Nous tenons à remercier le Dr Antonio Miranda-VIZUETE pour son aide précieuse. Nous aimons aussi à exprimer notre gratitude à Salma Boulayoune et Isabel Caballero pour l&#39;assistance technique précieuse. Ce travail a été financé par une subvention de la Junta de Andalucía (BIO-272). Cette recherche a été effectuée en accord avec les lois actuelles régissant l&#39;expérimentation génétique en Europe.

1: test d&#39;évitement osmotique. <ol><li> A propos de 16-24 heures avant le dosage, ramasser les animaux stade larvaire L4 de chaque génotype à une plaque de NGM frais contenant OP50 E. coli qu&#39;il andincubate à 20 ° C. Le lendemain, commencer l&#39;expérience avec de jeunes adultes. </li><li> Il est recommandé d&#39;effectuer le dosage &quot;aveuglément&quot;. Les plaques avec chaque souche à doser doivent être réétiquetés par un second expérimentateur, réaliser le dosage en utilisant les plaques réétiquetés qui serait démasqué lorsque l&#39;expérience est terminée. </li><li> Le jour du dosage, préparer une solution stock de 4M de fructose avec 1% rouge Congo solution et dissoudre complètement à température ambiante. Nous vous recommandons de vérifier la solution avant chaque essai, depuis le colorant pourrait précipiter avec le temps. </li><li> Anneau (1 cm de diamètre) sur la plaque NGM est décrit sur le centre du milieu solide, avec 15 ul de la solution de fructose 4M rouge. Laissez tremper la solution de fructose dans la gélose, ce qui prend généralement entre 2 et 5 minutes. </li><li> Place individuelle des animaux adultes jeunes de chaque souche dans le ring et suivre au cours des 10 prochaines minutes pour déterminer la réponse à la barrière osmotique. Animaux en évitant l&#39;anneau plus de six fois dans une rangée sont classés comme normaux, ceux de quitter le ring en moins de six tentatives sont considérés comme défectueux dans la sensibilité osmotique. Note: La souche de contrôle doit être utilisé dans chaque essai. Animaux N2 jeunes adultes sont utilisés comme contrôles positifs car ils décisive éviter la barrière anneau. Les animaux qui ont été préalablement affamés, traversé Dauer larves ou viennent de plaques qui sont trop secs ne doivent pas être utilisés. Au début, les animaux de contrôle devraient être évalués en double exemplaire, pour confirmer que les plaques de dosage et la solution sont corrects. </li></ol> 2: Génération des vers knockdown en alimentant l&#39;ARNi. <ol><li> ARNi plaques: les plaques d&#39;alimentation NGM RNAi contient par litre: 17 g d&#39;agar, 2,5 g de peptone, 3,0 g de NaCl, 1 ml de 5 mg de cholestérol ml -1; remplir le flacon à 1 litre avec H 2 O et autoclave. Après agar refroidit à environ 65 ° C, ajouter 25 ml de 1 M KPO 4, pH 6,0, 1 ml de 1 M CaCl 2, 1 ml de 1M MgSO 4, 0,5 ml de carbénicilline (50 mg ml -1) et 1 ml 1M IPTG. Si vous commencez avec le NGM solide préparé, faire fondre le milieu solide en micro-ondes et par la suite lieu NGM liquide sur le banc pour refroidir et ajouter ensuite KPO 4, pH 6,0, CaCl 2, MgSO 4, carbénicilline et IPTG comme indiqué précédemment. Ajouter 10 ml de NGM dans chaque boîte de Pétri de 60 mm. Laisser refroidir et inverser les plaques en les maintenant au jour au lendemain avant d&#39;utiliser RT. Les plaques peuvent être stockées dans un sac en plastique à 4 ° C pendant environ 4-5 jours. </li><li> Préparation bactérienne et de l&#39;induction: Isoler les colonies de E. coli HT115 (DE3) souche, transformé avec L4440 vecteur contenant un fragment correspondant au gène cible, sur Luria-Bertani (LB) gélose (17 g d&#39;agar, 10 g de tryptone, 5 g extrait de levure et 10 g de NaCl par litre) contenant de l&#39;ampicilline (50 ug / ml) et la tétracycline (15 pg / ml). Choisissez une colonie de bactéries et inoculer dans LB avec 50 pg / ml d&#39;ampicilline et cultivé pour 6 8 h avec agitation à 37 ° C; semences une goutte de cette culture sur les plaques préparées NGM et sécher les plaques à fond avant d&#39;être en incubation pendant la nuit ( 12 24 h) à température ambiante pour permettre aux bactéries de croître et de commencer à induction. L&#39;incubation a été dans l&#39;obscurité, car la tétracycline est sensible à la lumière et il peut affecter la variabilité des dosages ARNi dans les assiettes. Il est nécessaire d&#39;utiliser un positif et un contrôle négatif pour des expériences d&#39;alimentation ARNi. Le contrôle positif est E.coli HT115 cellule transformée avec le vecteur contenant la séquence L4440-22 du gène unc. Le knockdown de l&#39;UNC-22 gène produit un «phénotype tics». Le contrôle négatif est E.coli HT115 cellule transformée avec le vecteur L4440 vide. </li><li> La manipulation et la notation Worm: Le premier jour, place L4 vers l&#39;étape d&#39;une plaque de NGM ensemencé avec OP50 sur des plaques NGM sans bactéries et incuber à 20 ° C pendant 12 heures (à jeun). Le lendemain, le transfert des jeunes adultes hermaphrodites jeûné sur une plaque seedded avec les bactéries exprimant l&#39;ARNi spécifiques des gènes cibles. Laisser 40-48 heures à 20 ° C pour générer descendance F1. Ensuite, placez quelques vers pour adultes F1 sur une autre plaque ensemencée avec la même bactérie. Après 24-48 heures, sélectionner et isoler de la descendance F2, les jeunes adultes (entièrement formée vulve saillante avec quelques œufs) et le score pour les phénotypes. Remarque: l&#39;induction d&#39;ARNi dans les neurones ont quelques limitations en raison des propriétés réfractaires de la C elegans système nerveux de l&#39;ARNi.. Pour surmonter les problèmes de cette inefficacité dans le neurone, il est recommandé d&#39;utiliser la souche far-3, un fond d&#39;hypersensibilité aux effets de l&#39;ARNi dans les neurones 10. Dans nos expériences ne differences ont été trouvés entre Bristol N2 et far-3 souches pour les gènes et le phénotype analysé. </li></ol> 3: Les souches utilisées. Le C. elegans souches utilisées dans ce travail sont présentés au tableau 1. Des souches mutantes ont été hors traversé l&#39;encontre N2 sauvages - type au moins quatre fois pour enlever les mutations aléatoires qui auraient pu être générés au cours du protocole de mutagenèse. . OP50 E coli souche a été suppliied par Caenorhabditis génétique Center, University of Minnesota, USA E.coli HT115 (DE3) avec le plasmide portant pL4440 NRX-1 (JA: C29A12.5). Et NLG-1 (JA: C40C9.5 fragments de gènes) ont été fournies par le Dr Peter Askjaer, Centro Andaluz de Biología del Desarrollo (CABD), CSIC Universidad Pablo Olavide, Séville, Espagne. 4: Les résultats représentatifs. Résultats indicatifs sont représentés dans les figures 1 et 2. Dix animaux de chaque souche et un minimum de trois expériences répliques ont été réalisées. <img alt="Figure 1" src="/files/ftp_upload/1616/1616Fig1.jpg"/> Figure 1. Des expériences de comportement d&#39;évitement osmotique. Souches de contrôle et NRX-1 (ok1649 et tm1961) mutantworms V déficiente face en renversant en arrière quand ils rencontrent une barrière osmotique (4M fructose). NLG-1 (ok259 et tm474) X mutants ne parviennent pas à détecter cette barrière. Doubles mutants déficients en NLG-1 et NRX-1, les souches CRR21 (ok1649; ok259) VX et CRR23 (tm1961; ok259) VX récupéré le phénotype de type sauvage. Le * indique des différences significatives (P ≤ 0,001) par le t-test de d&#39;étudiants, dans la réponse de chaque souche de la N2 Bristol type sauvage par le t-test de d&#39;étudiants <img alt="Figure 2" src="/files/ftp_upload/1616/1616Fig2.jpg"/> Figure 2. Les expériences avec les vers knockdown en alimentant l&#39;ARNi. E.coli HT115 (DE3) transformées avec le vecteur vide ou contenant pL4440 un fragment correspondant aux gènes cibles NRX-1 ou NLG-1 ont été utilisés pour nourrir le ver souches différentes. Le * indique des différences significatives (P ≤ 0,001) par le t-test de d&#39;étudiants, dans la réponse de la souche N2 nourris avec des bactéries portant le vecteur pL4440 avec un fragment de ciblage des NLG-1 du gène en comparaison avec la souche N2 nourris avec des vides pL4440 vectoriel .

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Neurexines et effectuer des neuroligines rôle essentiel dans la transmission synaptique et la différenciation des 11 connexions synaptiques 12. Les deux molécules ont été identifiées comme des gènes candidats pour l&#39;autisme 13,14. Dans cette vidéo on montre une méthode simple qui nous permet d&#39;étudier l&#39;effet des gènes affectant la réponse d&#39;évitement osmotique dans C. elegans. Mutants déficients neuroligines sont défec...

Tableau 1. C. souches elegans. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Souche </td><td> Gene </td><td> Allèle </td><td> Source </td></tr><tr><td> Bristol N2 </td><td> - </td><td> - </td><td> CACG </td></tr><tr><td> VC228 </td><td> NLG-1 </td><td> ok259 </td><td> CCG </td></tr><tr><td> FX00474 </td><td> NLG-1 </td><td> tm474 </td><td> bNBP-JAPON </td></tr><tr><td> VC1416 </td><td> NRX-1 </td><td> ok1649 </td><td> CCG </td></tr><tr><td> FX1961 </td><td> NRX-1 </td><td> tm1961 </td><td> NBP-JAPON </td></tr><tr><td> NL2099 </td><td> far-3 </td><td> pk1436 </td><td> CCG </td></tr><tr><td> CRR21 </td><td> NRX-1; NLG-1 </td><td> ok1649; ok259 </td><td> Ce travail </td></tr><tr><td> CRR22 </td><td> NRX-1; NLG-1 </td><td> tm1961; ok259 </td><td> Ce travail </td></tr></tbody></table> a. Caenorhabditis génétique Center, University of Minnesota, USA. b. Projet bioressources nationale pour l&#39;expérimentation animale &quot;nématode C. elegans&quot;. Femmes de Tokyo Medical University, Japon.

osmotic avoidance in caenorhabditis elegans: synaptic function of two genes, orthologues of human nrxn1 and nlgn1, as candidates for autism

Neurexines et neuroligines sont des molécules d&#39;adhésion cellulaires présents dans les synapses excitateurs et inhibiteurs, et ils sont requis pour la fonction réseau correct neurone 1. Ces protéines se trouvent dans les membranes présynaptique et postsynaptique 2. Les études chez la souris indiquent que neurexines et neurologins ont un rôle essentiel dans la transmission synaptique 1. Des rapports récents ont montré que modifié connexions neuronales durant le développement du système nerveux humain pourrait constituer la base de l&#39;étiologie de nombreux cas de troubles du spectre autistique 3. Caenorhabditis elegans pourrait être utilisé comme un outil expérimental pour faciliter l&#39;étude du fonctionnement des composants synaptiques, en raison de sa simplicité pour l&#39;expérimentation en laboratoire, et étant donné que son système nerveux et le câblage synaptique a été complètement caractérisés. En C. Elegans NRX-1-1 et NLG gènes sont orthologues pour la santé humaine et de NRXN1 NLGN1 gènes qui codent l&#39;alpha-neurexine-1 et une neuroligine-protéines, respectivement. Chez les humains et les nématodes, l&#39;organisation de neurexines et neuroligines est similaire en ce qui concerne les domaines fonctionnels. La tête du nématode contient les amphid, un organe sensoriel du nématode, qui médiatise des réponses à des stimuli différents, y compris la force osmotique. Le amphid est constitué de 12 neurones sensoriels bipolaires avec des dendrites ciliées et un axone terminaison présynaptique 4. Deux de ces neurones, nommé ASHR et ASHL sont particulièrement importants dans la fonction sensorielle osmotique, la détection soluble dans l&#39;eau répulsifs avec force osmotique élevée 5. Les dendrites de ces deux neurones allonger à l&#39;extrémité de la bouche et les axones s&#39;étendent à l&#39;anneau nerveux, où ils font les connexions synaptiques avec d&#39;autres neurones déterminant la réponse comportementale 6. Pour évaluer les implications de la neurexine et neuroligines de l&#39;évitement à haute résistance osmotique, nous montrons la réponse différente des mutants de C. elegans défectueux dans NRX-1-1 et NLG gènes, en utilisant une méthode basée sur un anneau de fructose 4M 7. Les phénotypes comportementaux ont été confirmés en utilisant des clones spécifiques ARNi 8. Dans C. elegans, l&#39;ARNdb requis pour déclencher l&#39;ARNi peut être administré par une alimentation 9. La livraison des ARNdb induit par l&#39;alimentation de l&#39;interférence ARN interférence du gène d&#39;intérêt permettant ainsi l&#39;identification des composants génétiques et des voies du réseau.

Watch this Scientific Journal Video about Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism at JoVE.com

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexines et neuroligines sont des protéines d&#39;adhésion à la membrane des neurones, qui jouent des rôles essentiels dans la différenciation synaptique et la transmission. Mutants déficients neuroligines des<em&gt; C. elegans</em&gt; Sont défectueux dans la détection de la force osmotique, mais quand ils contiennent également une mutation dans le gène codant la neurexine, ils récupèrent le phénotype de type sauvage.

Évitement osmotique Caenorhabditis elegans: La fonction synaptique de deux gènes, orthologues de l&#39;homme NRXN1 Et NLGN1, Comme candidats pour l&#39;autisme

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network ...

osmotic-avoidance-caenorhabditis-elegans-synaptic-function-two-genes

Research

JoVE Journal

Biology

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

11.7K vues.  Facultad de Ciencias, Universidad de Córdoba. Neurexines et neuroligines sont des protéines d'adhésion à la membrane des neurones, qui jouent des rôles essentiels dans la différenciation synaptique et la transmission. Mutants déficients neuroligines des C. elegans Sont défectueux dans la détection de la force osmotique, mais quand ils contiennent également une mutation dans le gène codant la neurexine, ils récupèrent le phénotype de type sauvage.

Vidéo: Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

L&#39;isolement et la In vitro Activation des Caenorhabditis elegans Sperme

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

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Biologie

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Time-lapse de microscopie du début de l&#39;embryogenèse dans les Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

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 ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

Biochimique et High Throughput évaluation microscopique de la masse grasse dans<em&gt; Caenorhabditis elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Extraction de prostaglandine et de l&#39;analyse en<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

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 ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

La supplémentation alimentaire en acides gras polyinsaturés dans<em&gt; Caenorhabditis elegans</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

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 ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Préparation des échantillons et l’imagerie des Exosomes par microscopie électronique à Transmission

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Mesure des taux de consommation d’oxygène intactes de Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...