Gostaríamos de agradecer o Dr. Antonio Miranda-Vizuete para a sua valiosa ajuda. Nós também gostamos de expressar nossa gratidão a Salma Boulayoune e Isabel Caballero de assistência técnica valiosa. Este trabalho foi financiado por uma doação da Junta de Andalucía (BIO-272). Esta pesquisa foi realizada de acordo com as leis que regem a experimentação genética na Europa.

1: ensaio de evitar osmótica. <ol><li> Sobre 16-24 horas antes do ensaio, pick L4 animais estágio larval de cada genótipo a uma placa NGM fresco contendo OP50 E. coli andincubate-lo a 20 ° C. No dia seguinte, iniciar o experimento com adultos jovens. </li><li> Recomenda-se realizar o ensaio &quot;cegamente&quot;. As placas com cada estirpe a ser ensaiadas devem ser rotulados por um experimentador segundo, da realização do ensaio utilizando as placas rotulados que seria desmascarado quando o experimento foi concluída. </li><li> No dia do ensaio, prepare uma solução estoque 4M de frutose com 1% de solução Vermelho Congo e dissolver completamente em temperatura ambiente. Recomendamos que verifique a solução antes de cada ensaio uma vez que o corante poderia precipitar com o tempo. </li><li> Anel anular (1 cm de diâmetro) na placa NGM é esboçada no centro do meio sólido, com 15 mL da solução de vermelho de 4M frutose. Deixe a solução de frutose mergulhar no agar, o que normalmente leva entre 2 e 5 minutos. </li><li> Lugar individual animais adultos jovens de cada linhagem dentro do anel e siga ao longo dos próximos 10 minutos para determinar a resposta à barreira osmótica. Animais evitando o anel de mais de seis vezes em uma fileira são classificados como normais, aqueles sair do ringue em menos de seis tentativas são considerados defeitos de sensibilidade osmótica. Nota: A tensão de controle deve ser usado em cada ensaio. Animais adultos N2 jovens são utilizados como controle positivo uma vez que decisivamente evitar a barreira do anel. Animais que tenham sido previamente fome, passou por Dauer larvas ou são provenientes de placas que são demasiado seca não deve ser usado. No início, os animais de controle devem ser avaliadas em duplicata, para confirmar que as placas de ensaio ea solução estão corretas. </li></ol> 2: Geração de worms knockdown alimentando RNAi. <ol><li> RNAi placas: placas de alimentação NGM RNAi contém por litro: 17 g de ágar, 2,5 g de peptona, 3,0 g NaCl, 1 ml de 5 mg de colesterol -1 ml; encher o cantil de 1 litro com H 2 O e autoclave. Depois de agar esfria a cerca de 65 ° C, adicione 25 ml de 1 M KPO 4, pH 6,0, 1 ml de CaCl 2 1 M, 1 ml de 1M MgSO 4, 0,5 ml carbenicilina (50 mg ml -1) e 1 ml IPTG 1M. Se você está começando com NGM sólido preparado, derreta meio sólido por microondas e, posteriormente, lugar NGM líquido no banco para esfriar e adicione KPO 4, pH 6,0, CaCl 2, MgSO 4, carbenicilina e IPTG como indicado anteriormente. Adicionar 10 ml de NGM em cada placa de Petri 60 mm. Permitir o resfriamento e inverter as placas mantendo-as a RT noite antes de usar. As placas podem ser armazenadas em um saco plástico a 4 ° C por cerca de 4-5 dias. </li><li> Preparação bacteriana e indução: Isolar colônias de E.coli HT115 (DE3), estirpe transformada com L4440 vetor contendo um fragmento correspondente ao gene-alvo, para Luria-Bertani (LB) ágar (17 g de ágar, 10 g triptona, 5 g extrato de levedura e 10 g de NaCl por litro) contendo ampicilina (50 mcg / ml) e tetraciclina (15 mcg / ml). Escolha uma colônia de bactérias e inocular em LB com 50 mg / ml ampicilina, e cresceu para 6 8 h com agitação a 37 ° C; uma gota de semente desta cultura para as placas NGM preparada e secar os pratos cuidadosamente antes de ser incubada overnight ( 12 24 h) à temperatura ambiente para permitir que as bactérias para crescer e começar a indução. A incubação foi no escuro, porque a tetraciclina é sensível à luz e pode afetar a variabilidade dos ensaios RNAi entre placas. É necessário o uso de um positivo e um controle negativo para os experimentos de alimentação RNAi. O controle positivo é E.coli célula HT115 transformada com o vetor contendo a seqüência L4440 unc-22 gene. O knockdown da unc-22 gene produz um &quot;fenótipo espasmos&quot;. O controle negativo é E.coli HT115 célula transformada com o vetor L4440 vazio. </li><li> Manipulação de verme e de pontuação: O primeiro dia, coloque L4 worms palco de uma placa NGM semeados com OP50 em placas NGM sem bactérias e incubar a 20 ° C por 12 horas (em jejum). No dia seguinte, a transferência de jovens adultos hermafroditas jejum em uma placa seedded com as bactérias expressando o gene alvo específicos RNAi. Deixe 40-48 horas a 20 ° C para gerar progênie F1. Em seguida, coloque alguns vermes adultos F1 noutra placa semeada com a mesma bactéria. Após 24-48 horas, selecionar e isolar da progênie F2, adultos jovens (totalmente formado vulva protuberante com poucos ovos) e escore de fenótipos. Nota: a indução de RNAi em neurônios têm algumas limitações por causa das propriedades refratárias do sistema de C elegans nervoso para RNAi.. Para superar os problemas desta ineficiência no neurônio é recomendado o uso da cepa RRF-3, um fundo hipersensíveis aos efeitos da RNAi em neurônios 10. Em nossos experimentos não diferenes foram encontradas entre Bristol e RRF N2-3 cepas para os genes e do fenótipo analisado. </li></ol> 3: As cepas utilizadas. O C. elegans cepas utilizadas neste trabalho são apresentados na Tabela 1. Cepas mutantes estavam cruzados contra N2 selvagens - tipo, pelo menos, quatro vezes para remover indesejados mutações aleatórias que poderia ter sido gerada durante o protocolo de mutagênese. . OP50 E coli cepa foi suppliied por Caenorhabditis Genetic Center, University of Minnesota, EUA E.coli HT115 (DE3) com o plasmídeo carregando pL4440 NRX-1 (JA: C29A12.5). Florins e-1 (JA: C40C9.5 fragmentos de genes) foram fornecidos pelo Dr. Peter Askjaer, Centro Andaluz de Biologia del Desarrollo (&#39;Abd), Universidad Pablo Olavide CSIC, Sevilla, Espanha. 4: Os resultados representativos. Resultados ilustrativos são representados nas Figuras 1 e 2. Dez animais de cada cepa e um mínimo de três experimentos foram realizados réplica. <img alt="Figura 1" src="/files/ftp_upload/1616/1616Fig1.jpg"/> Figura 1. Experimentos de comportamento de evitação osmótica. Estirpes de controlo e NRX-1 (ok1649 e tm1961) mutantworms V deficiente responder, invertendo para trás quando se deparam com uma barreira osmótica (4M frutose). NLG-1 (ok259 e tm474) X mutantes não conseguem detectar essa barreira. Mutantes duplos deficiente em NLG-1 e NRX-1, cepas CRR21 (ok1649; ok259) VX e CRR23 (tm1961; ok259) VX recuperou o fenótipo de tipo selvagem. O * indica diferenças significativas (P ≤ 0,001) pelo teste t-student, na resposta de cada cepa para a N2 Bristol tipo selvagem por t-student <img alt="Figura 2" src="/files/ftp_upload/1616/1616Fig2.jpg"/> Figura 2. Experimentos com worms knockdown alimentando RNAi. E.coli HT115 (DE3) transformadas com o vetor pL4440 vazio ou contendo um fragmento correspondente aos genes alvo NRX-1 ou NLG-1 foram utilizados para cepas de vírus alimentados com diferentes. O * indica diferenças significativas (P ≤ 0,001) pelo teste t-student, na resposta da cepa N2 alimentados com bactérias carregando o vetor pL4440 com um fragmento do gene alvo NLG-1 em comparação com a cepa N2 alimentados com vector pL4440 vazia .

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Neurexinas e neuroliginas desempenhar papéis essenciais na transmissão sináptica 11 e diferenciação das conexões sinápticas 12. Ambas as moléculas têm sido identificadas como genes candidatos para o autismo 13,14. Neste vídeo mostramos um método simples que nos permite estudar o efeito de genes que afetam a resposta de esquiva osmótica em C. elegans. Neuroligina mutantes deficientes são defeituosos na detecção de resistência osmótica...

Tabela 1. C. cepas elegans. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Tensão </td><td> Gene </td><td> Alelo </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 n-JAPAN </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-JAPAN </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> Este trabalho </td></tr><tr><td> CRR22 </td><td> NRX-1; NLG-1 </td><td> tm1961; ok259 </td><td> Este trabalho </td></tr></tbody></table> a. Caenorhabditis Genetic Center, University of Minnesota, EUA. b. Projeto Nacional para a Bioresource Experimental Animal &quot;Nematóides C. elegans&quot;. Médica da Mulher de Tóquio University, no Japão.

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

Neurexinas e neuroliginas são moléculas de adesão de células presentes nas sinapses excitatórios e inibitórios, e eles são necessários para o funcionamento correto da rede neurônio 1. Estas proteínas são encontradas nas membranas pré-sinápticas e pós-sinápticos 2. Estudos em ratos indicam que neurexinas e neurologins têm um papel essencial na transmissão sináptica 1. Relatórios recentes têm mostrado que alterou conexões neuronais durante o desenvolvimento do sistema nervoso humano pode constituir a base da etiologia da numerosos casos de distúrbios do espectro do autismo 3. Caenorhabditis elegans pode ser usado como uma ferramenta experimental para facilitar o estudo do funcionamento dos componentes sináptica, devido à sua simplicidade para a experimentação de laboratório, e dado que seu sistema nervoso e fiação sináptica tem sido completamente caracterizado. Em C. Elegans NRX-1 e NLG-1 genes ortólogos são para humanos NRXN1 e NLGN1 genes que codificam a alfa-neurexin-1 e neuroligina-1 proteínas, respectivamente. Em humanos e nematóides, a organização de neurexinas e neuroliginas é semelhante no que respeita aos domínios funcionais. O chefe do nematóide contém o amphid, um órgão sensorial do nemátodo, que medeia as respostas a diferentes estímulos, incluindo a força osmótica. O amphid é feito de 12 neurônios sensoriais bipolares com dendritos e um axônio ciliadas terminal pré-sináptico 4. Dois desses neurônios, chamado ASHR e ASHL são particularmente importantes em função sensorial osmótica, detectando solúvel em água repelentes com força osmótica elevada 5. Os dendritos destes dois neurônios alongar até a ponta da boca e os axônios estendem ao anel nervoso, onde fazem conexões sinápticas com outros neurônios determinar a resposta comportamental 6. Para avaliar as implicações da neurexin e neuroligina em evitar alta resistência osmótica, mostramos a resposta diferente de mutantes C. elegans com defeito no NRX-1 e 1-NLG genes, usando um método baseado em um anel de frutose 4M 7. Os fenótipos comportamentais foram confirmados utilizando clones específicos RNAi 8. Em C. elegans, o dsRNA necessário para acionar RNAi pode ser administrado através da alimentação 9. A entrega de dsRNA através dos alimentos induz a interferência RNAi do gene de interesse permitindo assim a identificação dos componentes genéticos e caminhos de rede.

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

Neurexinas e neuroliginas são proteínas da membrana do neurônio-adesão que desempenham papéis essenciais na diferenciação e transmissão sináptica. Mutantes neuroligina deficiente de<em&gt; C. elegans</em&gt; Estão com defeito na detecção de resistência osmótica, mas quando eles contêm também uma mutação no gene que codifica neurexin, eles recuperar o fenótipo do tipo selvagem.

Evitar osmótica em Caenorhabditis elegans: Função Synaptic de dois genes, Orthologues dos Direitos Humanos NRXN1 E NLGN1, Como candidatos para o 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 visualizações.  Facultad de Ciencias, Universidad de Córdoba. Neurexinas e neuroliginas são proteínas da membrana do neurônio-adesão que desempenham papéis essenciais na diferenciação e transmissão sináptica. Mutantes neuroligina deficiente de C. elegans Estão com defeito na detecção de resistência osmótica, mas quando eles contêm também uma mutação no gene que codifica neurexin, eles recuperar o fenótipo do tipo selvagem.

Vídeo: 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 A ativação do 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 ...

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.

Lapso de tempo Microscopia de embriogênese inicial em 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.

Básico<em&gt; Caenorhabditis elegans</em&gt; Métodos: Sincronização e observação

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.

Bioquímica e Alto Rendimento Avaliação microscópica de massa gorda em<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.

Extração de prostaglandina e Análise em<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.

A suplementação dietética de ácidos graxos poliinsaturados em<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.

Medindo o Coeficiente de Permeabilidade osmótica de água (P<sub&gt; F</sub&gt;) De células esféricas: protoplastos planta isolada como um exemplo

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.

Preparação da amostra e a imagem latente de Exosomes por microscopia eletrônica de transmissão

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.

Modelagem de Doenças Neurodegenerativas Associadas à Idade em Caenorhabditis elegans

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