El autor agradece profundamente Dr. Asako Sugimoto (Tohoku University) para su discusión útil. wyIs251 fue un generoso regalo del Dr. Kang Shen (Stanford University). mnIs17 fue proporcionado por la CGC, que es financiada por los NIH oficina de programas de infraestructura de investigación (P40 OD010440). Este trabajo fue apoyado por JSPS KAKENHI grant #17 H 05010 y #16 H 06536 y Daiichi Sankyo Fundación, Brain Science Foundation y la Fundación de Naito.

1. preparación de la muestra <ol><li>generar un transgénico C. elegans cepa de interés utilizando metodologías transgénicas 18. Por otra parte, obtener cepas adecuadas desde el centro de genética de Caenorhabditis (CGC). Para visualizar el transporte axonal de los precursores de la vesícula sináptica, utilice la línea transgénica wyIs251 [Pmig-13::gfp::rab-3] 7. Para observar el IFT, obtener la línea transgénica mnIs17 [osm-6::gfp] 19. Estas cepas se utilizan en el presente Protocolo. 
	Nota: Ya sea matriz Extracromosómica, integración genómica o incluso GFP knock-en obras. Para reducir las señales de fondo, uso de promotores específicos de células, en lugar de pan-neuronal promotores. 
	Nota: Mantenimiento de gusanos se describe en otros protocolos 20. Las cepas se mantuvieron en el césped de Escherischia coli OP50 alimentador sobre el crecimiento de nematodos placas de medio (NGM) (agarosa al 1,7% (w/v), 50 mM NaCl, 0,25% (w/v) peptona, 1 mM CaCl 2, 5 mg/mL de colesterol, 25 m m KH 2 PO 4, 1 mM MgSO 4 en platos de plástico de diámetro 60 mm) a 20 ° C.</li>
	<li>Crecer gusanos a 20 ° C en las placas de NGM a cualquier fase del ciclo de vida de los gusanos. 
	Nota: Se pueden observar transporte axonal y IFT en cualquier etapa. L4 tarde a adulto joven es un buen control positivo ya que varias publicaciones han utilizado estas etapas y visualizar tráfico eventos 14 , 21 , 22 , 23.</li>
</ol> 2. Preparación del 10% agarosa Nota: muchos proveedores ofrecen productos similares tales como agar, polvo de agar y agarosa. Utilizar la electroforesis en agarosa de grado (fuerza del gel &#62; 1200 g/cm 2). Polvos de agar baratos no funcionan porque el gel resultante no es lo suficientemente fuerte para inmovilizar gusanos.
<ol><li>Agarosa de 0.4 g de mezcla de 4 mL agua destilada (DW) en un tubo de vidrio (1,5 cm x 10,5 cm). Ponga una tapa en el tubo de vidrio para evitar la evaporación.</li>
	<li>Poner el tubo de vidrio en un bloque de calor de 95 ° C durante 90 minutos. Además, preparar otro tubo de vidrio (1,5 cm x 10,5 cm) llenado de DW y mantener a 95 ° C. poner una pipeta de Pasteur en la 95 ° C DW ( figura 1 A). Se verá un montón de pequeñas burbujas en el tubo de la agarosa y la solución debe ser turbia. Dejarlo hasta que la solución sea clara. Luego, mezclar mediante pipeteo arriba y abajo hasta que la solución de agarosa sea homogénea. 
	Nota: Microondas no pueden utilizarse para preparar la solución de agarosa de 10% cuando hace de DW y agarosa. Pequeñas burbujas causadas por microondas evitar fusión de agarosa. Sin embargo, gel de agarosa de 10% solidificado puede fácilmente derretir otra vez usando un microondas. Tubos de vidrio que contiene gel de agarosa de 10% pueden ser preparados antes de tiempo y almacenados a 4 ° C durante al menos 6 meses. Si al hacerlo, derretir el caldo usando un microondas cuando sea necesario y comenzar desde el paso 3 en el presente Protocolo. 
	Nota: Agarosa gradualmente pierde la capacidad de solidificarse si se incuban a 95 ° C durante mucho tiempo o después de repetidos solidificación y fusión. Si esto sucede, preparar nuevas 10% agarosa.</li>
</ol> 3. Preparación de agarosa almohadilla <ol><li>utilizando la pipeta Pasteur caliente preparada en el paso 2.2, colocar 2 gotas de 10% de agarosa sobre un portaobjetos de 76 x 22 mm.</li>
	<li>Rápidamente cubierta con un segundo portaobjetos de 76 x 22 mm 2 antes de la caída de la agarosa se solidifica como se muestra en la figura 1 B y empuje hacia abajo en la segunda diapositiva para aplanar la solución de agarosa. El espesor debe ser de 0.5-1 mm.</li>
	<li>Rápidamente Limpie la pipeta de Pasteur pipetas 95 ° C DW hacia arriba y hacia abajo hasta que la solución de agarosa es eliminada. De lo contrario, la pipeta que se obstruyen por gel de agarosa. Salir de la situación de la pipeta en la 95 ° C DW.</li>
	<li>Espere 1 minuto. La agarosa forma y se convierte en turbia. Luego, deslice el portaobjetos apartes a mano ( figura 1 C).</li>
</ol> 4. Montaje de gusanos Nota: incluso las pequeñas cantidades de movimiento de gusano evitar buena observación. Levamisol se ha utilizado tradicionalmente para prevenir el movimiento del gusano en la agarosa pad 24 , 25. Sin embargo, levamisol inhibe los receptores neuronales en C. elegans y por lo tanto puede afectar tráfico eventos en neuronas 15. Utilizando microbeads del poliestireno descrito hereis una buena alternativa 17.
<ol><li>Microscopio estéreo equipado con una transiluminación espejo desplazable, transferencia de ~ 30 gusanos transgénicos que expresan los marcadores adecuados a un nuevo NGM placa sin bacterias usando un alambre de platino selección. 
	Nota: Una selección de alambre de platino se hace del alambre de platino y pipeta de Pasteur (5 pulgadas), y la punta del alambre de platino fue aplanada. La preparación de las selecciones de alambre de platino y manejo de lombrices con éstos se describen en el Wormbook (http://www.wormbook.org).</li>
	<li>Deja la placa durante 1 minuto las bacterias se eliminan automáticamente de las superficies de gusanos como los gusanos se mueven en gel de agarosa NGM sin bacterias.</li>
	<li>Puso un 0.5-1.0 μL caída de 2,6% microesferas poliestireno del diámetro de 100 nm en el agua sobre el bloque de agar ( figura 1 D). Transferencia de 10-20 gusanos de la placa NGM libre de bacterias a las microesferas. Gusano de gota y propagación de los gusanos usando el alambre de platino selección para evitar la superposición de cuerpos ( figura 1 E).</li>
	<li>Aplique 2 cubreobjetos 22 x 40 mm ( figura 1 F). Presionar suavemente para eliminar burbujas de aire, pero evitar la trituración de gusanos. La muestra está ahora lista para la proyección de imagen de. 
	Nota: Porque anestesia no es administrado, gusanos se fijan sólo por la fuerza de fricción generada por la almohadilla agar microbeads del poliestireno y cubreobjetos 17. La presencia de bacterias de alimentador o demasiada solución hará más débil la fuerza friccional. 
	Nota: Diferentes tamaños de trabajo cubreobjetos (p. ej., 22 x 22 mm 2, 18 x 18 mm 2). Sin embargo, pueden prevenir el cubreobjetos mayor inmersión agua o aceite de la lente del objetivo que se escapa en las muestras durante la observación.</li>
</ol> 5. Observación Nota: parámetros apropiados de la proyección de imagen (energía del laser, ganancia, binning, etc.) serán diferente para cada sistema de microscopio y cámara. Aquí, se utiliza un microscopio de campo amplio equipado con un escáner confocal de disco giratorio y una cámara CCD digital.
<ol><li>Regular la temperatura de la etapa a 20 ° C, o ajustar la temperatura a 20 ° C.</li>
	<li>Colocar la corredera de la muestra sobre la platina del microscopio. Utilizar 100 x (apertura numérica = 1.3 o mejor) objetivo.</li>
	<li>Busca de las esferas indicadas en la figura 2 A (para wyIs251 y transporte axonal) o figura 2 B (para mnIs17 y IFT) como controles positivos . Otras áreas pueden ser analizadas como bien 22.</li>
	<li>Conjunto de parámetros (aumento de CCD = 200, Binning = 1 o 2, energía del laser a 488 nm = 1.0-1.3 mW). Realizar la grabación de lapso de tiempo en 4 cuadros/s hasta 1 min guardar archivos en formato TIFF de páginas múltiples. 
	Nota: Si GFP señales desaparecen y es difícil de seguir observando OSM-6::GFP o GFP::RAB-3 por 30 s, la potencia del láser es demasiado fuerte. En ese caso, reduzca la potencia del láser. Por el contrario, si no se registra ningún movimiento vesicular, la potencia del láser es demasiado débil. En ese caso, aumentar la energía del laser.</li>
	<li>Observar un gusano diferente y repetir 5.3 y 5.4. Las observaciones pueden durar hasta 20 minutos, pero cada gusano debe registrarse una sola vez para evitar los efectos de photo daños. 
	Nota: Los gusanos se han observado por al menos 20 min sin defectos importantes 7 , 27 , 28. Una observación más larga posible pero asilo &#39; t sido probado todavía. Una clara señal de la muerte neuronal es el cambio de morfología neuronal como hinchazón de la neurita. Como pueden verse las señales débiles de GFP en axones y dendritas en wyIs251 y mnIs17, neurite morfología puede comprobarse fácilmente mirando a axones o dendritas. Morfología neuronal se muestra en la figura 2.</li>
	<li>Generar archivos de película.
	<ol><li>Abrir el multi-TIFF archivo utilizando el software de Fiji. Ir a archivo &#62; abrir Seleccione el archivo multi-TIFF guardó en el paso 5.4. 
		Nota: Fiji se puede descargar en jttps://fiji.sc. Imagen J y plugins también pueden usarse para generar kymographs. Si al hacer esto, siga las instrucciones para el plugin solicitadas.</li> <li>, haga clic en el &#34; selección Rectangular &#34; botón ( figura 3, flecha) y seleccione el área de interés por dibujar un rectángulo con un ratón de ordenador (rectángulo amarillo en la figura 3 A). Ir a la imagen &#62; pilas &#62; herramientas &#62; Substacks y seleccione los números de segmento que se incluyen en la película. Se genera un substack.</li>
		<li>Activar la ventana de substack haciendo clic y vamos a imagen &#62; ajuste &#62; brillo y contraste. Aparece una ventana para ajustar el brillo y contraste. En la ventana, deslice la parte superior de la barra (mínimo) a la derecha para que la señal de fondo desaparece y el segundo superior de la barra (máximo) a la izquierda así que el movimiento de vesículas y partículas pueden verse bastante bien sobre el fondo.</li>
		<li>Vamos a imagen &#62; pilas &#62; tiempo matriz. Como la película se graba en 4 cuadros/s en el paso 5.4, el intervalo de tiempo es de 0.25 s. Así, tipo &#34; 0,25 &#34; en intervalo de.</li>
		<li>Generar un archivo de película seleccionando Guardar como &#62; AVI desde el menú de archivo (pelicula 1 y 2). 
		Nota: Mediante el uso de plugins apropiados, usted puede guardar el archivo como otros formatos tales como &#34; mpeg4 &#34; o &#34; mov &#34; archivos.</li>
	</ol></li> <li>Genere kymographs.
	<ol><li>Abra los archivos originales de la película utilizando otra vez el software de FIji y repita los pasos 5.6.2 y 5.6.3 para ajustar el brillo y contraste.</li>
		<li>Clic &#34; segmentado línea &#34; ( figura 3 B, flecha). Utilizando un ratón, dibuje una línea segmentada a lo largo de la cilia o axón (línea amarilla, en la figura 3 B).</li>
		<li>Ir a analizar &#62; Multi Kymograph &#62; Multi Kymograph ( figura 3 C). Aparece la ventana de ancho de línea ( figura 3 D). Tipo &#34; 3 &#34; en la ventana de grosor de línea y haga clic en Aceptar ( figura 3 D).</li>
		Se crea la ventana de kymograph de <li>. Guardar la imagen en formato TIFF o JPEG.</li>
	</ol>
	</li>
</ol>

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat Methods. 2 (12), 905-909 (2005).</li><li>Bindels, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mScarlet:+a+bright+monomeric+red+fluorescent+protein+for+cellular+imaging.">mScarlet: a bright monomeric red fluorescent protein for cellular imaging.</a> Nat Methods. 14 (1), 53-56 (2017).</li><li>Prevo, B., Mangeol, P., Oswald, F., Scholey, J. M., Peterman, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+differentiation+of+cooperating+kinesin-2+motors+orchestrates+cargo+import+and+transport+in+C.+elegans+cilia.">Functional differentiation of cooperating kinesin-2 motors orchestrates cargo import and transport in C. elegans cilia.</a> Nat Cell Biol. 17 (12), 1536-1545 (2015).</li><li>Robison, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detyrosinated+microtubules+buckle+and+bear+load+in+contracting+cardiomyocytes.">Detyrosinated microtubules buckle and bear load in contracting cardiomyocytes.</a> Science. 352 (6284), 0659 (2016).</li><li>Hayashi, S., Okada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+superresolution+fluorescence+imaging+with+spinning+disk+confocal+microscope+optics.">Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics.</a> Mol Biol Cell. 26 (9), 1743-1751 (2015).</li></ol>

El autor no tiene nada que revelar.

Limitación con respecto a los métodos existentes El método descrito aquí está optimizado para observar eventos rápidas como transporte axonal y IFT. Así, la inmovilización se priorice más de incubación más largo. Aunque hemos podido observar eventos de tráfico por al menos 20 min sin perturbación significativa, este método no sea siempre conveniente observar eventos lentos que requieren más observaciones, como la elongación del axón y migración celular. Para más observaciones, es ne...

Proyección de imagen de células vivas es una herramienta esencial para el análisis de transporte intracelular. En biología de la célula neuronal, análisis de transporte axonal con proyección de imagen de células vivas son esenciales para la comprensión de la función y la morfogénesis neuronal1. Defectos en el transporte axonal subyacen varios neurodegenerativas trastornos2. Dynein y proteínas de Kinesin Superfamilia realicen transporte axonal anterogradely y retrogradely, respectivamente1,2.
Cilios son otro compartimento celular en el que la red de microtúbulos y tráfico de maquinaria son altamente desarrollada3. Maquinaria de síntesis de proteína no se localiza en los cilios, lo que significa que deberán transportarse ciliares proteínas desde el citoplasma a las puntas de los cilios. Cilios específicos quinesina y dineína, llamado kinesin-2 y citoplásmica dineína-2, respectivamente, el transporte de los componentes de los cilios4, en un fenómeno llamado Transporte intraflagelar (IFT)5. Deterioro de IFT causa un espectro de enfermedades llamadas ciliopathies6. Así, análisis del mecanismo IFT por proyección de imagen de células vivas es necesaria para entender los mecanismos básicos de formación ciliar y patogenia.
Elegans de Caenorhabditis (C. elegans) es un buen modelo para estudiar el transporte axonal y IFT7,8,9. Para observar el IFT, Chlamydomonas se ha utilizado ampliamente como un organismo modelo5,6. Chlamydomonas es un organismo unicelular, la relación de IFT con el envejecimiento, la función neuronal y comportamiento serían difícil de analizar. Además, no han aplicado técnicas genéticas esenciales como CRISPR/Cas9 a Chlamydomonas. En organismos más altos del modelo, tales como ratones y Drosophila, interrupción del transporte axonal y IFT a menudo causa fenotipos letal transporte axonal y IFT son esenciales para la morfogénesis y la homeostasis de los animales 10, 11. En el caso de ratones, transfección y el cultivo celular se necesita generalmente para observar el transporte axonal y IFT, que requiere muchas habilidades y tiempo extenso12,13. Además, se puede perder mucho del contexto fisiológico importante en líneas celulares y células cultivadas. Porque el sistema nervioso no es esencial para la supervivencia de las lombrices, se interrumpen los mutantes de C. elegans transporte axonal o IFT no son a menudo letales7,9,14. Transporte axonal y IFT pueden observarse directamente en vivo sin la disección ya que C. elegans es transparente y por lo tanto es fácil observar los marcadores etiquetados de GFP.
Hay varios protocolos para inmovilizar, C. elegans, como el uso de un dispositivo de microfluidos15, cojines de agarosa con anestesia16o17de microesferas. La inclusión de la anestesia puede inhibir los trata de personas eventos en neuronas15. Un claro inconveniente del método dispositivo de microfluidos es que preparar un dispositivo de microfluidos no siempre es fácil. En cambio, inmovilización por pastillas de agarosa y microesferas es una manera conveniente y fácil para realizar imágenes de lapso de tiempo en C. elegans. Aquí describo este protocolo básico para inmovilizar C. elegans y visualizar el transporte axonal y IFT en vivo en C. elegans. En comparación con los otros métodos, el método descrito aquí no requiere equipo especial y es mucho más barato y más fácil de realizar.

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			<td height="21" style="height:21px;width:263px;">Slideglass (76&#160; x 26 mm)</td>
			<td style="width:167px;">Matsunami</td>
			<td style="width:139px;">S1111</td>
			<td style="width:271px;"></td>
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			<td height="21" style="height:21px;width:263px;">Coverglass (22&#160; x 40 mm)</td>
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			<td style="width:139px;">C024401</td>
			<td></td>
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			<td height="21" style="height:21px;width:263px;">Agarose</td>
			<td style="width:167px;">Wako</td>
			<td style="width:139px;">318-01195</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Polystylene microbeads 0.1 micron</td>
			<td style="width:167px;">Polysciences</td>
			<td style="width:139px;">#00876</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Heat block</td>
			<td style="width:167px;">TAITEC</td>
			<td style="width:139px;">0063288-000</td>
			<td>CTU-mini</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Microscope</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:139px;">IX-71</td>
			<td>widefield microscope</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Spinning disk Scanner</td>
			<td style="width:167px;">Yokogawa</td>
			<td style="width:139px;">CSU-X1</td>
			<td>spinning disc confocal scanner&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Digital CCD camera</td>
			<td style="width:167px;">Hamamatsu Photonics</td>
			<td style="width:139px;">C10600-10B</td>
			<td>ORCA-R2 degital CCD camera</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Objective lens (x100, NA1.4)</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:139px;">UPLSAPO 100XO</td>
			<td></td>
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			<td height="21" style="height:21px;width:263px;">Pasteur pipette (5 inch)</td>
			<td style="width:167px;">IWAKI</td>
			<td style="width:139px;">IK-PAS-5P</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Glass tube (1.5 cm diameter x 10.5 cm)</td>
			<td style="width:167px;">IWAKI</td>
			<td style="width:139px;">9820TST15-105NP</td>
			<td></td>
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			<td height="21" style="height:21px;width:263px;">TV9211: wyIs251</td>
			<td style="width:167px;">Laboratory of Kang Shen</td>
			<td>N/A</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">OTL11: mnIs17</td>
			<td style="width:167px;">Ref. 27, 29</td>
			<td style="width:139px;">N/A</td>
			<td>SP2101 was backcrossed with wild type for 6 times</td>
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			<td height="21" style="height:21px;width:263px;">Stereo microscope</td>
			<td style="width:167px;">Carl Zeiss</td>
			<td style="width:139px;">435064-9000-000</td>
			<td>STEMI 508&#160;</td>
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			<td height="21" style="height:21px;width:263px;">Mirror transillumination unit</td>
			<td style="width:167px;">Carl Zeiss</td>
			<td>435425-9010-000</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">platinum wire (0.2 mm)</td>
			<td style="width:167px;">Nilaco Corporation</td>
			<td style="width:139px;">m78483501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mm plastic dish</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:139px;">#351007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Fiji</td>
			<td style="width:167px;">N/A</td>
			<td style="width:139px;">N/A</td>
			<td>https://fiji.sc/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">nematode growth medium (NGM)&#160;&#160;</td>
			<td style="width:167px;"></td>
			<td style="width:139px;"></td>
			<td>1.7% (w/v) agarose, 50mM NaCl, 0.25% (w/v) Peptone, 1 mM CaCl2, 5 mg/mL Cholesterol, 25 mM KH2PO4, 1 mM MgSO4&#160;</td>
		</tr>
	</tbody>
</table>

immobilization of caenorhabditis elegans to analyze intracellular transport in neurons

Transporte axonal y el Transporte intraflagelar (IFT) son esenciales para la función y la morfogénesis axon y cilios. Proteínas del superfamilia de quinesina y dineína son motores moleculares que regulan anterograde y retrógrada transporte, respectivamente. Estos motores utilizan redes de microtúbulos como rieles. Elegans de Caenorhabditis (C. elegans) es un organismo modelo de gran alcance para estudiar transporte axonal y IFT en vivo. Aquí describo un protocolo para observar el transporte axonal y IFT en la vida del C. elegans. Transporte de carga puede visualizarse por selección de proteínas de carga utilizando proteínas fluorescentes como la proteína verde fluorescente (GFP). C. elegans es transparente y proteínas GFP-tagged carga pueden expresarse en células específicas bajo promotores específicos de la célula. Gusanos vivos pueden ser fijos por microesferas en gel de agarosa de 10% sin matar o anestesiar los gusanos. En estas condiciones, movimiento de carga se puede observar directamente en los axones y cilios de la vida de C. elegans sin disección. Este método puede ser aplicado a la observación de cualquier molécula de carga en las células mediante la modificación de las proteínas de la blanco o las células que expresan. Proteínas básicas como motores moleculares y proteínas adaptadoras que intervienen en el transporte axonal y IFT se conservan en C. elegans. Comparado con otros organismos modelo, mutantes pueden ser obtenidos y mantiene más fácilmente en C. elegans. Combinando este método con varios mutantes de C. elegans puede aclarar los mecanismos moleculares del transporte axonal y IFT.

Transporte axonal de neuronas DA9 Utilizando la línea de wyIs251 , la anterógrada y el transporte axonal retrógrado de GFP::RAB-3 se puede grabar simultáneamente en la neurona de motor DA9. La velocidad media de anterograde y transporte retrógrado en el axón proximal dorsal de la neurona DA9 es aproximadamente 1,8 y 2,6 μm/s, respectivamente22. El número de vesículas móviles es 0.03 y 0.018 por μm del axón por s. Así, para ...

Watch this Scientific Journal Video about Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons at JoVE.com

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Elegans de Caenorhabditis (C. elegans) es un buen modelo para estudiar el transporte axonal e intracelular. Aquí describo un protocolo para el análisis del transporte axonal e intraflagelar en C. elegansy grabación en vivo .

Inmovilización de Caenorhabditis elegans para analizar el transporte intracelular en las neuronas

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

The overall goal of this time lapse recording is to observe the intracellular transport events in Caenorhabditis elegans neurons.This method can help answer key questions in the cell biology field such as axonal transport and interflagellar transport.The main advantage of this technique is that one can observe intracellular transport in C.Elegans without dissection.Start this experiment by growing transgenic C.elegans strain of interest at 20

immobilization-caenorhabditis-elegans-to-analyze-intracellular

Research

JoVE Journal

Developmental Biology

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

7.9K vistas.  Tohoku University. The overall goal of this time lapse recording is to observe the intracellular transport events in Caenorhabditis elegans neurons.This method can help answer key questions in the cell biology field such as axonal transport and interflagellar transport.The main advantage of this technique is that one can observe intracellular transport in C.Elegans without dissection.Start this experiment by growing transgenic C.elegans strain of interest at 20

Video: Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

Evaluación completa de la línea germinal Química de toxicidad de los nematodos<em&gt; Caenorhabditis elegans</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

Investigación

Biología del Desarrollo

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

Análisis espacial y temporal de ERK en el Activo<em&gt; C. elegans</em&gt; línea germinal

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods are described here to analyze distinct populations of MTs in the developing neural tube of the zebrafish embryo. While the focus is on neural tissue, this methodology is broadly applicable to other tissues. The procedures are optimized for early to mid-somitogenesis-stage embryos (1 somite to 12 somites), however they can be adapted to a range of other stages with relatively minor adjustments. The first protocol provides a method to assess the spatial distribution of stable and dynamic MTs and perform a quantitative analysis of these populations with image-processing software. This approach complements existing tools to image microtubule dynamics and distribution in real-time, using transgenic lines or transient expression of tagged constructs. Indeed, such tools are very useful, however they do not readily distinguish between dynamic and stable MTs. The ability to image and analyze these distinct microtubule populations has important implications for understanding mechanisms underlying cell polarization and morphogenesis. The second protocol outlines a technique to analyze nascent MTs specifically. This is accomplished by capturing the de novo growth properties of MTs over time, following microtubule depolymerization with the drug nocodazole and a recovery period after drug washout. This technique has not yet been applied to the study of MTs in zebrafish embryos, but is a valuable assay for investigating the in vivo function of proteins implicated in microtubule assembly.

Immunolabeling methods to analyze distinct populations of microtubules in the developing zebrafish brain are described here, which are broadly applicable to other tissues. The first protocol outlines an optimized method for immunolabeling stable and dynamic microtubules. The second protocol provides a method to image and quantify nascent microtubules specifically.

Uso de inmunomarcación para analizar microtúbulos estables, dinámicos y emergente en el embrión de pez cebra

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Disección y tinción inmunofluorescente de seta del cuerpo y las neuronas fotorreceptoras en cerebros de adultos de Drosophila melanogaster

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Aplicación de RNAi y expresión del Factor de transcripción inducida por choque térmico para reprogramar las células de germen para las neuronas en C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are facilitated by the transparency of the animal, the ability to do forward and reverse genetic assays, the relative ease of generating fluorescently labeled proteins, and the use of fluorescent dyes that can either be microinjected into the early embryo or incorporated into its food (E. coli strain OP50) to label cellular organelles (e.g. 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Here, we present the use of a fluorescent pH-sensitive dye that stains intestinal lysosomes, providing a visual readout of dynamic, physiological changes in lysosomal acidity in live worms. This protocol does not measure lysosomal pH, but rather aims to establish a reliable method of assessing physiological relevant variations in lysosomal acidity. cDCFDA is a cell-permeant compound that is converted to the fluorescent fluorophore 5-(and-6)-carboxy-2&#39;,7&#39;-dichlorofluorescein (cDCF) upon hydrolysis by intracellular esterases. Protonation inside lysosomes traps cDCF in these organelles, where it accumulates. Due to its low pKa of 4.8, this dye has been used as a pH sensor in yeast. Here we describe the use of cDCFDA as a food supplement to assess the acidity of intestinal lysosomes in C. elegans. This technique allows for the detection of alkalinizing lysosomes in live animals, and has a broad range of experimental applications including studies on aging, autophagy, and lysosomal biogenesis.

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2&#39;,7&#39;-dichlorofluorescein diacetate (cDCFDA)

Evaluación de alcalinización Lysosomal en el intestino de vivir Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Análisis computacional de lo elegans de Caenorhabditis del Germline para estudiar la distribución de núcleos, proteínas y citoesqueleto

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.

Estudiar el estrés oxidativo causado por los estreptococos del grupo Mitis en Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

Microscopía de lámina de luz isotrópica y análisis de linaje celular automatizado para catalogar Caenorhabditis elegans embriogénesis con resolución subcelular

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

Medición de la orientación y la motilidad del esperma dentro del tracto reproductivo de la Caenorhabditis elegans hermafrodita

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...