Agradecemos Jordan Davis, Yanyan Lin, Eric Schadler e Jimiao Zheng por sua ajuda técnica com este estudo. Este trabalho foi financiado pela NSF CARREIRA conceder MCB-1054962 para ALA

1. Preparando grandes da Apple Juice Placas-ágar <ol><li> Para fazer quatro placas, adicionar 375 ml de H 2 O, 11,25 g de agar mosca e uma barra de agitação para um balão de 1000 ml. Este é Mix A. Autoclave Mix Um frasco com a tampa solta tampado em um ciclo de 30 min-esterilização para produtos líquidos. </li><li> Adicione 125 ml de suco de maçã, 12,5 g de açúcar de mesa e uma barra de agitação para um copo de 500 ml. Esta mistura é B. Calor mistura B numa plataforma aquecida enquanto se agita e mantém a temperatura a cerca de 70 ° C até que a autoclave de mistura A é terminado. </li><li> Após a conclusão da Mix A autoclavagem, transferir Mix B para A. Misture Continue mexendo a mistura combinada com cuidado e deixe esfriar por 15 min. Evitar a sobre-arrefecimento e a solidificação de agar resultou antes da adição de conservante. </li><li> Adicione 10% (peso / volume) de solução de metil 4-hidroxibenzoato de metilo em etanol. Isto irá actuar como um conservante. Adicionar 3,75 mL da solução para a mistura de sumo de maçã-agar acima. Continue mexendo por another 5 min. </li><li> Despeje 125 ml mistura de suco de maçã-agar com conservante em cada 300 cm 2 de plástico ou placa de isopor. Deixe esfriar para RT e solidificar. Evite a formação de bolhas de ar ao derramar. Nota: As placas-ágar suco de maçã está pronto para usar imediatamente. Cobrir as placas não utilizadas com envoltórios claras e armazenar a 4 ° C durante até uma semana. </li></ol> 2. Colheita de embriões Drosophila e Tratamento de pré-reticulação <ol><li> Coloque uma gaiola população com cerca de 40.000 a 50.000 moscas a partir do qual os embriões serão coletados. </li><li> Faça duas placas, cada uma contendo 125 ml de farinha de milho-padrão médio melaço 12 e polvilhe aproximadamente 4 g de fermento de padeiro seco em cada placa. Coloque estas placas na gaiola população em uma incubadora a 25 ° C por 48 horas para engordar as moscas. </li><li> Aqueça 2 maçã placas suco-ágar a 25 ° C. Polvilhar aproximadamente 1 g de levedura de padeiro seca em cadaprato. Coloque as duas placas na gaiola população para começar a coletar 0-1 embriões hr de idade. </li><li> Retire das placas no final da recolha de 1 hora. Substitua com 2 placas novas, se for necessário mais uma rodada de coleta. </li><li> Adicionar água suficiente para cobrir as placas e ressuspender os embriões suavemente com um pincel fino. Despeje os embriões ressuspensão através de uma peneira de duas camadas feitas de malha de arame de aço inoxidável. Nota: A camada superior é feita de tamanho de poros médio (850 mm) de malha que reúne grande parte da mosca, deixando através de embriões. A camada inferior é feita de malha fina (75 mm) que recolhe embriões, deixando através de levedura e água. </li><li> Use o pincel fino tinta para transferir os embriões coletados em uma cesta coleção feita com um tubo de 50 ml e de malha fina de nylon. Lavar os embriões brevemente com água, em seguida, seque a malha em papel toalha para secar. </li><li> Mergulhe os embriões em 50% água sanitária com agitação suave por 3 min. Enxágüe completaly com água para remover qualquer remanescente de branqueamento. Blot a malha em papel toalha para secar. </li><li> Mergulhe os embriões dechorionated em isopropanol com agitação suave durante 15 segundos ou até que a quebra de aglomerados multi-embrião (isso não deve demorar mais do que 30 segundos). Blot a malha em papel toalha para secar completamente. </li><li> Mergulhe os embriões em heptano por 5 minutos enquanto estiver usando uma pipeta para transmitir heptano sobre os embriões para mantê-los ressuspenso. Blot a malha em papel toalha para secar. </li><li> Lavar os embriões com fluxos de fosfato salino tamponado, pH 7,4 (PBS) contendo 0,1% de Triton X-100 (PBST) durante 3 min. </li><li> Desmontar o cesto de recolha e transferência de embriões, juntamente com a malha para um tubo de 15 ml contendo 10 ml de PBST. Inverter o tubo suavemente para lavar os embriões fora da malha. </li><li> Retirar a malha do tubo. Colocar o tubo na posição vertical, e deixar os embriões afundar para o fundo do tubo por gravidade. Nota: Cerca de 100-300mL de embriões é esperado para uma coleção de 1 hora a partir de uma gaiola. </li><li> Remover o PBST e continuar a realizar a reticulação imediatamente. </li></ol> 3. In vivo A reticulação dos embriões colhidos <ol><li> Prepare a 0,2% de formaldeído em PBST imediatamente antes da utilização. Adicionar 10 ml de formaldeído para o fixador os embriões e incubar a 25 ° C com agitação suave num agitador rotativo durante 10 min. Descarte fixador não utilizados até o final do dia. </li><li> Extingue-se a reacção de reticulação <ol><li> No final da reticulação, deixar os embriões afundar para o fundo do tubo. </li><li> Remover a solução de formaldeído. Imediatamente adicionar 10 ml de 0,25 M de glicina em PBST para extinguir a reacção de reticulação. Incubar a 25 ° C com agitação suave num agitador rotativo, durante 5 min. </li><li> Deixe os embriões afundar para o fundo do tubo. Remover a solução de resfriamento. Lavar os embriões com 10 mL de PBST três vezes. </li><li> Remover completamente o PSolução de lavagem BST com uma pipeta. Armazenar embriões a -80 ° C até à sua utilização. </li></ol></li></ol> 4. Proteína Preparação de Amostras e Immunoprecipitation <ol><li> Homogeneizar 500 ul embriões reticulados utilizando um homogeneizador Dounce, em 2 ml de tampão de lise (0,5 M de ureia, 0,01% de SDS, 2% de Triton-X 100, fluoreto de fenilmetanossulfonilo 2 mM [FPMS], e cocktail inibidor de protease em PBS). Nota: Execute este e os seguintes passos na RT, a menos que especificado em contrário. </li><li> Transferir o lisado para tubos de 1,5 ml de centrífuga e agitar suavemente sobre uma plataforma rotativa durante 15 minutos para assegurar a lise completa. Centrifuga-se o lisado a 16.000 x g durante 5 min para sedimentar os detritos celulares. Salve o sobrenadante em um tubo de 15 ml limpo. </li><li> Adicionar 40 ul de contas de agarose anti-HA lama para o sobrenadante e incubar numa plataforma rotativa durante 2,5 horas. </li><li> Sedimentar as pérolas por centrifugação a 2500 xg durante 5 min. Remover o sobrenadante, mas deixar adeximately 250 mL no tubo. Ressuspender as esferas no sobrenadante remanescente e transferidos para uma coluna de centrifugação de 1,5 ml com 10 mM de filtro poroso. </li><li> Centrifugar a coluna girar a 12.000 xg por 10 segundos e descartar o fluxo através. </li><li> Lave os grânulos por adição de 200 ul de tampão de lavagem (0,1 M de glicina, 1 M de NaCl, 1% IGEPAL CA-630, 0,1% de Tween-20, em PBS). Centrifugar a coluna a 12.000 xg por 10 segundos e descartar a lavagem. Repita a lavagem mais duas vezes. </li><li> Adicionar 40 ul de tampão de eluição (2 mg / ml de HA-péptido em PBS) às pérolas e incubar a 37 ° C durante 15 min com agitação suave a cada 5 min. Centrifugar a 12000 xg durante 10 seg e recolher o eluato. </li><li> Confirmar eluição do complexo por análise de Western-blot, utilizando um anticorpo anti-HA 13. </li></ol>

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Os autores não têm nada a revelar.

O formaldeído foi utilizada como um reagente de ligação cruzada para a identificação da proteína-proteína e interacções proteína-ácido nucleico. A sua boa solubilidade e permeabilidade da membrana da célula, juntamente com a compatibilidade com os procedimentos de espectrometria de massa a jusante, fazer formaldeído um agente candidato ideal para aplicações de reticulação intracelulares 3,15-17. Em particular, foi utilizado com sucesso para identificar mRNAs associados com vasa, uma helicase ...

Isolamento de conjuntos de proteínas de subunidades múltiplas e complexos de ADN ou ARN-proteína é realizada para identificar complexos proteicos, locus genómico reconhecidos pelas proteínas reguladoras de ligação de ADN ou de ARN alvos de proteínas de ligação a ARN. Diferentes métodos permitem a identificação do genoma de sites de DNA reconhecidos por fatores de transcrição ou proteínas da cromatina (Chip-seq) 1 e metas de RNA associadas a uma determinada proteína de ligação de RNA (CLIP-seq) 2. As bibliotecas do ADNc derivado de ARN-ou alvos de DNA são então profundamente sequenciado. Estes métodos utilizam química ou reticulação para estabilizar os complexos seguido por imunoprecipitação (IP), com um anticorpo contra um componente proteico do complexo estudado induzida por UV. Durante o desenvolvimento de um organismo, muitos complexos de proteínas formam transitoriamente. Portanto, é essencial analisar a composição e função dos complexos in vivo para compreender os mecanismos moleculares que controlam devolvimento. Tal análise in vivo seria superior à abordagem in vitro, uma vez que é virtualmente impossível reproduzir concentrações nativas dos componentes que interagem e ambiente bioquímico celular in vitro. Aqui demonstramos uma abordagem in vivo que utilizam com sucesso para isolar grandes complexos de proteínas a partir de embriões de Drosophila. Neste método, os complexos de proteína em embriões vivos são reticulados com um baixo teor de formaldeído e, subsequentemente, os complexos de proteína de interesse é isolado por IP com um anticorpo contra um componente conhecido dos complexos, seguida de purificação em gel dos complexos e análise de espectrometria de massa para identificar componentes complexos desconhecidos. Uma vez que o formaldeído é capaz de permear a membrana celular e possui um intervalo de reticulação de 2,3-2,7 Å 3, complexos de proteína, pode ser reticulado in vivo, e os componentes do complexo são susceptíveis de estar perto um do outro. Neste artigo,descrever este método usando o isolamento de Tudor (Tud) complexo de proteína como um exemplo. Tud é uma proteína da linha germinativa, que é essencial para o desenvolvimento da linha germinativa 4-7. Esta proteína contém 11 domínios TuD conhecidas por interagirem com argininas metiladas ou lisinas de outros polipeptídeos 8-10. Anteriormente, têm gerado uma linha de Drosophila transgênica que expressa HA-marcado funcional Tud 5 e, portanto, o anticorpo anti-HA específico é utilizado para puxar para baixo complexo Tud após reticulação. Em adição a ligações cruzadas de proteína-proteína, o formaldeído pode gerar ácido nucleico ligações cruzadas em proteína e é usado em experiências de ChIP-seq. Além disso, em Drosophila, in vivo, de ligação cruzada com formaldeído, permitiu a identificação de um alvo de ARN da proteína helicase vasa ARN 11. Enquanto neste artigo descrevemos um método para a reticulação vivo e purificação de complexos de proteínas a partir de embriões de Drosophila, este método pode ser adaptado para outros organismos e os tecidos.

<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

Muitos processos celulares são controlados por complexos de proteína de múltiplas subunidades. Freqüentemente, esses complexos formam transitoriamente e exigem ambiente nativo de montar. Portanto, para identificar estes complexos proteicos funcionais, é importante para estabilizar in vivo antes da lise das células e purificação subsequente. Aqui nós descrevemos um método utilizado para isolar grandes complexos de proteínas de boa-fé a partir de embriões de Drosophila. Este método baseia-se na permeabilização embrião e estabilização dos complexos no interior dos embriões por reticulação in vivo, utilizando uma baixa concentração de formaldeído, o que pode facilmente atravessar a membrana celular. Subsequentemente, o complexo de proteína de interesse é imunopurificada seguido de purificação em gel e analisados ​​por espectrometria de massa. Ilustraremos este método utilizando a purificação de uma proteína complexa Tudor, que é essencial para o desenvolvimento da linha germinativa. Tudor é uma grande proteína, que contém vários domínios Tudor - pequenomódulos que interagem com argininas metiladas ou lisinas de proteínas alvo. Este método pode ser adaptado para o isolamento de complexos de proteína nativa a partir de diferentes organismos e tecidos.

A eficácia da reticulação e a purificação com sucesso do complexo proteico reticulado Tud foram analisadas por SDS-PAGE em um 3% - gel de 7% etapa (ilustrada na Figura 1), seguido por transferência de Western (Figura 2). O objectivo de utilizar a 3% - 7% de gel passo é baseado na separação eficaz de complexo de proteína reticulada Tud da proteína Tud reticulada remanescente e a concentração do complexo. Sob as nossas condições in vivo...

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

Complexos de proteínas multi-componentes desempenham um papel crucial durante a função celular e desenvolvimento. Aqui, descrevemos um método usado para isolar complexos proteína nativa a partir de embriões de Drosophila após reticulação in vivo seguido de purificação dos complexos de ligações cruzadas para a subsequente análise da estrutura-função.

Uma In vivo A reticulação Abordagem para isolar Complexos de proteínas a partir de Drosophila Os embriões

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 visualizações.  Murray State University.  Complexos de proteínas multi-componentes desempenham um papel crucial durante a função celular e desenvolvimento. Aqui, descrevemos um método usado para isolar complexos proteína nativa a partir de embriões de Drosophila após reticulação in vivo seguido de purificação dos complexos de ligações cruzadas para a subsequente análise da estrutura-função. 

Vídeo: 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.

Preparação de culturas de neurônios a partir de embriões Midgastrula Stage Drosophila

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

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.

Microfabricated Pós-Array-detectores (mPADs): uma abordagem para isolar Forças Mecânica

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.

Preparação de embriões de Microscopia Eletrônica do Drosophila Tubo cardíaco embrionário

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 de proteínas lábeis Multi-Complexos por In vivo Controlled Cellular Cross-Linking and Imuno-magnética cromatografia de afinidade

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 Centrifugação de Embriões 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.

Imagem interações proteína-proteína 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.

Imagem vertical de Drosophila Os embriões

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.

Culturas de células primárias de Drosophila Os embriões Gástrula

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.

Imagem Alterar Forma Celular em Viver Drosophila Os embriões

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 Purificação de cinesina partir Drosophila Os embriões

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