Vorremmo ringraziare il dottor Antonio Miranda-Vizuete per il suo prezioso aiuto. Ci piace anche esprimere la nostra gratitudine a Salma Boulayoune e Isabel Caballero per una valida assistenza tecnica. Questo lavoro è stato finanziato da una sovvenzione della Junta de Andalucía (BIO-272). Questa ricerca è stata effettuata in accordo con le leggi vigenti che disciplinano la sperimentazione genetica in Europa.

1: test di evitamento osmotica. <ol><li> Circa 16-24 ore prima del test, raccogliere L4 animali stadio larvale di ciascun genotipo per un piatto fresco NGM contenente OP50 E. coli che andincubate a 20 ° C. Il giorno dopo inizia l&#39;esperimento con i giovani adulti. </li><li> Si consiglia di effettuare il test &quot;alla cieca&quot;. Le piastre con ogni sforzo per essere analizzati devono essere rietichettate da uno sperimentatore secondo, eseguendo il test con le piastre rietichettati che sarebbe stato smascherato quando l&#39;esperimento è terminato. </li><li> Il giorno del test, preparare una soluzione 4M stock di fruttosio con 1% di soluzione di Rosso Congo e sciogliere completamente a temperatura ambiente. Si consiglia di verificare la soluzione prima di ogni test in quanto il colorante potrebbe precipitare con il tempo. </li><li> Anello anulare (1 cm di diametro) sulla piastra NGM è delineata al centro del terreno solido, con 15 microlitri della soluzione rosso fruttosio 4M. Lasciate che la soluzione di fruttosio penetrare nel agar, questo avviene di solito tra i 2 ei 5 minuti. </li><li> Luogo singoli animali adulti giovani di ogni ceppo all&#39;interno della circonvallazione e seguire nei prossimi 10 minuti per determinare la risposta alla barriera osmotica. Animali evitando l&#39;anello più di sei volte di fila sono classificati come normali, quelli in uscita l&#39;anello in meno di sei tentativi sono considerati difettosi nella sensibilità osmotica. Nota: il ceppo di controllo deve essere utilizzato in ogni test. Animali adulti N2 giovani vengono utilizzati come controlli positivi in ​​quanto decisamente evitare la barriera anello. Gli animali che sono stati precedentemente fame, passato attraverso Dauer larve o sono provenienti da piatti che sono troppo secche non deve essere utilizzato. All&#39;inizio, animali di controllo devono essere valutati in duplice copia, a confermare che le piastre di analisi e la soluzione siano corretti. </li></ol> 2: Generazione di vermi knockdown alimentando RNAi. <ol><li> RNAi piastre: piastre NGM alimentazione RNAi contiene per litro: 17 g di agar, 2,5 g di peptone, 3,0 g NaCl, 1 ml di 5 mg di colesterolo ml -1; riempire il pallone a 1 litro con H 2 O e autoclave. Dopo agar si raffredda fino a circa 65 ° C, aggiungere 25 ml di 1 M KPO 4, pH 6.0, 1 ml di 1 M CaCl 2, 1 ml di 1M MgSO 4, carbenicillina 0,5 ml (50 mg ml -1) e 1 ml 1M IPTG. Se siete pronti a partire con NGM solide, si fondono terreno solido da microonde e successivamente posto NGM liquido in panchina per raffreddare e poi aggiungere KPO 4, pH 6.0, CaCl 2, MgSO 4, carbenicillina e IPTG come indicato in precedenza. Aggiungere 10 ml di NGM in ogni piatto 60 millimetri Petri. Lasciare raffreddare e capovolgere le piastre mantenimento delle stesse in una notte prima dell&#39;uso RT. Le piastre possono essere conservate in un sacchetto di plastica a 4 ° C per circa 4-5 giorni. </li><li> Preparazione batterica e induzione: Isolare le colonie di E.coli HT115 (DE3) ceppo, trasformato con L4440 vettore contenente un frammento corrispondente al gene bersaglio, su Luria-Bertani (LB) piastre di agar (17 agar g, 10 g di triptone, 5 g estratto di lievito e 10 g di NaCl per litro) contenente ampicillina (50 mg / ml) e tetraciclina (15 mg / ml). Scegli una colonia di batteri e inoculare in LB con 50 mg / ml ampicillina, e destinata alla produzione di 6 8 ore con agitazione a 37 ° C; seme una goccia di questa cultura sulle piastre NGM preparato e asciugare i piatti accuratamente prima di essere incubazione overnight ( 12 24 h) a temperatura ambiente per permettere ai batteri di crescere e di iniziare a induzione. L&#39;incubazione era al buio perché tetracicline è sensibile alla luce e può influenzare la variabilità dei saggi RNAi tra i piatti. E &#39;necessario utilizzare un positivo e un controllo negativo per esperimenti di alimentazione RNAi. Il controllo positivo è E.coli HT115 cellule trasformate con il vettore L4440 contenente la sequenza del gene unc-22. L&#39;atterramento di unc-22 gene produce un &quot;fenotipo spasmi&quot;. Il controllo negativo è E.coli HT115 cellule trasformate con vuoto vettore L4440. </li><li> Gestione verme e punteggio: Il primo giorno, luogo L4 vermi palco da una piastra NGM seminato con OP50 su piastre NGM senza batteri e incubare a 20 ° C per 12 ore (a digiuno). Il giorno dopo, il trasferimento di giovani adulti ermafroditi digiuno su un piatto seedded con i batteri che esprimono il gene bersaglio specifico RNAi. Lascia 40-48 ore a 20 ° C per generare progenie F1. Poi inserire un paio di vermi adulti F1 su un altro piatto seminato con i batteri stessi. Dopo 24-48 ore, selezionare e isolare dalla progenie F2, i giovani adulti (completamente formato vulva sporgente con le uova pochi) e punteggio per fenotipi. Nota: l&#39;induzione RNAi nei neuroni hanno alcune limitazioni a causa delle proprietà refrattario del sistema nervoso C. elegans a RNAi.. Per superare i problemi di questa inefficienza nel neurone si raccomanda di utilizzare il rif-3 ceppo, uno sfondo ipersensibili agli effetti di RNAi nei neuroni 10. Nei nostri esperimenti non differences sono stati trovati tra Bristol N2 e rif-3 ceppi per i geni e il fenotipo analizzati. </li></ol> 3: I ceppi utilizzati. Il C. elegans ceppi utilizzati in questo lavoro sono riportati nella tabella 1. Ceppi mutanti erano fuori incrociata contro N2 selvaggio - Tipo di almeno quattro volte per rimuovere i mutazioni casuali che avrebbero potuto essere generati durante il protocollo di mutagenesi. . OP50 E coli ceppo è stato suppliied da Caenorhabditis Genetic Center, University of Minnesota, USA E.coli HT115 (DE3) con il plasmide pL4440 portando NRX-1 (JA: C29A12.5). NLG e-1 (JA: C40C9.5 frammenti di geni) sono stati forniti dal Dott. Peter Askjaer, Centro Andaluz de Biologia del Desarrollo (Abd), CSIC Universidad Pablo Olavide, Siviglia, Spagna. 4: I risultati rappresentativi. Risultati illustrativo sono rappresentati nelle figure 1 e 2. Dieci animali di ogni ceppo e un minimo di tre esperimenti replica sono state effettuate. <img alt="Figura 1" src="/files/ftp_upload/1616/1616Fig1.jpg"/> Figura 1. Esperimenti di comportamento di evitamento osmotica. Ceppi di controllo e NRX-1 (ok1649 e tm1961) mutantworms V carenti rispondere invertendo indietro quando incontrano una barriera osmotica (4M fruttosio). NLG-1 (ok259 e tm474) X mutanti non riescono a rilevare questa barriera. Doppi mutanti carenti di NLG-1 e NRX-1, ceppi CRR21 (ok1649; ok259) VX e CRR23 (tm1961; ok259) VX recuperato il fenotipo wild type. Il simbolo * indica differenze significative (p ≤ 0,001) da t-student test, nella risposta di ogni sforzo per la N2 Bristol wild-type da t-studente di prova <img alt="Figura 2" src="/files/ftp_upload/1616/1616Fig2.jpg"/> Figura 2. Gli esperimenti con i vermi knockdown alimentando RNAi. E.coli HT115 (DE3) trasformato con vuoti vettore pL4440 o contenente un frammento corrispondente ai geni bersaglio NRX-1 o NLG-1 sono stati utilizzati per ceppi di vite senza fine alimentato diverse. Il simbolo * indica differenze significative (p ≤ 0,001) da t-student test, nella risposta del ceppo N2 alimentato con i batteri portando la pL4440 vettore con un frammento intese a promuovere l&#39;NLG-1 gene in confronto con il ceppo N2 alimentato con vuoto pL4440 vettore .

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	<li>Sudhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 455 (7215), 903-903 (2008).</li><li>Fabrichny, I. P., Leone, P., Sulzenbacher, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuron. 56 (6), 979-979 (2007).</li><li>Garber, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Science. 317 (5835), 190-190 (2007).</li><li>Wang, K., Zhang, H., Ma, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 459 (7246), 528-528 (2009).</li><li>Ward, S., Thomson, N., White, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Journal of comparative neurology. 160 (3), 313-313 (1975).</li><li>Bargmann, C. I., Thomas, J. H., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+Spring+Harbor+symposia+on+quantitative+biology.">Cold Spring Harbor symposia on quantitative biology.</a> 55, 529-529 (1990).</li><li>White, J. G., Southgate, E., Thomsom, J. N., 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=.">.</a> Philos. Trans. R. Soc. Lond. B Biol. Sci. 314, 1-1 (1986).</li><li>Culotti, J. G., Russell, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genetics. 90 (2), 243-243 (1978).</li><li>Fire, A., Xu, S., Montgomery, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 391 (6669), 806-806 (1998).</li><li>Timmons, L., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 395 (6705), 854-854 (1998).</li><li>Simmer, F., Tijsterman, M., Parrish, S., Koushika, S. P., Nonet, M. L., Fire, A., Ahringer, J., Plasterk, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Curr Biol. 12, 1317-1317 (2002).</li><li>Missler, M., Zhang, W., Rohlmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 423 (6943), 939-939 (2003).</li><li>Varoqueaux, F., Aramuni, G., Rawson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuron. 51 (6), 741-741 (2006).</li><li>Graf, E. R., Zhang, X., Jin, S. X., Scheiffele, P., Fan, J., Choih, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell. 119 (7), 1013-1013 (2004).</li><li>Scheiffele, P., Fan, J., Choih, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell. 101 (6), 657-657 (2000).</li><li>Jamain, S., Quach, H., Betancur, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature genetics. 34 (1), 27-27 (2003).</li><li>Szatmari, P., Paterson, A. D., Zwaigenbaum , L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature genetics. 39 (3), 319-319 (2007).</li></ol>

Neurexins e neuroligins svolgono ruoli fondamentali nella trasmissione sinaptica 11 e la differenziazione delle connessioni sinaptiche 12. Entrambe le molecole sono stati identificati come geni candidati per l&#39;autismo 13,14. In questo video vi mostriamo un metodo semplice che ci permette di studiare l&#39;effetto di geni che influenzano la risposta osmotica evitare in C. elegans. Neuroligin mutanti carenti sono difettosi nel rilevare la forza osm...

Tabella 1. C. elegans ceppi. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Tensione </td><td> Gene </td><td> Allele </td><td> Fonte </td></tr><tr><td> Bristol N2 </td><td> - </td><td> - </td><td> aCGC </td></tr><tr><td> VC228 </td><td> NLG-1 </td><td> ok259 </td><td> CGC </td></tr><tr><td> FX00474 </td><td> NLG-1 </td><td> tm474 </td><td> bNBP-GIAPPONE </td></tr><tr><td> VC1416 </td><td> NRX-1 </td><td> ok1649 </td><td> CGC </td></tr><tr><td> FX1961 </td><td> NRX-1 </td><td> tm1961 </td><td> NBP-GIAPPONE </td></tr><tr><td> NL2099 </td><td> RRF-3 </td><td> pk1436 </td><td> CGC </td></tr><tr><td> CRR21 </td><td> NRX-1; NLG-1 </td><td> ok1649; ok259 </td><td> Questo lavoro </td></tr><tr><td> CRR22 </td><td> NRX-1; NLG-1 </td><td> tm1961; ok259 </td><td> Questo lavoro </td></tr></tbody></table> a. Caenorhabditis Genetic Center, Università del Minnesota, USA. b. Progetto nazionale per la Bioresource sperimentale degli animali &quot;nematode C. elegans&quot;. Tokyo donna Medical University, in Giappone.

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

Neurexins e neuroligins sono molecole di adesione delle cellule presenti nelle sinapsi eccitatorie e inibitorie, e sono tenuti per un corretto funzionamento della rete neuronale 1. Queste proteine ​​si trovano al membrane presinaptico e postsinaptico 2. Studi condotti su topi indicano che neurexins e neurologins hanno un ruolo essenziale nella trasmissione sinaptica 1. Recenti studi hanno dimostrato che le connessioni neuronali alterate nel corso dello sviluppo del sistema nervoso umano potrebbe costituire la base della eziologia di numerosi casi di disturbi dello spettro autistico 3. Caenorhabditis elegans potrebbe essere utilizzato come uno strumento sperimentale per facilitare lo studio del funzionamento dei componenti sinaptica, per la sua semplicità per la sperimentazione di laboratorio, e dato che il suo sistema nervoso e il cablaggio sinaptica è stato pienamente caratterizzato. In C. Elegans NRX-1 e NLG-1 geni ortologhi di uomo e NRXN1 NLGN1 geni che codificano neurexin-alfa-1-1 e neuroligin proteine, rispettivamente. Negli esseri umani e nematodi, l&#39;organizzazione di neurexins e neuroligins è simile rispetto a domini funzionali. Il capo del nematode contiene il amphid, un organo sensoriale del nematode, che media le risposte a stimoli diversi, tra cui la forza osmotica. Il amphid è composto da 12 neuroni bipolari con dendriti sensoriali ciliate e un assone terminale presinaptico 4. Due di questi neuroni, chiamato ASHR e ASHL sono particolarmente importanti in osmotica funzione sensoriale, rilevando idrosolubili repellenti con elevata resistenza osmotica 5. I dendriti di queste due neuroni allungare fino alla punta della bocca e gli assoni estendere all&#39;anello nervose, dove effettuare i collegamenti sinaptici con altri neuroni determinare la risposta comportamentale 6. Per valutare le implicazioni di neurexin e neuroligin in alto evitamento forza osmotica, ci mostrano la diversa risposta di C. elegans mutanti difettosi in NRX-1 e NLG-1 geni, usando un metodo basato su un anello di fruttosio 4M 7. I fenotipi comportamentali sono stati confermati utilizzando specifici cloni RNAi 8. In C. elegans, il dsRNA necessaria per attivare RNAi può essere somministrato per alimentazione 9. La consegna dei dsRNA attraverso il cibo induce l&#39;interferenza di RNAi del gene di interesse permettendo così l&#39;identificazione di componenti genetiche e percorsi di rete.

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

Neurexins e neuroligins sono membrane dei neuroni proteine ​​di adesione che svolgono ruoli essenziali nella differenziazione e la trasmissione sinaptica. Neuroligin mutanti carenti di<em&gt; C. elegans</em&gt; Sono difettosi nel rilevare la forza osmotica, ma quando contengono anche una mutazione nel gene che codifica neurexin, si riprendono il fenotipo wild type.

Evitare osmotica Caenorhabditis elegans: Funzione sinaptica di due geni, ortologhi di Human NRXN1 E NLGN1, Come candidati per l&#39;autismo

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 Views.  Facultad de Ciencias, Universidad de Córdoba. Neurexins e neuroligins sono membrane dei neuroni proteine ​​di adesione che svolgono ruoli essenziali nella differenziazione e la trasmissione sinaptica. Neuroligin mutanti carenti di C. elegans Sono difettosi nel rilevare la forza osmotica, ma quando contengono anche una mutazione nel gene che codifica neurexin, si riprendono il fenotipo wild type.

Video: 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.

Isolamento e In vitro Attivazione di 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 ...

Ricerca

Biologia

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.

Microscopia time-lapse di Embriogenesi All&#39;inizio del 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.

Di base<em&gt; Caenorhabditis elegans</em&gt; Metodi: sincronizzazione e di osservazione

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.

Biochimica e High Throughput valutazione microscopica di massa grassa in<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.

Prostaglandina estrazione e l&#39;analisi in<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 supplementazione alimentare di acidi grassi polinsaturi a<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, ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Misurare il osmotica dell&#39;acqua Permeabilità Coefficiente (P<sub&gt; F</sub&gt;) Di celle sferiche: protoplasti impianto isolato come un esempio

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

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.

Preparazione del campione e la formazione immagine di esosomi da microscopia elettronica di trasmissione

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

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

Modellazione di malattie neurodegenerative associate all'età in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...