine nylon mesh</td>
			<td style="width:235px;">Flystuff (http://www.flystuff.com/)</td>
			<td style="width:119px;">57-102</td>
			<td style="width:289px;">When making the collection basket, cut the mesh slightly larger than the opening of the falcon tube cap to ensure a tight seal</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dounce homogenizer</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">D8938-1SET</td>
			<td style="width:289px;">Chill the homogenizer on ice and prerinse with cold lysis buffer before use to prevent protein degradation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Protease inhibitor cocktail&#160;</td>
			<td style="width:235px;">Roche (http://www.rocheusa.com/portal/usa)</td>
			<td style="width:119px;">4693132001</td>
			<td style="width:289px;">PBS could be used to prepare concentrated protease inhibitor stock solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-HA agarose beads</td>
			<td style="width:235px;">MBL international (http://www.mblintl.com/)</td>
			<td style="width:119px;">561-8</td>
			<td style="width:289px;">The kit also includes HA-peptide and spin columns</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HA-peptide</td>
			<td style="width:235px;">MBL international (http://www.mblintl.com/)</td>
			<td style="width:119px;">561-8</td>
			<td style="width:289px;">Prepare to 2 mg/ml with PBS&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spin Column</td>
			<td style="width:235px;">MBL international (http://www.mblintl.com/)</td>
			<td style="width:119px;">561-8</td>
			<td style="width:289px;">Spin columns are included as part of the kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Isopropanol</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP26324</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Triton X-100</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP151-500</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Heptane</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">H350-4</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PBS</td>
			<td style="width:235px;">Invitrogen (https://www.lifetechnologies.com/us/en/home.html)</td>
			<td style="width:119px;">AM9625</td>
			<td style="width:289px;">Dilute from 10 X to 1 X with nanopure water before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Formaldehyde</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP531-500</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycine</td>
			<td style="width:235px;">BioRad (www.bio-rad.com/&#8206;)</td>
			<td style="width:119px;">161-0724</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:235px;">BioRad (www.bio-rad.com/&#8206;)</td>
			<td style="width:119px;">161-0301</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:235px;">BioRad (www.bio-rad.com/&#8206;)</td>
			<td style="width:119px;">161-0730</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Phenylmethanesulfonyl fluoride</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">P7626-1G</td>
			<td style="width:289px;">Prepare 200 mM stock solution in isopropanol then dilute to working concentration of 2 mM in lysis buffer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">IGEPAL CA-630</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">I8896-50ML</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tween 20</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP337-100</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">15-ml tubes</td>
			<td style="width:235px;">USA Scientific (http://www.usascientific.com/)</td>
			<td style="width:119px;">1475-0511</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bleach</td>
			<td style="width:235px;">Clorox brand</td>
			<td style="width:119px;"></td>
			<td style="width:289px;">Dilute with equal volume of nanopure water to make 50% bleach</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">50-ml tubes</td>
			<td style="width:235px;">BD Biosciences (http://www.bdbiosciences.com/home.jsp)</td>
			<td style="width:119px;">352098</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Top layer sieve</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">04-884-1AK</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Bottom layer sieve</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">04-884-1BA</td>
			<td style="width:289px;"></td>
		</tr>
	</tbody>
</table>

an in vivo crosslinking approach to isolate protein complexes from drosophila embryos

Molti processi cellulari sono controllati da complessi proteici multisubunit. Spesso questi complessi formano transitoriamente e richiedono ambiente nativo da montare. Pertanto, per identificare questi complessi proteici funzionali, è importante stabilizzarli in vivo prima lisi cellulare e successiva purificazione. Qui si descrive un metodo utilizzato per isolare grandi complessi proteici in buona fede da embrioni di Drosophila. Questo metodo si basa su embrione permeabilizzazione e stabilizzazione dei complessi all&#39;interno degli embrioni in vivo reticolazione usando una bassa concentrazione di formaldeide, che può facilmente attraversare la membrana cellulare. Successivamente, il complesso proteina di interesse è immunopurified seguita da purificazione gel e analizzato mediante spettrometria di massa. Illustriamo questo metodo utilizzando la purificazione di un complesso proteico Tudor, che è essenziale per lo sviluppo della linea germinale. Tudor è una grande proteina, che contiene più domini Tudor - piccolomoduli che interagiscono con arginine metilate o lysines delle proteine ​​target. Questo metodo può essere adattato per l&#39;isolamento di complessi proteina nativa da organismi e tessuti differenti.

L&#39;efficacia della reticolazione e la purificazione successo del reticolato complesso proteico Tud sono stati analizzati mediante SDS-PAGE su un 3% - 7% gel fase (illustrata in figura 1), seguita da Western blot (Figura 2). Lo scopo di utilizzare il 3% - gel passaggio 7% si basa sulla separazione effettiva del reticolato complesso proteico Tud dalla restante proteina non reticolato Tud e concentrazione del complesso. Nelle nostre condizioni di reticolazio...

Watch this Scientific Journal Video about An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos at JoVE.com

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

Complessi proteici Multi-componenti giocano un ruolo cruciale durante la funzione e lo sviluppo cellulare. Qui si descrive un metodo utilizzato per isolare complessi proteina nativa da embrioni di Drosophila in vivo dopo reticolazione seguita da purificazione dei complessi reticolati per successiva analisi struttura-funzione.

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 ...

an-vivo-crosslinking-approach-to-isolate-protein-complexes-from

Research

JoVE Journal

Biology

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

16.6K Views.  Murray State University.  Complessi proteici Multi-componenti giocano un ruolo cruciale durante la funzione e lo sviluppo cellulare. Qui si descrive un metodo utilizzato per isolare complessi proteina nativa da embrioni di Drosophila in vivo dopo reticolazione seguita da purificazione dei complessi reticolati per successiva analisi struttura-funzione. 

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

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Preparazione delle Culture neuronale da Midgastrula fase embrioni di Drosophila

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Ricerca

Biologia

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

Microfabbricazione post-Array-Detector (mPADs): un approccio per isolare le forze meccaniche

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

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 ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Isolamento di Labile Multi-proteina complessi da In vivo Controllato Cellular Cross-Linking e Immuno-magnetica Cromatografia di affinità

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

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 ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Imaging interazioni proteina-proteina In vivo</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Imaging verticale di Drosophila Embrioni

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Colture cellulari primarie da Drosophila Gastrula embrioni

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

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 ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Isolamento e purificazione di chinesina dal Drosophila Embrioni

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...