Agradecemos ao Bloomington Drosophila da Center for fornecendo stocks de voar. Agradecemos também a estudos de desenvolvimento Hybridoma Banco e Dr. Lynn Cooley (Yale University) para a prestação de anticorpos. Um agradecimento especial vai para AhHyun Yoo por sua ajuda no manuscrito. Este trabalho foi financiado por doações de Ciências Naturais e Engenharia Research Council of Canada (Krieger), o William e Ada Fundo Aço Isabelle (Krieger), e os Institutos Canadenses de Pesquisa em Saúde (Harden).

1. Corpo de parede Preparação NOTA: Preparação do terceiro estádio larvar paredes do corpo (para estudo de NMJs que enervam os músculos da parede corporal) foi realizada como previamente descrito em Brent et al 10, ou Ramachandran e Budnik 11,12, mas com algumas modificações.. <ol><li> Dissecação <ol><li> Elevar existências mosca e cruzes, a 25 ° C durante cinco a seis dias, utilizando 13 procedimentos standard. </li><li> Escolha rastejando larvas de terceiro instar de frascos ou garrafas usando uma pinça fina. </li><li> Lavar as larvas numa pequena placa de Petri contendo tampão de fosfato salino (PBS) para remover quaisquer partículas de alimento. </li><li> Coloque uma única larva em um disco Sylgard e mergulhá-lo em algumas gotas de PBS gelado. Usando PBS gelado vai ajudar atordoar o larva tornando mais fácil de manipular. Ao longo do esvaziamento, assegurar que a preparação está sempre submersa em PBS para evitar que sequem. </li><li> Posicione a larva com o seu lado dorsal voltada para cima, de modo que as duas vias de traqueais são visíveis sob um microscópio de dissecção (Figura 2A). Usando a pinça para agarrar um pino minutien, fixar a larva para baixo na extremidade posterior, perto das spiracles (Figura 2B). Com um outro pino, furar através da cutícula na extremidade anterior perto dos ganchos na boca. Delicadamente, esticar a larva no sentido longitudinal, em seguida, fixá-lo para baixo (Figura 2B). </li><li> Com uma tesoura microdissecção, aperte a extremidade posterior perto do pino para criar uma pequena abertura. A incisão deve ser superficial apenas o suficiente para passar através da cutícula. </li><li> Colocar a ponta da lâmina inferior das tesouras na incisão, cortado ao longo de todo o comprimento da linha média dorsal entre as duas vias de traqueal (Figura 2C). Aponte as lâminas da tesoura ligeiramente para cima quando o corte para evitar danificar os músculos da parede ventral do corpo. </li><li> Faça uma pequena incisão horizontal ligeiramente anterior para o cargoeriorly pino colocado (Figura 2C). Fazer uma outra incisão semelhante ligeiramente posterior ao pino colocado anteriormente (Figura 2C). As incisões deve apenas passar através da cutícula. NOTA: As três incisões de passos 1.1.7 e 1.1.8 combinados deve assemelhar-se um &quot; <img fo:content-width="0.09in" src="/files/ftp_upload/52139/52139_letter_I.jpg" width="15px" /> &quot;Quando concluído, ou seja, uma aba esquerda e da direita à mão no lado dorsal do corpo larval deve ser produzido. </li><li> Limpe cuidadosamente os órgãos internos com a pinça. Adicionando algumas gotas contundentes de PBS vai ajudar a substituir os órgãos fora do corpo larval, tornando assim mais fácil para removê-los. Evite cutucando o corpo larval como ele irá causar danos aos músculos da parede do corpo. </li><li> Desfraldar o corpo larval aberto e fixam os cantos para baixo (Figura 2D). Quando fixando, esticar a parede do corpo, tanto horizontal como verticalmente para formar um rectângulo uniformemente-tensionada (SEe Figura 2G para a forma), tomando cuidado para não rasgar os músculos da parede do corpo no processo. </li><li> Terminar de remover qualquer restantes órgãos internos (Figura 2E). </li></ol></li><li> Fixação e permeabilização <ol><li> Mergulhe as paredes do corpo preso em várias gotas de solução de Bouin. Incubar por 15 min no gelo. Alternativamente, usar 4% de paraformaldeído (PFA) como um fixador alternativo; incube durante 30 min. </li><li> Lavar três vezes com tampão fosfato salina com Triton (PBT). </li><li> Usando uma pinça fina, retire cuidadosamente os pinos e transferir as paredes do corpo por seus cantos em um siliconized 0,65 ml tubo de microcentrífuga. </li><li> Armazenar as paredes do corpo em PBT a 4 ° C até estar pronto para o PLA. Para melhores resultados, comece imunocoloraï¿½o as paredes do corpo dentro de um ou dois dias de dissecção. NOTA: Para economizar nos reagentes e para assegurar que todas as paredes do corpo são tratados da mesma forma durante o ensaio, diferentes genótipos podem ser colocados em uma únicatubo. Os genótipos podem ser distinguidos através do corte dos cantos das paredes do corpo de forma diferente (ver Figura 2F para exemplos). </li></ol></li></ol> 2. A imuno-histoquímica NOTA: A imunocoloração de terceiro estádio larvar paredes do corpo foi realizada como previamente descrito em Brent et al 14 e Ramachandran e Budnik 11, mas com algumas modificações 11,14.. NOTA: Execute todas as etapas à temperatura ambiente e com agitação suave, salvo indicação contrária. <ol><li> Bloqueio <ol><li> Lavam-se as paredes do corpo com PBT três vezes durante 10 minutos cada. </li><li> Bloquear com 1% de Albumina de Soro Bovino (BSA) durante 1 h. </li></ol></li><li> Immunostaining <ol><li> Incubar as paredes do corpo com anticorpos de ratinho e de coelho primárias contra as duas proteínas de interesse (diluídos em 1% de BSA) durante 2 horas à temperatura ambiente, ou durante a noite a 4 ° C. Neste caso, usar 1:10 rato anti-Dlg e 1: 250 de um coelhonti-HtsM 15,16. Os anticorpos contra os marcadores que não são feitos em rato ou coelho também podem ser incluídos - por exemplo, usar 1: 200 de cabra anti-HRP para delinear as membranas neuronais. </li><li> Lavar três vezes com PBT durante 10 min cada. </li><li> Incubar com anticorpos secundários conjugados com um fluoróforo para detectar os marcadores (diluídos em 1% de BSA) durante 2 horas à temperatura ambiente, ou durante a noite a 4 ° C. Como o sinal de PLA é depois visualizada com um fluoróforo vermelho, outro fluoróforo deve ser usada para detectar o marcador - por exemplo, usar 1: 200 conjugado com FITC anti-cabra para detectar o anticorpo de cabra anti-HRP. Devido ao uso de reagentes sensíveis à luz, manter os tubos no escuro a partir deste ponto. NOTA: O kit que é usado para PLA permite a ser realizada entre os anticorpos primários desenvolvidos no rato e coelho, com o sinal visualizado no canal vermelho sob microscopia confocal. Se desejado, outros kits disponíveis que permitem o ensaio deve ser feito com primary anticorpos desenvolvidos em outras espécies, e o sinal visualizado em outros canais. </li></ol></li></ol> 3. Proximidade Ligation Assay NOTA: Execute todas as etapas à temperatura ambiente e com agitação suave, salvo indicação contrária. <ol><li> PLA Sondas <ol><li> Lavam-se as paredes do corpo com PBT três vezes durante 10 minutos cada. </li><li> Incubar com sondas PLA (1: 5 de cada diluição em 1% de BSA) durante 2 horas a 37 ° C. Neste caso, usar 40 ul de sonda de PLA anti-ratinho MENOS, 40 ul de sonda de PLA anti-coelho PLUS e 120 ul de BSA a 1% para assegurar a imersão adequada e mistura de 5-10 paredes do corpo. Até uma diluição das sondas PLA 01:25 ainda pode resultar em uma proporção adequada de sinal-para-ruído (isto é, para esta experiência). </li></ol></li><li> Ligadura <ol><li> Lave as paredes do corpo com Wash Buffer A duas vezes por 5 min cada. </li><li> Incubar com solução Ligadura (diluição 1:40 de ligase em tampão de ligação) durante 1 hora umat 37 ° C. Neste caso, utilizar 5 ul de ligase, 40 ul de tampão de 5 ligadura e 155 ul de água de pureza elevada para assegurar a imersão adequada e mistura de 5-10 paredes do corpo. </li></ol></li><li> Amplificação <ol><li> Lave as paredes do corpo com Wash Buffer A duas vezes para 2 min cada. </li><li> Incubar com solução Amplificação (diluição 1:80 em tampão de polimerase da amplificação) durante 2 horas a 37 ° C. Neste caso, usar 2,5 ul de polimerase, 40 ul de tampão de 5 Amplificação e 157,5 uL de água de pureza elevada para assegurar a imersão adequada e mistura de 5-10 paredes do corpo. </li></ol></li><li> Preparação para criação de imagens <ol><li> Lave as paredes do corpo com tampão de lavagem B duas vezes por 10 min cada. </li><li> Lavar com 0.01x Wash Buffer B uma vez para 1 min. </li><li> Equilibrar com algumas gotas de solução de montagem para, pelo menos, 30 min antes da montagem, ou armazenar durante a noite a 4 ° C. </li><li> Usando uma pinça fina, transferir cuidadosamente as paredes do corpo sobre uma lâmina de plataforma wom suas cutículas voltado para baixo. Posicione as paredes do corpo em fileiras e com a mesma orientação dentro de uma gota ou duas de montagem. Coloque uma 22 mm x 40 milímetros lamela sobre a preparação tomando cuidado para não gerar bolhas de ar, em seguida, selar o slide com unhas polonês claro. </li><li> Guarde os slides no escuro a -20 ° C até que esteja pronto para a imagem latente confocal. </li></ol></li></ol>

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Este relatório demonstra como PLA pode ser aplicado para a Drosophila larval JNM. O ensaio é realizado em preparações de músculo da parede do corpo de larvas para a finalidade de detecção de interacções proteína-proteína endógenos presentes na NMJ. Com esta técnica, Dlg e Hts mostram-se em estreita proximidade uns dos outros, e assim existir em um complexo, especialmente na região pós-sináptica 27. Em apoio a esse resultado, um estudo anterior tenha apresentado provas de sua associaç...

Discos de Drosophila grande (Dlg) é um membro conservada da guanilato-quinase associada à membrana da família de proteínas de andaimes que ajudam a orquestrar a montagem de grandes complexos de proteínas em sítios específicos da membrana plasmática. Originalmente identificado como uma proteína supressora de tumor, Dlg serve como um importante determinante da epitelial 1,2,3 apicobasal polaridade. Dlg também serve como um importante módulo de andaime na junção neuromuscular (JNM) dos neurônios motores glutamatérgicos durante o desenvolvimento larval 4. Dlg desempenha diversos papéis na JNM larval, e a sua pleiotropismo depende da sua capacidade de se associar com várias proteínas de 5,6. Uma dessas proteínas é Hu-li tai Shao (HTS), um homólogo às adducins mamíferos que foram descritos principalmente no que diz respeito às suas funções na regulação da actina-spectrin citoesqueleto 7. Foi previamente mostrado que Dlg e Hts pode formar um complexo com os outros com base em testes in vitro </em&gt; experimentos co-imunoprecipitação envolvendo toda mosca adulta lisados ​​8. Uma desvantagem destes resultados, no entanto, é que elas não indicam onde este formas complexas. Com a utilização de imuno-histoquímica, as distribuições de Dlg e Hts são observados a sobrepor-se na membrana pós-sináptica da NMJs larvas, mas são eles em um complexo na região 8? Como mostrado recentemente e detalhado mais aqui, Proximity Ligation Assay (PLA) é usado para procurar um em associação situ entre Dlg e Hts especificamente no larval NMJ 27. PLA é uma técnica relativamente nova usado principalmente em cultura de células e de tecidos que podem detectar interacções proteína-proteína in situ 9. Neste ensaio, os anticorpos primários contra as duas proteínas de interesse são detectados com um par de anticorpos secundários específicos da espécie, denominados sondas de PLA, que são conjugados com oligonucleótidos (Figura 1A, B). Se as duas proteínass estão em estreita proximidade uns dos outros (isto é, dentro de poucas dezenas de nanómetros), a distância entre as sondas de PLA podem ser ligados em ponte através de hibridação de dois oligonucleótidos de conectores adicionais (Figura 1C). Neste conformação, as extremidades livres dos oligonucleótidos do conector está perto o suficiente para entrar em contacto uns com os outros, e uma molécula de ADN circular, fechada pode ser formada a partir de ligadura situ (Figura 1D). A molécula de ADN circular, serve como molde para a amplificação in situ círculo rolante, o qual é preparado por um dos oligonucleótidos conjugados com as sondas de PLA (Figura 1E). Sequências dentro do amplificado, produto de ADN concatemeric resultante pode então ser visualizado com marcada com fluorescência, as sondas de oligonucleótidos complementares (Figura 1F). Uma vez que o ADN amplificado permanece ligado a uma das sondas de PLA, a localização subcelular da interacção proteína-proteína withind tecido pode ser facilmente determinada. Vários métodos são comumente usados ​​para detectar interacções proteína-proteína, incluindo técnicas in vitro tais como a co-imunoprecipitação, ensaios de pull-down e levedura de rastreio de dois híbridos, e em técnicas in vivo, tais como transferência de energia de ressonância de Förster (FRET) e Bimolecular Complementação de fluorescência ( BiFC). Um problema das técnicas in vitro é que eles não identificam onde a interacção ocorre endogenamente, enquanto o acima mencionados nas técnicas in vivo envolvem a expressão artificial de proteínas de fusão que pode não reflectir o comportamento nativo dos seus homólogos endógenos. Uma grande vantagem de PLA é que ele é capaz de determinar dentro de um tecido a localização subcelular de proteínas de interactores endógenos que estão em estreita proximidade uns dos outros e provável que formam um complexo, com o grau de proximidade necessária para gerar um sinal a ser comparáveis ​​aos FRET e BiFC. PLA podem detectar interacções com elevada especificidade e sensibilidade devido ao acoplamento de reconhecimento do anticorpo e a amplificação de ADN. Assim, o ensaio pode gerar sinais luminosos discretas, em forma de pontos lacrimais que indicam a posição exacta da interacção. Além disso, os antigénios dificilmente visíveis pode ser detectada. Finalmente, PLA é uma técnica relativamente simples de executar, e já não tem do que um procedimento padrão imuno-histoquímico para ser concluído. Portanto, PLA fornece uma vantagem técnica sobre outros ensaios de interação proteína-proteína que muitas vezes são atormentados com tempos de preparação longa e extensa solução de problemas. Este protocolo demonstra como PLA pode ser aplicado para a Drosophila larval JNM para a finalidade de detecção de interacções proteína-proteína endógenos, in situ. Aqui, PLA é realizada em preparações de músculos da parede do corpo larval onde Dlg e Hts são mostrados para realmente existem em um complexo na região pós-sináptica da NMJs. PLA não tenha sido previamente utilizado para estudar a NMJ larval, e existem, actualmente, apenas um punhado de artigos publicados que utilizaram este ensaio no tecido Drosophila. Espera-se que uma maior exposição do PLA para a comunidade Drosophila resultará em aumento da sua utilização como uma ferramenta adicional para complementar outras, mais comumente utilizados ensaios de interação proteína-proteína.

<table border="0" cellpadding="0" cellspacing="0" width="630">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Name of Material/Equipment</td>
			<td style="width:120px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:191px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Forceps (fine #5)</td>
			<td style="width:120px;">Almedic</td>
			<td>A10-704</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="133">
			<td height="133" style="height:133px;width:211px;">Sylgard Disc</td>
			<td style="width:120px;">World Precision Instruments</td>
			<td>SYLG184</td>
			<td style="width:191px;">Mix elastomer base and curing agent in a 10:1 ratio. Set for 30 min. Pour into a mold (e.g. use a 12-well cell culture plate). Let cure for at least 24 hr. Adhere to the lid of a 60x15mm petri dish lid when dissecting.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Minutien Pins (0.0125 mm tip diameter)</td>
			<td style="width:120px;">Fine Science Tools</td>
			<td>26002-10</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">Microdissection Scissors (ultra fine)</td>
			<td style="width:120px;">Fine Science Tools</td>
			<td style="width:111px;">15200-00</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;width:211px;">Platform Slides</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">Glue two 22x22mm coverslips onto a microscope slide with clear nail polish, leaving a &#60;20mm gap in between for sample mounting.</td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;width:211px;">w1118</td>
			<td style="width:120px;">Bloomington Drosophila Stock Center</td>
			<td style="width:111px;">3605</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;width:211px;">hts01103</td>
			<td style="width:120px;">Bloomington Drosophila Stock Center</td>
			<td style="width:111px;">10989</td>
			<td style="width:191px;">Stock was re-balanced over a GFP balancer so that homozygous mutants can be selected based on the absence of GFP signal.</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:211px;">1x PBS (Phosphate Buffered Saline): 3mM NaH2PO4, 7mM Na2HPO4, 130mM NaCl, pH 7.0</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">NaH2PO4 (Caledon Laboratories - 8180-1), Na2HPO4 (Caledon - 8120-1), NaCl (Caledon - 7560-1)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Bouin&#39;s Solution</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">HT10132</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="114">
			<td height="114" style="height:114px;width:211px;">4% PFA (Paraformaldehyde): 4% PFA in 1x PBS</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">PFA (Anachemia Science - 66194-300). See doi:10.1101/pdb.rec9959 Cold Spring Harb Protoc 2006 for instructions on how to make the solution.&#160;</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">1x PBT (Phosphate Buffered Saline with Triton): 1x PBS with 0.01% Triton</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">Triton X-100 (Sigma-Aldrich - T8787)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">1% BSA (Bovine Serum Albumin): 1% BSA in 1x PBT</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">BSA (Bioshop Canada - ALB001). Store at 4 &#176;C.</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">mouse anti-Dlg (Discs large)</td>
			<td style="width:120px;">Developmental Studies Hybridoma Bank</td>
			<td>4F3</td>
			<td style="width:191px;">Use at a 1:10 dilution in 1% BSA.</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">rabbit anti-HtsM (Hu-li tai shao)</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">Provided by Dr. Lynn Cooley (Yale University). Use at a 1:250 dilution in 1% BSA.</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:211px;">rabbit anti-Pak (p21-activated kinase)</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">Provided by Dr. Nicholas Harden (Simon Fraser University). Use at a 1:500 dilution in 1% BSA.</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">mouse anti-Wg (Wingless)</td>
			<td style="width:120px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:111px;">4D4</td>
			<td style="width:191px;">Use at a 1:5 dilution in 1% BSA.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">goat anti-Hrp (Horseradish peroxidase)</td>
			<td style="width:120px;">Jackson&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; ImmunoResearch</td>
			<td style="width:111px;">123-065-021</td>
			<td style="width:191px;">Use at a 1:200 dilution in 1% BSA.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">FITC-conjugated donkey anti-goat</td>
			<td style="width:120px;">Jackson&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; ImmunoResearch</td>
			<td style="width:111px;">705-095-003</td>
			<td style="width:191px;">Use at a 1:200 dilution in 1% BSA.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Duolink In Situ PLA Probe anti-mouse MINUS</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO92004</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Duolink In Situ PLA Probe anti-rabbit PLUS</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO92002</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Duolink In Situ Detection Reagents Red</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td>DUO92008</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:211px;">1x Wash Buffer A: 0.01M Tris, 0.15M NaCl, 0.05% Tween 20, pH 7.4</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO82049</td>
			<td style="width:191px;">Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1), Tween 20 (Fisher Scientific - BP337)</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">1x Wash Buffer B: 0.2M Tris, 0.1M NaCl, pH 7.5</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO82049</td>
			<td style="width:191px;">Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1)</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">0.01x Wash Buffer B: 2mM Tris, 1mM NaCl, pH 7.5</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO82049</td>
			<td style="width:191px;">Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Duolink In Situ Mounting Medium with DAPI</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO82040</td>
			<td style="width:191px;"></td>
		</tr>
	</tbody>
</table>

detection of in situ protein-protein complexes at the drosophila larval neuromuscular junction using proximity ligation assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Em selvagens terceiro estádio NMJs larvais, Dlg é predominantemente encontrado na membrana pós-sináptica de tipo I boutons glutamatérgicos, com níveis de imunorreatividade Dlg sendo mais pronunciada na boutons tipo Ib que o tipo é boutons (Figura 3A) 4. Hts está presente em todo o músculo, mas concentra-se na região pós-sináptica com Hts níveis imunorreatividade aparecendo igual em ambos os tipos I boutons, e também é encontrada pré-sináptico (Figura 3A &#39;)</strong...

Watch this Scientific Journal Video about Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay at JoVE.com

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Este protocolo demonstra como Ensaio de Proximidade A ligadura pode ser utilizada para detectar em interacções proteína-proteína in situ na junção neuromuscular, de larvas de Drosophila. Com esta técnica, os discos grande e Hu-li Shao tai são apresentados para formar um complexo na região pós-sináptica, uma associação previamente identificado por meio de co-imunoprecipitação.

Detecção de<em&gt; In Situ</em&gt; Complexos proteína-proteína no<em&gt; Drosophila</em&gt; Larval Junção Neuromuscular Usando Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

detection-situ-protein-protein-complexes-at-drosophila-larval

Research

JoVE Journal

Neuroscience

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

13.0K visualizações.  Simon Fraser University.  Este protocolo demonstra como Ensaio de Proximidade A ligadura pode ser utilizada para detectar em interacções proteína-proteína in situ na junção neuromuscular, de larvas de Drosophila. Com esta técnica, os discos grande e Hu-li Shao tai são apresentados para formar um complexo na região pós-sináptica, uma associação previamente identificado por meio de co-imunoprecipitação. 

Vídeo: Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

Visualização de Larval Nervos segmentar em 3 Rd Instar Drosophila Preparações larval

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

Neurociência

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Em Visualization vivo das vesículas sinápticas Dentro Drosophila larval Axônios segmentar

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

Dissecção e imagem das Zonas de Ativos no Drosophila Junção neuromuscular

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Sequencial de foto-branqueamento de delinear células de Schwann individuais na junção neuromuscular

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

Por Quantificação Matérias: Caracterização de fenótipos na<em&gt; Drosophila</em&gt; Larval Junção Neuromuscular

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Medir a funcionalidade da junção Neuromuscular

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

Dois Algoritmos para métodos de alta capacidade e Multi-paramétrico Quantificação de<em&gt; Drosophila</em&gt; Junção Neuromuscular Morfologia

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

Macropatch focal gravações das correntes sinápticas da junção Neuromuscular Larval da drosófila

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The well characterized larval neuromuscular junction is often used for such studies. However, rapid larval development followed by muscle histolysis and nervous system remodeling during metamorphosis makes this model problematic for the study of slow age-dependent degenerative changes like those occurring in amyotrophic lateral sclerosis. Alternatively, adult flies live for 90 days and the adult leg can be used to study motor neuron changes over the course of adult lifespan using in vivo fluorescent imaging through the cuticle. Here, we describe a leg dissection technique coupled with immunocytochemistry, which allows for the study of molecular changes at the neuromuscular junction of identified adult leg motor neurons. These techniques can be coupled with a myriad of antibodies labeling both pre- and post-synaptic structures. Together these procedures allow a more complete characterization of slow age-dependent changes in adult flies and can be applied across multiple motor neuron disease models.

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

We describe a dissection technique that preserves the architecture of the neuromuscular junction and enables a detailed immunocytochemical study of motor neurons in the adult Drosophila leg.

Dissecção e Imunohistoquímica da Perna Adulta Drosophila para detectar alterações na junção neuromuscular para um neurônio motor identificado

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The ...

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting the expression of G protein-coupled receptors (GPCRs). Formalin-fixed jugular-nodose ganglia were processed with the RNAscope technology to simultaneously detect the expression of two representative GPCRs (cholecystokinin and ghrelin receptors) in combination with one marker gene of either nodose (paired-like homeobox 2b, Phox2b) or jugular afferent neurons (PR domain zinc finger protein 12, Prdm12). Labeled ganglia were imaged using confocal microscopy to determine the distribution and expression patterns of the aforementioned transcripts. Briefly, Phox2b afferent neurons were found to abundantly express the cholecystokinin receptor (Cck1r) but not the ghrelin receptor (Ghsr). A small subset of Prdm12 afferent neurons was also found to express Ghsr and/or Cck1r. Potential technical caveats in the design, processing, and interpretation of multiplex ISH are discussed. The approach described in this article may help scientists in generating accurate maps of the transcriptional profiles of vagal afferent neurons.

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

Multiplex in situ hybridization (ISH) was employed to simultaneously visualize the transcripts for two G protein-coupled receptors and one transcription factor in the entire vagal ganglionic complex of the adult mouse. This protocol could be used to generate accurate maps of the transcriptional profiles of vagal afferent neurons.

Detecção de expressão receptora acoplada à proteína G em neurônios aferentes do mouse vagal usando multiplex in situ hibridização

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting ...