Agradecemos a la Bloomington Drosophila Stock Center para proporcionar reservas de la mosca. También agradecemos al hibridoma Estudios del Desarrollo del Banco y el Dr. Lynn Cooley (Universidad de Yale) para proporcionar anticuerpos. Un agradecimiento especial a AhHyun Yoo por su ayuda en el manuscrito. Este trabajo fue apoyado por becas de Ciencias Naturales e Ingeniería de Investigación de Canadá (Krieger), el William y Fondo de acero Isabelle Ada (Krieger), y los Institutos Canadienses de Investigación en Salud (Harden).

1. Cuerpo Preparación de la pared NOTA: Preparación de tercer estadio larval paredes del cuerpo (para el estudio de los NMJs que inervan los músculos de la pared corporal) se realizó como se ha descrito previamente en Brent et al 10, o Ramachandran y Budnik 11,12, pero con algunas modificaciones.. <ol><li> Disección <ol><li> Elevar las existencias de moscas y cruces a 25 ° C durante cinco o seis días, utilizando procedimientos estándar 13. </li><li> Escoja arrastrándose larvas de tercer estadio de frascos o botellas utilizando unas pinzas finas. </li><li> Lavar las larvas en una pequeña placa Petri que contenía tampón fosfato salino (PBS) para eliminar las partículas de comida. </li><li> Coloque una sola larva en un disco Sylgard y sumergirlo en unas gotas de helado de PBS. El uso de helado de PBS ayudará aturdir la larva por lo que es más fácil de manipular. A lo largo de la disección, asegúrese de que la preparación siempre se sumerge en PBS para evitar que se seque. </li><li> Coloque la larva con el lado dorsal de modo que las dos secciones traqueales son visibles bajo un microscopio de disección (Figura 2A). Usando las pinzas para agarrar un alfiler minutien, Pin de la larva hacia abajo en el extremo posterior cerca de los espiráculos (Figura 2B). Con otro pin, perforar a través de la cutícula en el extremo anterior cerca de los ganchos de la boca. Estire suavemente la larva a cabo a lo largo, entonces pin abajo (Figura 2B). </li><li> Con unas tijeras de microdisección, pellizcar el extremo posterior cerca del pasador para crear una pequeña abertura. La incisión debe ser lo suficientemente superficial sólo tiene que pasar a través de la cutícula. </li><li> La colocación de la punta de la cuchilla inferior de las tijeras en la incisión, corte a lo largo de toda la longitud de la línea media dorsal entre las dos secciones traqueales (Figura 2C). Señalar las cuchillas de la tijera ligeramente hacia arriba cuando se corta para evitar dañar los músculos de la pared ventral del cuerpo. </li><li> Hacer una pequeña incisión horizontal ligeramente anterior al puestopin colocado eriorly (Figura 2C). Haz otra incisión similares posterior ligeramente a la aguja colocada anteriormente (Figura 2C). Las incisiones deben sólo tiene que pasar a través de la cutícula. NOTA: Los tres incisiones de los pasos 1.1.7 y 1.1.8 combinados debe ser similar a un &quot; <img fo:content-width="0.09in" src="/files/ftp_upload/52139/52139_letter_I.jpg" width="15px" /> &quot;Una vez terminado, es decir, una aleta izquierda y derecha en la parte dorsal del cuerpo de la larva debe ser producido. </li><li> Limpiar cuidadosamente los órganos internos con los fórceps. Añadir unas gotas contundentes de PBS ayudará a desplazar a los órganos fuera del cuerpo de la larva, por lo que es más fácil para eliminarlos. Evite meter el cuerpo de las larvas, ya que puede causar daños en los músculos de la pared corporal. </li><li> Desplegar el cuerpo larval abierta y fijar las esquinas hacia abajo (Figura 2D). Cuando fijación, estirar la pared del cuerpo, tanto horizontal como verticalmente para formar un rectángulo uniformemente tensado (see Figura 2G para la forma), teniendo cuidado de no romper los músculos de la pared del cuerpo en el proceso. </li><li> Termine de retirar cualquiera de los órganos internos restantes (Figura 2E). </li></ol></li><li> La fijación y permeabilización <ol><li> Sumerja las paredes del cuerpo clavado en varias gotas de solución de Bouin. Incubar durante 15 min en hielo. Como alternativa, utilice el 4% de paraformaldehído (PFA) como una solución fijadora alternativa; incubar durante 30 min. </li><li> Enjuague tres veces con tampón fosfato salino con Triton (PBT). </li><li> Con unas pinzas finas, retire con cuidado los pasadores y transferir las paredes del cuerpo por sus esquinas en un tubo de microcentrífuga de 0,65 ml siliconado. </li><li> Guarde las paredes del cuerpo de PBT a 4 ° C hasta el momento de PLA. Para obtener resultados óptimos, comience inmunotinción las paredes del cuerpo dentro de un día o dos de disección. NOTA: Para ahorrar en reactivos y para asegurar que todas las paredes del cuerpo son tratados por igual durante el ensayo, diferentes genotipos se pueden colocar en una solatubo. Los genotipos se pueden distinguir por el corte de las esquinas de las paredes del cuerpo de forma diferente (véase la Figura 2F para los ejemplos). </li></ol></li></ol> 2. La inmunohistoquímica NOTA: La inmunotinción de tercer instar paredes del cuerpo de las larvas se realizó como se ha descrito previamente en Brent et al 14 y Ramachandran y Budnik 11, pero con algunas modificaciones 11,14.. NOTA: Realice todos los pasos a temperatura ambiente y con agitación suave a menos que se indique lo contrario. <ol><li> Bloqueo <ol><li> Lavar las paredes del cuerpo con PBT tres veces durante 10 minutos cada uno. </li><li> Bloque con 1% albúmina de suero bovino (BSA) durante 1 hr. </li></ol></li><li> Inmunoticción <ol><li> Incubar las paredes del cuerpo con anticuerpos de ratón y primarios de conejo contra las dos proteínas de interés (diluido en 1% de BSA) durante 2 horas a temperatura ambiente, o durante la noche a 4 ° C. En este caso, utilice 1:10 de ratón anti-Dlg y 1: 250 de conejo unnti-HtsM 15,16. Los anticuerpos contra los marcadores que no se hacen en el ratón o conejo también se pueden incluir - por ejemplo, utilizar 1: 200 cabra anti-HRP para delinear las membranas neuronales. </li><li> Lavar con PBT tres veces durante 10 minutos cada uno. </li><li> Incubar con anticuerpos secundarios conjugado con fluoróforo para detectar los marcadores (diluido en 1% de BSA) durante 2 horas a temperatura ambiente, o durante la noche a 4 ° C. Como la señal de PLA se visualizó más tarde con un fluoróforo rojo, otro fluoróforo debe ser utilizado para detectar el marcador - por ejemplo, usar 1: 200 anti-cabra conjugado con FITC para detectar el anticuerpo de cabra anti-Hrp. Debido a la utilización de los reactivos sensibles a la luz, mantener los tubos en la oscuridad desde este punto en adelante. NOTA: El kit que se utiliza para PLA permite que se realice entre los anticuerpos primarios planteadas en ratón y conejo, con la señal visualizado en el canal rojo bajo microscopía confocal. Si se desea, otros kits están disponibles que permiten que el ensayo se realiza con primaanticuerpos ry criados en otras especies, y la señal visualizaron en otros canales. </li></ol></li></ol> 3. Proximidad Ligadura Ensayo NOTA: Realice todos los pasos a temperatura ambiente y con agitación suave a menos que se indique lo contrario. <ol><li> PLA Sondas <ol><li> Lavar las paredes del cuerpo con PBT tres veces durante 10 minutos cada uno. </li><li> Incubar con sondas de PLA (dilución 1: 5 cada uno en 1% BSA) durante 2 horas a 37 ° C. En este caso, utilizar 40 l de sonda de PLA MENOS anti-ratón, 40 l de sonda de PLA anti-conejo PLUS y 120 l de BSA al 1% para asegurar la inmersión adecuado y mezcla de 5-10 paredes del cuerpo. Hasta una dilución 01:25 de las sondas de PLA todavía puede resultar en una adecuada relación señal a ruido (es decir, para este experimento). </li></ol></li><li> Ligadura <ol><li> Lavar las paredes del cuerpo con el tampón de lavado A dos veces durante 5 minutos cada uno. </li><li> Incubar con solución de ligación (dilución 1:40 de ligasa en tampón de ligación) durante 1 hr unat 37 ° C. En este caso, utilizar 5 l de ligasa, 40 l de tampón de ligación y 5 155 l de agua de alta pureza para garantizar la inmersión adecuado y mezcla de 5-10 paredes del cuerpo. </li></ol></li><li> Amplificación <ol><li> Lavar las paredes del cuerpo con el tampón de lavado A dos veces durante 2 minutos cada uno. </li><li> Incubar con solución de amplificación (1:80 dilución de la polimerasa en tampón de amplificación) durante 2 horas a 37 ° C. En este caso, utilizar 2,5 l de polimerasa, 40 l de tampón de amplificación 5 y 157,5 l de agua de alta pureza para garantizar la inmersión adecuado y mezcla de 5-10 paredes del cuerpo. </li></ol></li><li> Preparación de la Imagen <ol><li> Lavar las paredes del cuerpo con el tampón de lavado B dos veces durante 10 minutos cada uno. </li><li> Lavar con 0,01x Wash Buffer B una vez durante 1 min. </li><li> Equilibrar en unas pocas gotas de solución de montaje por lo menos 30 min antes del montaje, o almacenar durante la noche a 4 ° C. </li><li> Con unas pinzas finas, transferir cuidadosamente las paredes del cuerpo en un portaobjetos plataforma wITH sus cutículas hacia abajo. Coloque las paredes del cuerpo en filas y en la misma orientación dentro de una o dos gotas de medio de montaje. Coloque un 22 mm x 40 mm cubreobjetos sobre la preparación con cuidado de no generar burbujas de aire, y luego sellar la diapositiva con esmalte de uñas transparente. </li><li> Guarde las diapositivas de la oscuridad a -20 ° C hasta el momento de la imagen confocal. </li></ol></li></ol>

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Este informe demuestra cómo PLA se puede aplicar a la Drosophila larval NMJ. El ensayo se realizó en preparaciones de músculo de la pared corporal de larvas para el propósito de la detección de interacciones proteína-proteína endógenos presentes en la NMJ. Con esta técnica, Dlg y Hts se muestran a estar en estrecha proximidad entre sí, y por lo tanto existir en un complejo, específicamente en la región postsináptica 27. En apoyo de este resultado, un estudio previo ha proporcionado prueb...

Drosophila discos grandes (DLG) es un miembro conservada de la guanilato quinasa de la familia asociada a la membrana de andamios proteínas que ayudan a orquestar el ensamblaje de complejos de proteínas grandes en sitios específicos de la membrana plasmática. Originalmente identificado como una proteína supresora de tumores, Dlg sirve como un determinante importante de la polaridad epitelial 1,2,3 apicobasal. Dlg también sirve como un módulo de andamio importante en la unión neuromuscular (NMJ) de las neuronas motoras glutamatérgicas durante el desarrollo larvario 4. Dlg juega diversos papeles en la larval NMJ, y su pleiotropism se basa en su capacidad para asociarse con múltiples proteínas de 5,6. Una de estas proteínas es shao Hu-li tai (HTS), un homólogo de los mamíferos adducins que principalmente se han descrito en lo que respecta a sus funciones en la regulación de la actina-espectrina citoesqueleto 7. Previamente se ha demostrado que Dlg y Hts pueden formar un complejo con el uno al otro sobre la base de in vitro </em&gt; experimentos de co-inmunoprecipitación que participe toda adultos lisados ​​mosca 8. Una deficiencia de estos resultados, sin embargo, es que no indican donde este complejo formas. Con el uso de inmunohistoquímica, se observan las distribuciones de Dlg y Hts a superponerse en la membrana postsináptica de NMJs larvas, pero son en un complejo en esta región 8? Como se muestra recientemente y se detalla más adelante aquí, ligadura de ensayo de proximidad (PLA) se utiliza para buscar una en asociación situ entre Dlg y Hts específicamente en el larval NMJ 27. PLA es una técnica relativamente nueva se utiliza principalmente en las células y el tejido cultural que puede detectar interacciones proteína-proteína in situ 9. En este ensayo, los anticuerpos primarios contra las dos proteínas de interés se detectan con un par de anticuerpos secundarios específicos de especie, denominados sondas de PLA, que están conjugados a oligonucleótidos (Figura 1A, B). Si la proteína de doss están muy cerca entre sí (es decir, dentro de unas pocas decenas de nanómetros), la distancia entre las sondas PLA adjuntos puede salvarse a través de la hibridación de dos oligonucleótidos de conectores adicionales (Figura 1C). En esta conformación, los extremos libres de los oligonucleótidos de conector son lo suficientemente cerca para hacer contacto uno con otro, y una molécula de ADN circular cerrado se pueden formar sobre la ligadura en situ (Figura 1D). La molécula de ADN circular sirve como una plantilla para in situ amplificación por círculo rodante, que se prepara por uno de los oligonucleótidos conjugados con las sondas PLA (Figura 1E). Las secuencias dentro de la amplificado, producto de ADN resultante concatemeric entonces se pueden visualizar con, sondas de oligonucleótido complementario marcado con fluorescencia (Figura 1f). Dado que el ADN amplificado permanece unida a una de las sondas de PLA, la localización subcelular de la interacción proteína-proteína withitejido na puede determinarse fácilmente. Varios métodos se usan comúnmente para detectar interacciones proteína-proteína incluyendo técnicas in vitro tales como co-inmunoprecipitación, ensayos de pull-down y la levadura de dos híbridos, y en técnicas in vivo tales como Förster de transferencia de energía de resonancia (FRET) y bimolecular de fluorescencia de Complementación ( BiFC). Un error de las técnicas in vitro es que no se identifican donde la interacción se produce de manera endógena, mientras que la mencionada en técnicas in vivo implican la expresión de proteínas de fusión artificial que puede no reflejar el comportamiento natural de sus homólogos endógenos. Una ventaja importante de PLA es que es capaz de determinar dentro de un tejido la localización subcelular de interactores de proteínas endógenas que se encuentran en estrecha proximidad entre sí y probablemente formando un complejo, con el grado de cercanía requerido para generar una señal comparable a FRET y BiFC. PLA puede detectar interacciones con alta especificidad y sensibilidad debido al acoplamiento de reconocimiento de anticuerpos y la amplificación de ADN. Por lo tanto, el ensayo puede generar, señales luminosas discretas en forma de puntos lagrimales que revelan la posición exacta de la interacción. Además, los antígenos apenas visibles pueden ser detectados. Finalmente, PLA es una técnica relativamente sencilla de realizar, y ya no tiene que un procedimiento inmunohistoquímico estándar para completar. Por lo tanto, PLA ofrece una ventaja técnica sobre otros ensayos de interacción proteína-proteína que a menudo se ven afectadas con largos tiempos de preparación y amplia solución de problemas. Este protocolo se muestra cómo PLA se puede aplicar a la Drosophila larval NMJ para el propósito de la detección de interacciones proteína-proteína endógenos in situ. Aquí, el PLA se realiza en preparaciones de músculo de la pared corporal larvarios donde se muestran Dlg y Hts de existir de hecho en un complejo en la región postsináptica de NMJs. PLA no se ha utilizado previamente para estudiar la larval NMJ, y existen en la actualidad sólo un puñado de trabajos publicados que han utilizado este ensayo en el tejido Drosophila. Se espera que una mayor exposición de PLA a la comunidad de Drosophila se traducirá en el aumento de su uso como una herramienta adicional para complementar otros ensayos de interacción proteína-proteína más comúnmente utilizados,.

<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.

En los de tipo salvaje tercer instar NMJs larvales, Dlg es predominantemente encontrar en la membrana postsináptica de boutons glutamatérgicos tipo I, con niveles de inmunorreactividad Dlg siendo más pronunciada en boutons tipo Ib que el tipo es boutons (Figura 3) 4. Hts está presente en todo el músculo sino que se concentra en la región postsináptica con Hts niveles de inmunorreactividad aparece igual en ambos tipo I boutons, y también se encuentra presinápticamente (Figura ...

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 demuestra cómo la proximidad Ligadura ensayo se puede usar para detectar in situ en las interacciones proteína-proteína en la unión neuromuscular Drosophila larval. Con esta técnica, los discos grandes y Hu-li shao tai se muestra para formar un complejo en la región postsináptica, una asociación identificado previamente a través de co-inmunoprecipitación.

Detección de<em&gt; In Situ</em&gt; Complejos proteína-proteína en la<em&gt; Drosophila</em&gt; Larval unión neuromuscular Usando proximidad Ligadura Ensayo

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 vistas.  Simon Fraser University.  Este protocolo demuestra cómo la proximidad Ligadura ensayo se puede usar para detectar in situ en las interacciones proteína-proteína en la unión neuromuscular Drosophila larval. Con esta técnica, los discos grandes y Hu-li shao tai se muestra para formar un complejo en la región postsináptica, una asociación identificado previamente a través de co-inmunoprecipitación. 

Video: 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.

La visualización de larvas Nervios segmentarios en 3 Rd Instar Drosophila Preparación de larvas

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

Investigación

Neurociencias

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.

Visualización en vivo de las vesículas sinápticas En Drosophila segmentaria axones larvas

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.

La disección y la imagen de zonas activas en el Drosophila La unión 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.

Secuencial Foto-blanqueamiento para Precisar las células individuales de Schwann en la unión 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 qué importa la cuantificación: Caracterización de fenotipos en el<em&gt; Drosophila</em&gt; Larval unión 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 la funcionalidad de la placa 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).

Dos algoritmos para de alto rendimiento y multi-paramétrico Cuantificación de<em&gt; Drosophila</em&gt; La unión neuromuscular Morfología

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.

Grabaciones de Macropatch focal de corrientes sinápticas de la Unión Neuromuscular larvaria de Drosophila

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.

Disección e inmunohistoquímica de la pierna adulta de Drosophila para detectar cambios en la unión neuromuscular de una neurona motora identificada

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.

Detección de la expresión del receptor acoplado a la proteína G en neuronas aferentes vagas de ratón mediante hibridación multiplex in situ

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