Nous tenons à remercier S. Taneja-Bageshwar, K. Beifuss, S. Kedroske, et HC. Hsiao pour les discussions utiles. Ce travail a été financé par des fonds de démarrage du ministère TAMHSC de médecine moléculaire et cellulaire. Le champ composé et disque rotatif ont été achetés avec des fonds fournis par le ministère et le Bureau du doyen TAMHSC. Certaines souches ont été fournis par le Centre de Génétique Caenorhabditis, qui est financé par le National Center for Research Resources. pRF4 (rol-6 (su1006)) a été un don de feu A..

1. Préparation de DiI tache <ol><li> Préparer une solution stock de 20 mg / mL DII (Biotium, Inc, Hayward, CA) dans le DMF. Dii est sensible à la lumière, afin de protéger de la lumière DiI en enveloppant dans du papier. </li><li> Créer une dilution de travail de DiI en ajoutant 0,6 uL DiI stocks d&#39;399,4 uL M9 pour chaque population. Cela devrait donner une dilution finale de travail de 30 ug / ml dans le DiI M9. Cela peut être étendu pour la coloration des populations multiples simultanément. Bouclier DiI de la lumière en enveloppant le tube (s) au fleuret. </li></ol> 2. Préparation de nématodes <ol><li> Utilisez une plaque de 60 mm contenant une population de nématodes non contaminée. Lavez les animaux de la plaque en utilisant une solution de 0,5% de Triton X-100 dans le tampon M9 en remuant doucement de liquide dans un mouvement circulaire sur la surface de la plaque à desserrer tous les animaux larvaire et adulte. Transfert de lavage dans une stérile de 1,5 ml tube. </li><li> Immédiatement spin-down des animaux à 2000 rpm pendant 30 sec. Retirez et jetez autant soutenirernatant que possible sans déranger la masse des animaux au fond du tube. </li><li> Afin de réduire résiduelle de Triton X-100, rincez les animaux en utilisant M9 tampon, spin, et enlever le surnageant. Répétez cette étape une fois. </li><li> Ajouter 400 ul de solution de travail dans DiI M9 dans le tube et brièvement au vortex pour remettre les animaux dans la solution. </li><li> Agiter le tube à 20 ° C à l&#39;horizontale pendant 3 heures à 350 rpm dans un environnement lumineux protégé. Si désiré, les animaux peuvent être mis à incuber jusqu&#39;à 16 heures pour la coloration. </li><li> Pour réduire la quantité de colorant non lié, ralentit les animaux à 2000 rpm pendant 20 sec. Retirez et jetez autant que possible le surnageant sans déranger la masse des animaux. </li><li> Remettre en suspension dans 400 animaux M9 pi de tampon et de verser le liquide sur une partie libre de bactéries d&#39;une plaque de gélose ensemencés avec NGM OP50 E. coli. Permettre aux animaux de récupérer au moins 30 minutes. Pendant le temps de récupération, les animaux doivent ramper dans le liquide coloration DII et sur les aliments. Cette étaperéduit la fluorescence de fond auprès de DII libre. </li></ol> 3. Montage et l&#39;observation des spécimens <ol><li> Faire fondre la gélose de 4% dans de l&#39;eau en utilisant un autoclave ou un four micro-ondes. </li><li> Créer entretoises réutilisable, qui peut être utilisé pour assurer une épaisseur uniforme de la plaquette de gélose, par superposition de deux morceaux de ruban de laboratoire sur une lame de verre. Faites deux diapositives entretoise total. </li><li> Convenir d&#39;une lame de verre propre entre deux lames d&#39;espacement. Pipeter environ 150 uL (quatre gouttes) de 4% agar fondu sur le centre de la lame de verre propre. Couvrir rapidement l&#39;agar fondu en utilisant une lame supplémentaire pour former un coussin d&#39;agar. Retirez délicatement la lamelle, en gardant le pad centrée sur le haut de la montage des diapositives. </li><li> Pipeter environ 5 pi de nématodes anesthésie (100 uM - 1 lévamisole mM, par exemple) sur le pavé. </li><li> Mont de 8 à 12 animaux dans l&#39;anesthésie et couvrir avec une lamelle de microscope. </li><li> Observez les animaux en utilisant un microscope confocal composé ou équipés d&#39;au moins une l&#39;objectivation 40x<html> ve et une DsRed / TRITC (ou autre compatible) filtre. Le maximum d&#39;excitation de fluorescence de Dii est 549 nm et son émission maximum est de 565 nm pour le colorant lié (Biotium, Inc, Hayward, CA). </li></ol> 4. Les résultats représentatifs Taches DiI la cuticule de type sauvage et mutant C. elegans. La surface cuticulaire contient anneaux séparés par des sillons circonférentielle et, dans certains stades, des crêtes longitudinales appelées ailes. Chaque phase de développement a des structures cuticulaires avec des compositions distinctes 2. Crêtes ou sillons des deux ailes du nez et anneaux fluorescence tache, selon la composition de surface, à travers les stades larvaire et adulte et reste visible jusqu&#39;à une journée après la récupération en utilisant cette méthode. Taches de fond fluorescents sont parfois observées (figures 2F, G), mais pas systématiquement (Figure 1, Figure 2A-E, H, I). Toutes les images ont été prises avec disque rotatif confocale ou, lorsqu&#39;il est apposé, à la microscopie à champ large composé (WF). ent &quot;&gt; <img alt="Figure 1" src="/files/ftp_upload/3362/3362fig1.jpg" /> Figure 1. DiI taches cuticules et les neurones écologiquement exposés. Bête de scène L2. Un grossissement de 630x. Parties d&#39;image mosaïque ont été capturées à l&#39;aide iVision-logiciels Mac (Technologies de BioVision, Exton, PA). Les images ont été jointes à l&#39;aide d&#39;Adobe Photoshop CS3 (Adobe Systems, Inc, San Jose, CA). Barre d&#39;échelle = 10 um. DiI aussi par fluorescence des taches et des amphid phasmide neurones sensoriels de la tête et la queue, respectivement (flèches indiquent certains). <img alt="Figure 2" src="/files/ftp_upload/3362/3362fig2.jpg" /> Figure 2. DiI fluorescence des taches C. cuticules elegans à toutes les étapes du développement post-embryonnaire. Un grossissement de 630x. Barre d&#39;échelle = 10 um. Coloration des animaux de type sauvage dans une L1); B) L2; C) Dauer; D) L3, E) L4 et F) stades adultes. Les arêtes du nez et annulaire sont tachés par fluorescence chez les animaux L1 et Dauer (A, C). DiI taches nervures annulaires chez les animaux L2(B). Annulaire sillons tache dans L3 et L4 animaux (D, E). Les sillons des ailes et des anneaux sont colorées chez les animaux adultes (FH). Alae sont composées de deux, cinq ou trois crêtes (en L1, Dauer, ou les animaux adultes, respectivement) qui courent le long de l&#39;animal (flèches) 16. Annuli créer des arêtes circonférentielles autour de l&#39;animal (pointes de flèches, F et J). La cuticule de l&#39;adulte des animaux mutants affichage modérée défauts organisation cuticulaires (GH). G) Ridges des ailes sont discontinus (WF). H) arêtes du nez surnuméraires sont fondus et ramifiés ou bifurquée (WF). Mutants du gène du collagène présentent ailes et les défauts de l&#39;organisation annulaire (IJ). I) Chez les animaux transgéniques surexprimant pRF4 (rol-6 (su1006)), les crêtes du mensonge ailes à un angle à la longueur de l&#39;animal. J) anneaux au transgéniques surexprimant pRF4 animaux (rol-6 (su1006)) affiche un motif irrégulier. <img alt="Figure 3" src="/files/ftp_upload/3362/3362fig3.jpg" /> Figure 3. Externestructures morphologiques al sont illuminés par coloration DII. grossissement de 630x. Barres d&#39;échelle = 10 um. DiI souligne également d&#39;autres caractéristiques extérieures, y compris A) pour adultes hermaphrodites vulve, B) les rayons pour adultes queue du mâle et de ventilateur, et C) hermaphrodites queue pointe (fourchue dans ce contexte mutant). <img alt="Figure 4" src="/files/ftp_upload/3362/3362fig4.jpg" /> Cuticule Figure 4. Prend plus de tache avec DiI que les neurones écologiquement exposés. Un grossissement de 630x. Barre d&#39;échelle = 10 um. Après deux heures de coloration, amphid (non représenté) et phasmide neurones sensoriels (flèches) sont suffisamment colorés. En revanche, la cuticule des animaux plus jeunes n&#39;est que partiellement teinté en plaques (tête de flèche). <table border="1"><tbody><tr><td> Laver </td><td> Solution de coloration (+ DII) </td><td> Le temps d&#39;incubation </td><td> Tachés cuticules </td></tr><tr><td> M9 + 0,5% TrIton X-100 </td><td> M9 + 0,5% de Triton X-100 </td><td> 2 heures </td><td> pas </td></tr><tr><td> M9 + 0,5% de Triton X-100 </td><td> M9 + 0,5% de Triton X-100 </td><td> 3 heures </td><td> pas </td></tr><tr><td> M9 + 0,5% de Triton X-100 </td><td> M9 </td><td> 2 heures </td><td> partielle </td></tr><tr><td> M9 + 0,5% de Triton X-100 </td><td> M9 </td><td> 3 heures </td><td> oui </td></tr><tr><td> M9 + 0,5% de Triton X-100 </td><td> H 2 0 </td><td> 2 heures </td><td> partielle </td></tr><tr><td> M9 + 0,5% de Triton X-100 </td><td> H 2 0 </td><td> 3 heures </td><td> oui </td></tr></tbody></table> Tableau 1. Coloration cuticulaire dans des conditions différentes. Différentes solutions d&#39;incubation et les délais ont été testés afin d&#39;optimiser coloration cuticulaires chez les animaux. H 2 0, l&#39;eau distillée stérile. Coloration partielle indique coloration inégale de la cuticule larvaire (figure 4), bien que les taches de la cuticule adulte consistently.

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+actin+filaments+in+patterning+the+Caenorhabditis+elegans+cuticle.">The role of actin filaments in patterning the Caenorhabditis elegans cuticle.</a> Dev. Biol. 184, 373-384 (1997).</li><li>Sapio, M. R., Hilliard, M. A., Cermola, M., Favre, R., Bazzicalupo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zona+Pellucida+domain+containing+proteins,+CUT-1,+CUT-3+and+CUT-5,+play+essential+roles+in+the+development+of+the+larval+alae+in+Caenorhabditis+elegans.">The Zona Pellucida domain containing proteins, CUT-1, CUT-3 and CUT-5, play essential roles in the development of the larval alae in Caenorhabditis elegans.</a> Dev. Biol.. 282, 231-245 (2005).</li><li>Johnstone, I. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+exoskeleton+collagen+COL-19:+an+adult-specific+marker+for+collagen+modification+and+assembly,+and+the+analysis+of+organismal+morphology.">Caenorhabditis elegans exoskeleton collagen COL-19: an adult-specific marker for collagen modification and assembly, and the analysis of organismal morphology.</a> Dev. Dyn. 226, 523-539 (2003).</li><li>McMahon, L., Muriel, J. M., Roberts, B., Quinn, M., Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+sets+of+interacting+collagens+form+functionally+distinct+substructures+within+a+Caenorhabditis+elegans+extracellular+matrix.">Two sets of interacting collagens form functionally distinct substructures within a Caenorhabditis elegans extracellular matrix.</a> Molecular Biology of the Cell. 14, 1366-1378 (2003).</li><li>Link, C. D., Ehrenfels, C. W., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+expression+of+male+copulatory+bursa+surface+markers+in+Caenorhabditis+elegans.">Mutant expression of male copulatory bursa surface markers in Caenorhabditis elegans.</a> Development. 103, 485-495 (1988).</li><li>Tong, Y. G., Burglin, T. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+osm-6,+a+gene+that+affects+sensory+cilium+structure+and+sensory+neuron+function+in+Caenorhabditis+elegans.">Analysis of osm-6, a gene that affects sensory cilium structure and sensory neuron function in Caenorhabditis elegans.</a> Genetics. 148, 187-200 (1998).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li></ol>

Nous n&#39;avons rien à révéler.

La méthode de coloration DiI présenté ici permet d&#39;une façon relativement rapide et pratique pour visualiser la cuticule chez C. elegans. En réorientation et l&#39;optimisation d&#39;une méthode couramment utilisée pour l&#39;image écologiquement exposés neurones sensoriels 15,17, Dii peut être utilisé pour fluorescence tache deux ailes du nez et des structures annulaires (figures 1 et 2), ainsi que la vulve, queue du mâle, et la queue hermaphrodites pic (Figure 3). Nous avons trouvé...

<table border="1"><tbody><tr><td> Réactifs </td><td> Synonymes </td><td> Société </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> DiI </td><td> 1,1 &#39;-dioctadécyl-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine perchlorate </td><td> Biotium, Inc </td><td> 60010 </td><td> Dilution de stock: 20 mg / ml dans le DMF dilution de travail: 30 mg / ml </td></tr><tr><td> DMF </td><td> Diméthylformamide </td><td> Sigma-Aldrich, Inc </td><td> D4551 </td><td></td></tr><tr><td> Triton X-100 </td><td> Octylphénoxypolyéthoxyéthanol </td><td> VWR International, LLC. </td><td> EM-9410 </td><td></td></tr><tr><td> M9 </td><td> 22mm KH 2 PO 4, 42mm Na 2 HPO 4, 86mm de NaCl, 1 mM MgSO 4 </td><td></td><td></td><td></td></tr><tr><td> NGM &lt;/ Td&gt; <td> Milieu de croissance des nématodes </td><td> IPM Scientific, Inc </td><td> 11006-501 </td><td> Peut être préparé en suivant le protocole de gélose NGM 18 </td></tr><tr><td> L&#39;agar-agar </td><td></td><td> EMD Chemicals, Inc </td><td> 1,01614 </td><td> 4% dans de l&#39;eau </td></tr><tr><td> Lévamisole </td><td> Chlorhydrate de lévamisole </td><td> Sigma-Aldrich, Inc </td><td> 31742 </td><td> 100 M - 1 mM lévamisole tel que requis </td></tr><tr><td> Lames de microscope </td><td></td><td> VWR International, LLC. </td><td> 16005-106 </td><td></td></tr><tr><td> Lamelles de microscope </td><td></td><td> VWR International, LLC. </td><td> 16004-302 </td><td></td></tr><tr><td> Champ composé </td><td></td><td> Carl Zeiss, Inc </td><td> A1m </td><td> Utiliser des objectifs pour répondre aux besoins de l&#39;expérience </td></tr><tr><td> TRITC ou autre filtre compatibles </td><td> &amp;nbsp; </td><td> Chroma Technology Corp </td><td> 49005 </td><td> HE - DsRed (TRITC/Cy3) a des ratés ensemble de filtres </td></tr></tbody></table>

visualization of caenorhabditis elegans cuticular structures using the lipophilic vital dye dii

La cuticule de C. elegans est une structure hautement résistante qui entoure l&#39;extérieur de 1-4 animaux. La cuticule protège non seulement l&#39;animal de l&#39;environnement, mais détermine aussi la forme du corps et joue un rôle dans la motilité 4-6. Plusieurs couches sécrétées par les cellules épidermiques comprennent la cuticule, y compris une couche lipidique externe 7. Crêtes circonférentielle dans la cuticule appelé pattern anneaux de la longueur de l&#39;animal et sont présents pendant toutes les étapes du développement 8. Alae sont des crêtes longitudinales qui sont présents lors des étapes spécifiques du développement, y compris L1, Dauer, et les stades adultes 2,9. Des mutations dans les gènes qui affectent l&#39;organisation du collagène cuticulaire peut modifier la structure et la morphologie cuticulaires corps animal 5,6,10,11. Alors que l&#39;imagerie par microscopie cuticulaires composé avec l&#39;optique DIC est possible, les méthodes actuelles qui mettent en évidence les structures cuticulaires comprennent fluorescentescent l&#39;expression du transgène 12, 13 coloration des anticorps, et la microscopie électronique 1. Étiqueté de germe de blé agglutinine (WGA) a également été utilisé pour visualiser les glycoprotéines cuticulaires, mais il est limité dans la résolution plus fine des structures cuticulaires 14. Coloration de la surface de la cuticule en utilisant un colorant fluorescent a été observée, mais jamais caractérisés en détail 15. Nous présentons une méthode pour visualiser en direct la cuticule C. elegans utilisant le colorant rouge fluorescent lipophile DII (1,1 &#39;-dioctadécyl-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine perchlorate), qui est couramment utilisé en C. elegans pour visualiser les neurones écologiquement exposés. Ce protocole optimisé pour la coloration de Dii est une méthode simple et robuste pour la visualisation haute résolution fluorescentes d&#39;anneaux, du nez, vulve, queue du mâle, et la queue hermaphrodites pic en C. elegans.

Watch this Scientific Journal Video about Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI at JoVE.com

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Nous présentons une méthode pour visualiser les cuticules en direct<em&gt; C. elegans</em&gt; En utilisant les colorants lipophiles rouge fluorescent DII (1,1 &#39;-dioctadécyl-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine perchlorate), qui est couramment utilisé dans<em&gt; C. elegans</em&gt; Pour visualiser les neurones écologiquement exposés. Avec ce protocole optimisé, les structures des ailes du nez et annulaire cuticulaires sont colorés par DII et observées par microscopie composé.

Visualisation des Caenorhabditis elegans</emOuvrages cuticules> Utilisation du colorant lipophile Vital, Dii

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

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Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

17.0K vues.  Texas A&M University System Health Science Center, College of Medicine. Nous présentons une méthode pour visualiser les cuticules en direct C. elegans En utilisant les colorants lipophiles rouge fluorescent DII (1,1 '-dioctadécyl-3, 3,3', 3'-tetramethylindocarbocyanine perchlorate), qui est couramment utilisé dans C. elegans Pour visualiser les neurones écologiquement exposés. Avec ce protocole optimisé, les structures des ailes du nez et annulaire cuticulaires sont colorés par DII et observé...

Vidéo: Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Mesure Caenorhabditis elegans</emDurée de vie> à 96 plaques de microtitration bien

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

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Biologie

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

De base<em&gt; Caenorhabditis elegans</em&gt; Méthodes: Synchronisation et d&#39;observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

la culture de<em&gt; Caenorhabditis elegans</em&gt; En trois dimensions dans le laboratoire

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1&#8217;-Dioctadecy-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl&#8217;s blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

La perfusion DiI comme méthode de visualisation vasculaire dans<em&gt; Ambystoma mexicanum</em

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Approches quantitatives pour l'étude des structures cellulaires et de la morphologie d'organite à Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

Purification et analyse de Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

Utilisation de Caenorhabditis elegans pour dépister les interactions tissulaires spécifiques des chaperons

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

Automatisation de la quantification agrégée chez Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCR-based applications.

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Presented here is a description of the straightforward and relatively rapid isolation of Caenorhabditis elegans genomic DNA from one or a few animals using a commercially available tissue kit. The resulting gDNA preparation is a suitable template for PCR.

Extraction à petite échelle de l’ADN génomique de Caenorhabditis elegans

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and ...

Visualisation des Caenorhabditis elegans Utilisation du colorant lipophile Vital, Dii

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Mesure Caenorhabditis elegans à 96 plaques de microtitration bien

De base

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

la culture de

La perfusion DiI comme méthode de visualisation vasculaire dans

Approches quantitatives pour l'étude des structures cellulaires et de la morphologie d'organite à Caenorhabditis elegans

Purification et analyse de Caenorhabditis elegans Extracellular Vesicles

Utilisation de Caenorhabditis elegans pour dépister les interactions tissulaires spécifiques des chaperons

Automatisation de la quantification agrégée chez Caenorhabditis elegans

Extraction à petite échelle de l’ADN génomique de Caenorhabditis elegans