Nos gustaría agradecer a centro Caenorhabditis genética de las cepas, las ciencias naturales y Consejo de investigación Ingeniería (NSERC) y la Fundación de Canadá para la innovación (CFI) para la financiación. Nos gustaría agradecer a Dr. Lizhen Chen (Departamento de sistemas celular y anatomía, UT salud San Antonio) para permitir el uso irrestricto de sus instalaciones de laboratorio para todos los experimentos de C. elegans , así como el Dr. Exing Wang (Director asociado, centro de la proyección de imagen óptica UT salud San Antonio) para obtener ayuda con microscopía confocal. También nos gustaría agradecer a Dr. Myron Ignatius para proporcionar apoyo y aliento para facilitar la sesión de video.

1. Mancha y lisosomas intestinales de la imagen
<ol><li>Medios de cultivo nematodos de semilla (NGM) placas con OP50
	<ol>
<li>Preparar platos para NGM según el protocolo recomendado19 y permita que las placas cerradas secar por 2 días a temperatura ambiente.</li>
		<li>Inocular las bacterias OP50 en caldo estéril caldo de Luria (LB) y crecen en un baño incubador o agitación a 37 ° C durante 36 h o hasta que el OD es entre 0.2 y 0.4. Evitar el uso de un cultivo bacteriano con OD > 1 o OD < 0,2.</li>
		<li>Una vez seco planchas NGM, coloque una gota (30 μL) del inóculo OP50 en el centro de la placa y luego la gota en un parche más o menos que es de 2 cm x 2 cm de tamaño. Nota: Cualquier inóculo OP50 restantes puede almacenarse a 4 ° C por hasta un mes.</li>
		<li>Invertir las placas OP50 una vez que el inóculo ha secado (aproximadamente 10 o 15 min) y luego incubar las placas a 37 ° C durante 36 horas.</li>
		<li>Después de 36 h, quitar las placas de la incubadora y almacenar a 4 ° C hasta su uso posterior. Nota: Las placas deben ser buenas para un máximo de 1 mes a 4 ° C.</li>
	</ol></li>
	<li>CDCFDA que suple a OP50
	<ol>
<li>Para complementar cDCFDA sobre placas OP50, preparar una acción de trabajo de cDCFDA de 10 mM en dimetil sulfóxido (DMSO). Mantener la solución de cDCFDA protegida de la luz y almacenar a-20 ° C para su uso posterior.</li>
		<li>Suavemente coloque 100 μl de solución de cDCFDA de 10 mM sobre la superficie del parche OP50 2 cm x 2 cm, tal que la totalidad del parche está uniformemente cubierto con cDCFDA. Nota: Es muy importante que la totalidad del parche de OP50 está cubierto con cDCFDA. Si es necesario, utilizar hasta 150 μL de cDCFDA para cada placa. También es imprescindible que la solución de cDCFDA no se extiende más allá de las fronteras del parche OP50, puesto que cualquier cDCFDA que sale el parche OP50 no será incorporado a los alimentos y se traducirá en menor intensidad de la tinción.</li>
		<li>Coloque las placas OP50 en una caja oscura en la mesa hasta que todos los de la solución cDCFDA es absorbido en el parche bacteriano (generalmente alrededor de 25 a 30 minutos).</li>
	</ol></li>
	<li>Tinción de gusanos usando cDCFDA
	<ol>
<li>Una vez que el cDCFDA ha permeado totalmente el parche OP50, colocar no más de 20 gusanos por placa e incubar las placas invertidas a 20 ° C durante un mínimo de 14 h. Nota: Se recomienda colocar las placas de cDCFDA en una caja opaca para minimizar la exposición a la luz.</li>
		<li>Controlar por las diferencias de consumo entre experimentos y normalizar las señales cDCFDA, (recomiendan) Mancha Co con () 3-{2-[(1H,1'H-2,2'-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato) Difluoro) boro (2,5 μl de una solución recién hecho de 1 mM), un colorante de acidotropic amina débil cuya fluorescencia es en gran parte no variante sobre el espectro de pH ácido (véase Tabla de materiales para los nombres comunes de reactivos). Nota: No mancha gusanos de menos de 14 h, ya que esto puede proporcionar la intensidad de la tinción inadecuada cDCFDA y dar una falsa interpretación del pH lisosomal.</li>
	</ol></li>
	<li>Preparación de portaobjetos para microscopía
	<ol>
<li>Coloque dos tiras paralelas de etiquetado cinta unas 3 pulgadas aparte en una superficie plana lisa, como un espejo (figura 1).</li>
		<li>Capa de la superficie del espejo con spray repelente al agua y limpie el exceso de líquido.</li>
		<li>Derretir la agarosa al 2% (disuelto en agua destilada H2O) en el microondas hasta que totalmente licuado, luego coloque una gota 50 de μl de agarosa entre las dos tiras de cinta adhesiva y cubrir rápidamente la gota con un portaobjetos de microscopio, que es perpendicular a las tiras de la diapositiva cinta. Después de la agarosa ha solidificado, formando una almohadilla circular debajo de la diapositiva, voltee la diapositiva y agregar 10-15 μl de azida de sodio 5mM (NaN3) en la almohadilla de agarosa. Nota: NaN3 es un inhibidor del citocromo C que cuando se utiliza en bajas concentraciones, anestesia reversible gusanos para que no se mueva durante la proyección de imagen.</li>
		<li>Recoger gusanos de la placa OP50 complementado con cDCFDA y transferirlas a una placa limpia de NGM sin cualquier OP50. Que los gusanos a mover durante unos segundos permitir que la mayoría de los OP50 a eliminarse de la superficie de los gusanos y luego se transfieren los gusanos a la almohadilla de agarosa que contiene azida de sodio 5 mM.</li>
		<li>Cubra inmediatamente la almohadilla de agarosa con un cubreobjetos. Asegúrese de colocar suavemente el cubreobjetos sin aplicar demasiada presión, ya que esto puede resultar en explosión de gusanos. Nota: Bacterias OP50 complementadas con cDCFDA fluorescencia cuando es visualizado por microscopía, por lo tanto es importante limpiar los gusanos lo mejor posible antes de colocar sobre la almohadilla de agarosa que contiene azida de sodio. Si preparando más de 6 cepas de C. elegans (incluyendo N2, control de tipo salvaje), es recomendable preparar las muestras en lotes de 6, con el fin de asegurar que los gusanos no seque sobre la almohadilla de agarosa. En algunas cepas, la vulva comienza a romperse después de largos períodos de tiempo debido a la presión de la hoja de cubierta, por lo que es importante planificar en consecuencia.</li>
	</ol></li>
	<li>Microscopía confocal Nota: Algunos microscopios tienen una función incorporada que automáticamente aumenta la intensidad de fluorescencia para compensar niveles variables de fluorescencia. Estos microscopios pueden no funcionar bien para gusanos teñidas con cDCFDA, puesto que intrínsecamente aumentan la intensidad de fluorescencia de las muestras de la proyección de imagen.<ol>
<li>Realizar la proyección de imagen en un microscopio confocal estándar con longitudes de onda de excitación/emisión a 488/530 nm para cDCFDA. Solo plano imágenes (no Z-stacks) de lisosomas intestinales y utilizar sólo los lisosomas que se encuentran en foco (intensidad máxima) para la cuantificación de la intensidad. Nota: Tal vez debido a diferencias en la disponibilidad de tinte, lisosomas de las células intestinales directamente posteriores a la faringe mantienen cDCFDA tinción intensidad independientemente del genotipo o edad, por lo tanto debe tenerse cuidado para evitar imágenes de lisosomas de estas células.</li>
		<li>Imagen de la diapositiva que contiene 2 viejo tipo wild que N2 gusanos primero. Al hacerlo, ajustar los parámetros de proyección de imagen confocales como energía del laser, agujero de alfiler y apertura para garantizar que la cDCFDA intensidad de tinción no es sobresaturado. Nota: Una vez que estos ajustes se establecen, no cambiar para toda la duración de la proyección de imagen para ese día.</li>
		<li>Capturar imágenes de 1024 x 1024 píxeles de un solo plano del intestino (3 o 4 imágenes por gusano) usando un 60 X lente de aumento. Nota: El espectro de emisión de cDCFDA en gusanos producirá un solo pico prominente en 520 nm coincidiendo con su divulgado fluorescencia espectro20, proporcionando una lectura de la señal específica que es distinta de Autofluorescencia intestinal (figura 3).</li>
		<li>Exportar imagen confocal (ficheros .lsm) como archivos .tiff para cada canal.</li>
		<li>A continuación, abra ImageJ y haga clic en archivo | Abrir abrir el archivo de imagen para ser cuantificada. Una vez que se carga la imagen, utilice la herramienta de selección de forma ovalada en ImageJ para seleccionar una región de interés (ROI).</li>
		<li>Después de eso, haga clic en analizar | Medida para cuantificar la intensidad de la fluorescencia para ese retorno de la inversión. Seleccione 4 – 5 diferentes foco lisosomas por imagen y calcular la intensidad de fluorescencia relativa promedio de cada región de interés (ROI).</li>
		<li>Para cada imagen, medir la intensidad de la fluorescencia de fondo en una región del intestino entre lisosomas. Restar los valores de intensidad de fluorescencia de fondo de los valores de intensidad de fluorescencia lysosomal.</li>
		<li>En total, recogen alrededor de 30 a 50 valores normalizados individuales de intensidad cDCFDA (animales de 6 a 10) para cada cepa ensayada. Use estos valores para trazar un diagrama de caja y bigotes utilizando cualquier software estadístico apropiado. Nota: La coloración puede variar considerablemente dependiendo de factores como temperatura, tiempo de incubación y la salud general, edad de los animales que sólo son relevantes en proceso de muestras en el mismo experimento tinción comparaciones de intensidad de fluorescencia. Por lo tanto, es importante procesar controles en cada experimento de tinción. Calcular diferencias estadísticas (t-pruebas) utilizando cualquier software estadístico apropiado.</li>
		<li>Imagen todos las cepas del mismo experimento (teñido en el mismo día) en secuencia, teniendo cuidado de no para cambiar a cualquiera de los parámetros de la proyección de imagen.</li>
	</ol></li>
</ol>

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Los autores no declaran conflicto de intereses.

Una variedad de eventos celulares y moleculares que contribuyen al envejecimiento, influenciado por características de historia de vida y factores genéticos. Nuestro reciente estudio22 sugiere que el ciclo reproductivo juega un papel importante en el control de la aptitud del soma a través de la regulación de la dinámica de pH lisosomal. Hemos mostrado que proteolisis lisosomal mediada es promovido mientras que animales se reproducen activamente upregulation de la transcripción de la v-ATPas...

La aparición de agregados proteicos es ampliamente aceptada que un sello de envejecimiento en las células eucariotas1,2,3y la formación de los cuales se cree que entre los conductores de principio de senescencia celular4 , 5 , 6 , 7. Existe creciente evidencia que como células edad, catabolismo proteico se deteriora, provocando un aumento en la agregación de la proteína. El colapso de la proteólisis en el envejecimiento de las células conlleva una debilitación de la autofagia8 , así como de la degradación de proteínas mediada por proteasoma9. Finalmente, oxidación irreversible de la proteína aumenta en las células viejas, deteriorando aún más proteína catabolismo10.
Autofagia fue pensada inicialmente para ser un proceso no selectivo para la degradación de la mayor parte de proteínas dañadas, pero estudios recientes han indicado que la autofagia es altamente selectiva para el catabolismo de los agregados de proteínas y organelos disfuncionales que no son susceptibles a la degradación a través de otras proteínas separación mecanismos11. Durante el proceso de autofagia, proteínas dañadas y agregadas son secuestradas en una vesícula de membrana doble llamada el autophagosome. Este autophagosome entonces se funde con los ácidos organelos llamados lisosomas, que conduce a la degradación de la carga de autophagosome12. Lisosomas representan el punto final de la vía de la autofagia y participan en diversos procesos celulares tales como reparación de membrana, control transcripcional y detección de nutrientes; destacando su papel centralizado en la homeostasis celular (revisada en Ref. 13). Varios estudios han demostrado una asociación entre una disminución dependiente de la edad en la función lisosomal y de trastornos neurodegenerativos diversos13. Constantemente, restaurar la función lysosomal en células mayores puede retrasar la aparición de fenotipos relacionados con el envejecimiento14,15. Estudios de la composición del medio intralumen sugieren que el colapso de la función lysosomal en células mayores no es debido a una reducción en la producción de proteasas lisosomales16. Por otra parte, se ha propuesto que la pérdida de acidez produce, un requisito fundamental de su actividad enzimática, puede ser la base la disminución de la proteólisis mediada por el lisosoma17. Para poder explorar esta hipótesis, es esencial desarrollar los reactivos y protocolos para cambios dinámicos en pH lisosomal en células vivas de una manera coherente y replicable.
El intestino de C. elegans es el tejido metabólico importante en gusanos y es un regulador crítico de la homeostasis sistémica y la vida útil. Desarrollamos análisis para evaluar los cambios en la acidez del lumen intestinales lisosomas de gusanos para determinar cómo lisosoma-mediada de la proteólisis contribuye al envejecimiento. Aunque fluoróforos sensibles al pH han sido utilizados previamente en C. elegans para marcar lisosomas intestinales, sin embargo no ha habido un esfuerzo por establecer un protocolo exitoso que puede detectar pequeños incrementos en el pH lisosomal en vivo18. Presentamos un protocolo que puede utilizarse para detectar pérdida de acidez lisosomal de las células intestinales de C. elegans mediante un protocolo de alimentación sencillo y cómodo que incorpora un fluoróforo sensible al pH (cDCFDA) OP50 alimentos.

<table><tbody><tr><td>OP50 (&lt;em&gt;E. coli&lt;/em&gt;)</td><td>Caenorhabditis Genetics Center</td><td></td><td>Order online at https://cgc.umn.edu/strain/OP50</td></tr><tr><td>5(6)-carboxy-2&amp;rsquo;,7&amp;rsquo;-dichlorofluorescein diacetate&amp;nbsp;</td><td>ThermoFisher</td><td>C369</td><td>Commonly known as cDCFDA</td></tr><tr><td>9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#039;H-2,2&#039;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron</td><td>ThermoFisher</td><td>L7528</td><td>Commonly known as Lysotracker Red</td></tr><tr><td>Confocal microscope (e.g. Zeiss LSM 510)</td><td></td><td></td><td></td></tr><tr><td>ImageJ</td><td></td><td></td><td>Download for free from https://imagej.nih.gov/ij/download.html</td></tr><tr><td>LB Broth powder</td><td>ThermoFisher</td><td>22700041</td><td></td></tr><tr><td>Bacto Agar</td><td>Sigma</td><td>A5306-1KG</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>Bacto Peptone</td><td>Fisher Scientific</td><td>S71604</td><td></td></tr><tr><td>Cholesterol powder</td><td>Sigma</td><td>C3045&amp;nbsp;</td><td></td></tr><tr><td>CaCl2</td><td>Sigma</td><td>449709</td><td></td></tr><tr><td>MgSO4</td><td>Sigma</td><td>M7506</td><td></td></tr><tr><td>K3PO4</td><td>Sigma</td><td>P5629</td><td></td></tr><tr><td>Sodium Azide</td><td>Sigma</td><td>S2002</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418</td><td></td></tr><tr><td>Microscope Slides</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>Cover Slips</td><td>ThermoFisher</td><td>3406</td><td></td></tr><tr><td>Agarose</td><td>Sigma</td><td>A6013</td><td></td></tr><tr><td>Incubator</td><td></td><td></td><td></td></tr><tr><td>Mirror or other smooth flat surface</td><td></td><td></td><td></td></tr></tbody></table>

assessing lysosomal alkalinization in the intestine of live caenorhabditis elegans

El nematodo Caenorhabditis elegans (C. elegans) es un modelo que es ampliamente utilizado para el estudio de longevidad y las vías de desarrollo. Tales estudios son facilitados por la transparencia de los animales, la capacidad de marcha adelante y atrás ensayos genéticos, la relativa facilidad de generar proteínas fluorescencia de etiquetado y el uso de tintes fluorescentes que tampoco puede ser microinyectados en el temprano embrión o incorporado en su alimento (e. coli cepa OP50) etiquetar organelos celulares (e.g. 9-dietilamino-5 H-benzo (a) fenoxazina-5-one y (3-{2-[(1H,1'H-2,2'-bipyrrol-5-yl-kappaN(1)) methylidene]-2 H-pyrrol-5-yl-kappaN} - N - [2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Aquí, presentamos el uso de un fluorescente tinte pH-sensible que tiñe lisosomas intestinales, proporcionando una lectura visual de los cambios dinámicas, fisiológicas en acidez lysosomal en gusanos vivos. Este protocolo no mide pH lisosomal, sino más bien tiene por objeto establecer un método confiable de evaluación fisiológicas variaciones relevantes en acidez lysosomal. cDCFDA es un compuesto celular florescente que es convertido a fluorescente fluoróforo 5-(and-6)-carboxy-2',7'-dichlorofluorescein (cDCF) al producirse la hidrólisis por esterasas intracelulares. Protonación dentro de lisosomas trampas cDCF en estos orgánulos, donde se acumula. Debido a su bajo pKa de 4.8, este colorante se ha utilizado como un sensor de pH en la levadura. Aquí describimos el uso de cDCFDA como suplemento del alimento para determinar la acidez de lisosomas intestinales en C. elegans. Esta técnica permite la detección de alcalinizantes lisosomas en animales vivos, y tiene una amplia gama de aplicaciones experimentales, incluyendo estudios sobre envejecimiento, autofagia y biogénesis lisosomal.

cDCFDA lisosomas tiñe de manera dependiente del pH y su pKa bajo y lista absorción en lisosomas es un sensor de pH ideal21. cDCFDA coloración de intensidad es inversamente proporcional al pH lisosomal (es decir, tinción intensidad los aumentos como disminuciones de pH)18,22. las señales cDCFDA son siempre débiles en los lisosomas de los animales tratados con 20 mM de la cloroquina, un inhibid...

Watch this Scientific Journal Video about Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans at JoVE.com

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

Una guía paso a paso para la pérdida de acidez lysosomal en el intestino de C. elegans con el colorante vital sensible a pH 6 - carboxi - 2', 7'-Diclorofluoresceína diacetato (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 ...

assessing-lysosomal-alkalinization-intestine-live-caenorhabditis

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

5.9K vistas.  University of Saskatchewan. Una guía paso a paso para la pérdida de acidez lysosomal en el intestino de C. elegans con el colorante vital sensible a pH 6 - carboxi - 2', 7'-Diclorofluoresceína diacetato (cDCFDA)

Video: Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

Análisis colorimétrico de lactato intracelular en pequeña escala y del piruvato en el nematodo Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Investigación

Bioquímica

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a&#160;model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism&#39;s genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

El intestino C. elegans como modelo una morfogénesis Lumen intercelular y En Vivo biogénesis de la membrana polarizada a nivel unicelular: etiquetado de anticuerpos, ARNi pérdida-de-función análisis y proyección de imagen de

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

Biología del Desarrollo

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

El Canal de Excretory C. elegans como modelo de morfogénesis Lumen intracelular y En Vivo biogénesis de la membrana polarizada en una sola celda: etiquetado por fusiones de GFP, ARNi interacción pantalla e imagen

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

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 ...

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

La<em&gt; Caenorhabditis elegans</em&gt; Sistema Modelo para el Estudio Amylopathy

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Neurociencias

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.

Perfiles de isótopos estables de flujo intermediarios metabólicos en fase de desarrollo y de Adultos Caenorhabditis elegans</em

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

Biología

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

Hibridación in situ de fluorescencia de ARN (FISH) para visualizar la colonización microbiana y la infección en los intestinos de Caenorhabditis elegans

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

Inmunología e Infección

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

Un Modelo Anoxia inanición por isquemia / reperfusión en el<em&gt; C. elegans</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a nonmammalian animal model that is widely used as an assay system due to its short lifespan, transparency, cost-effectiveness, and lack of animal ethics issues. In this study, a method was developed to investigate the effects of different bacteria and 3,3'-diindolylmethane (DIM) on the intestinal permeability of C. elegans with a high-throughput image analysis system. The worms were infected with different gut bacteria or cotreated with DIM for 48 h and fed with fluorescein isothiocyanate (FITC)-dextran overnight. Then, the intestinal permeability was examined by comparing the fluorescence images and the fluorescence intensity inside the worm bodies. This method may also have the potential to identify probiotic and pathogenic intestinal bacteria that affect intestinal permeability in the animal model and is effective for examining the effects of harmful or health-promoting chemicals on intestinal permeability and intestinal health. However, this protocol also has some considerable limitations at the genetic level, especially for determining which genes are altered to control illness, because this method is mostly used for phenotypic determination. In addition, this method is limited to determining exactly which pathogenic substrates cause inflammation or increase the permeability of the worms' intestines during infection. Therefore, further in-depth studies, including investigation of the molecular genetic mechanism using mutant bacteria and nematodes as well as chemical component analysis of bacteria, are required to fully evaluate the function of bacteria and chemicals in determining intestinal permeability.

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

This protocol describes how to measure intestinal permeability of Caenorhabditis elegans. This method is helpful for basic biological research on intestinal health related to the interaction between intestinal bacteria and their host and for screening to identify probiotic and chemical agents to cure leaky gut syndrome and inflammatory bowel diseases.

Medición de los efectos de las bacterias y los productos químicos en la permeabilidad intestinal de los ceganes

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a ...

Medicina

Hand Dissection of Caenorhabditis elegans Intestines

Comprised of only 20 cells, the Caenorhabditis elegans intestine&#160;is the nexus of many life-supporting functions, including digestion, metabolism, aging, immunity, and environmental response. Critical interactions between the C. elegans host and its environment converge within the intestine, where gut microbiota concentrate. Therefore, the ability to isolate intestine tissue away from the rest of the worm is necessary to assess intestine-specific processes. This protocol describes a method for hand dissecting adult C. elegans intestines. The procedure can be performed in fluorescently labeled strains for ease or training purposes. Once the technique is perfected, intestines can be collected from unlabeled worms of any genotype. This microdissection approach allows for the simultaneous capture of host intestinal tissue and gut microbiota, a benefit to many microbiome studies. As such, downstream applications for the intestinal preparations generated by this protocol can include but are not limited to RNA isolation from intestinal cells and DNA isolation from captured microbiota. Overall, hand dissection of C. elegans intestines affords a simple and robust method to investigate critical aspects of intestine biology.

Hand Dissection of Caenorhabditis elegans Intestines

The present protocol describes a procedure for isolating intestines from adult Caenorhabditis elegans nematode worms by hand for input in genomics, proteomics, microbiome, or other assays.

Disección manual de los intestinos de Caenorhabditis elegans

Comprised of only 20 cells, the Caenorhabditis elegans intestine&#160;is the nexus of many life-supporting functions, including digestion, metabolism, ...

Evaluación de alcalinización Lysosomal en el intestino de vivir Caenorhabditis elegans

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