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1.
Geroscience ; 46(2): 2281-2293, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-37940787

RESUMO

Finding new interventions that slow ageing and maintain human health is a huge challenge of our time. The nematode Caenorhabditis elegans offers a rapid in vivo method to determine whether a compound extends its 2 to 3-week lifespan. Measuring lifespan is the standard method to monitor ageing, but a compound that extends lifespan will not necessarily maintain health. Here, we describe the automated monitoring of C. elegans movement from early to mid-adulthood as a faster healthspan-based method to measure ageing. Using the WormGazer™ technology, multiple Petri dishes each containing several C. elegans worms are imaged simultaneously and non-invasively by an array of cameras that can be scaled easily. This approach demonstrates that most functional decline in C. elegans occurs during the first week of adulthood. We find 7 days of imaging is sufficient to measure the dose-dependent efficacy of sulfamethoxazole to slow ageing, compared to 40 days required for a parallel lifespan experiment. Understanding any negative consequences of interventions that slow ageing is important. We show that the long-lived mutant age-1(hx546) stays active for longer than the wild type but it moves slower in early adulthood. Thus, continuous analysis of movement can rapidly identify interventions that slow ageing while simultaneously revealing any negative effects on health.


Assuntos
Envelhecimento , Caenorhabditis elegans , Animais , Longevidade
2.
Nutrients ; 14(9)2022 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-35565952

RESUMO

Guarana (Paullinia cupana) is a widely consumed nutraceutical with various health benefits supported by scientific evidence. However, its indirect health impacts through the gut microbiota have not been studied. Caenorhabditis elegans is a useful model to study both the direct and indirect effects of nutraceuticals, as the intimate association of the worm with the metabolites produced by Escherichia coli is a prototypic simplified model of our gut microbiota. We prepared an ethanoic extract of guarana seeds and assessed its antioxidant capacity in vitro, with a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and in vivo, utilizing C. elegans. Additionally, we studied the impact of this extract on C. elegans lifespan, utilizing both viable and non-viable E. coli, and assessed the impact of guarana on E. coli folate production. The extract showed high antioxidant capacity, and it extended worm lifespan. However, the antioxidant and life-extending effects did not correlate in terms of the extract concentration. The extract-induced life extension was also less significant when utilizing dead E. coli, which may indicate that the effects of guarana on the worms work partly through modifications on E. coli metabolism. Following this observation, guarana was found to decrease E. coli folate production, revealing one possible route for its beneficial effects.


Assuntos
Paullinia , Animais , Antioxidantes/farmacologia , Caenorhabditis elegans , Escherichia coli , Ácido Fólico/farmacologia , Longevidade , Paullinia/química , Extratos Vegetais/farmacologia
3.
Methods Mol Biol ; 2502: 373-393, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35412251

RESUMO

C. elegans is a well-characterized and relatively simple model organism, making it attractive for studying nuclear pore complex proteins in cell and developmental biology. C. elegans is transparent and highly amendable to genetic manipulation. Therefore, it is possible to generate fluorescently tagged proteins and combine this with various light microscopy techniques to study protein behavior in space and time. Here, we provide protocols to prepare both fixed and live C. elegans for confocal and light sheet microscopy. This enables the analysis of nuclear pore complex proteins from embryonic stages to the aging adult.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Complexo de Proteínas Formadoras de Poros Nucleares , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Microscopia de Fluorescência/métodos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo
4.
Biology (Basel) ; 10(5)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34069854

RESUMO

In traditional medicine, Jasonia glutinosa (L.) DC or rock tea (RT) has been mainly used to treat digestive and respiratory pathologies but also as an antimicrobial or an antidepressant herbal remedy. An ethanolic extract of RT has been demonstrated to have antioxidant and anti-inflammatory effects, which may be explained by its phytochemical profile, rich in polyphenols and pigments. The aim of this study is to investigate the neuroprotective potential of RT. For this purpose, the ethanolic extract of RT is assayed in Caenorhabditis elegans (C. elegans) as an in vivo model, and through in vitro assays using monoamine oxidase A, tyrosinase and acetylcholinesterase as enzymes. The RT extract reduces juglone-induced oxidative stress in worms and increases the lifespan and prevents paralysis of C. elegans CL4176, a model of Alzheimer's disease; the extract is also able to inhibit enzymes such as acetylcholinesterase, monoamine oxidase A and tyrosinase in vitro. Together these results demonstrate that Jasonia glutinosa is a good candidate with antioxidant and neuroprotective potential for the development of new products with pharmaceutical interests.

5.
PLoS Genet ; 17(3): e1009358, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33661901

RESUMO

The feeling of hunger or satiety results from integration of the sensory nervous system with other physiological and metabolic cues. This regulates food intake, maintains homeostasis and prevents disease. In C. elegans, chemosensory neurons sense food and relay information to the rest of the animal via hormones to control food-related behaviour and physiology. Here we identify a new component of this system, SKN-1B which acts as a central food-responsive node, ultimately controlling satiety and metabolic homeostasis. SKN-1B, an ortholog of mammalian NF-E2 related transcription factors (Nrfs), has previously been implicated with metabolism, respiration and the increased lifespan incurred by dietary restriction. Here we show that SKN-1B acts in two hypothalamus-like ASI neurons to sense food, communicate nutritional status to the organism, and control satiety and exploratory behaviours. This is achieved by SKN-1B modulating endocrine signalling pathways (IIS and TGF-ß), and by promoting a robust mitochondrial network. Our data suggest a food-sensing and satiety role for mammalian Nrf proteins.


Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Comportamento Animal , Caenorhabditis elegans/genética , Modelos Biológicos , Músculos/metabolismo , Fator de Crescimento Transformador beta/metabolismo
6.
Front Aging ; 2: 740582, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35821999

RESUMO

The increase in our molecular understanding of the biology of aging, coupled with a recent surge in investment, has led to the formation of several companies developing pharmaceuticals to slow aging. Research using the tiny nematode worm Caenorhabditis elegans was the first to show that mutations in single genes can extend lifespan, and subsequent research has shown that this model organism is uniquely suited to testing interventions to slow aging. Yet, with a few notable exceptions, C. elegans is not in the standard toolkit of longevity companies. Here we discuss the paths to overcome the barriers to using C. elegans in industrial drug discovery. We address the predictive power of C. elegans for human aging, how C. elegans research can be applied to specific challenges in the typical drug discovery pipeline, and how standardised and quantitative assays will help C. elegans fulfil its potential in the biotech and pharmaceutical industry. We argue that correct application of this model and its knowledge base will significantly accelerate progress to slow human aging.

7.
Genes Nutr ; 15(1): 4, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-32138646

RESUMO

Micronutrients cannot be synthesized by humans and are obtained from three different sources: diet, gut microbiota, and oral supplements. The microbiota generates significant quantities of micronutrients, but the contribution of these compounds to total uptake is unclear. The role of bacteria in the synthesis and uptake of micronutrients and supplements is widely unexplored and may have important implications for human health. The efficacy and safety of several micronutrient supplements, including folic acid, have been questioned due to some evidence of adverse effects on health. The use of the simplified animal-microbe model, Caenorhabditis elegans, and its bacterial food source, Escherichia coli, provides a controllable system to explore the underlying mechanisms by which bacterial metabolism impacts host micronutrient status. These studies have revealed mechanisms by which bacteria may increase the bioavailability of folic acid, B12, and iron. These routes of uptake interact with bacterial metabolism, with the potential to increase bacterial pathogenesis, and thus may be both beneficial and detrimental to host health.

8.
J Med Food ; 23(1): 72-78, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31545123

RESUMO

The causative relationship between oxidative stress and aging remains controversial, but it is a fact that many of the pathologies of age-related diseases are associated with oxidative stress. Phytochemicals may reduce damage from oxidative stress; the intake of these through diet could represent a strategy to lessen their pathological consequences. The popular and widely consumed licorice (Glycyrrhiza glabra) is a rich source of potential antioxidants. The aim of this study was to investigate whether licorice increases the oxidative stress resistance and lifespan of the animal model Caenorhabditis elegans. Licorice roots ethanolic extract showed in vitro antioxidant activity, with an IC50 of 51.17 µg/mL using 2,2-diphenyl-1-picrylhydrazyl (DPPH) as free radical. C. elegans pretreated with licorice showed an increase of survival rate when exposed to the oxidant juglone, being this increase up to ∼33.56%. This pretreated population also showed an increase in lifespan of 14.28% at a concentration of 250 µg/mL. In conclusion, we suggest that licorice has a high antioxidant capability both in vitro and in vivo and that this activity may explain the observed extension of lifespan.


Assuntos
Antioxidantes/farmacologia , Glycyrrhiza/química , Longevidade/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Caenorhabditis elegans , Naftoquinonas
9.
BMC Biol ; 16(1): 125, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30382925

RESUMO

David Weinkove is an associate professor at Durham University, UK, studying host-microbe interactions in the model organism Caenorhabditis elegans. David has been focusing on the way microbes affect the physiology of their hosts, including the process of aging. In this interview, he discusses the questions shaping his research, how they evolved over the years, and his guiding principles for leading a lab.


Assuntos
Envelhecimento , Caenorhabditis elegans/microbiologia , Caenorhabditis elegans/fisiologia , Escherichia coli/fisiologia , Pesquisa/história , Animais , Inglaterra , História do Século XXI
10.
BMC Biol ; 16(1): 67, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29903004

RESUMO

BACKGROUND: To prevent folate deficiencies, many countries supplement various foodstuffs with folic acid. This compound is a synthetic oxidised folate that differs from naturally occurring reduced folates in its metabolism and uptake. Notably, safety reviews of folic acid supplementation have not considered interactions with gut bacteria. Here, we use the Caenorhabditis elegans - Escherichia coli animal- microbe model to examine a possible bacterial route for folic acid uptake. It has been assumed that supplements are taken up directly by the worm, especially because E. coli is unable to take up folates. However, E. coli, like many other bacteria, can transport the folate breakdown product, para-aminobenzoate-glutamate (PABA-glu), via AbgT and use it for bacterial folate synthesis. This pathway may impact host health because inhibition of bacterial folate synthesis increases C. elegans lifespan. RESULTS: Folic acid supplementation was found to rescue a C. elegans developmental folate-deficient mutant; however, a much higher concentration was required compared to folinic acid, a reduced folate. Unlike folinic acid, the effectiveness of folic acid supplementation was dependent on the E. coli gene, abgT, suggesting a bacterial route with PABA-glu uptake by E. coli as a first step. Surprisingly, we found up to 4% PABA-glu in folic acid preparations, including in a commercial supplement. Via breakdown to PABA-glu, folic acid increases E. coli folate synthesis. This pathway restores folate synthesis in a bacterial mutant defective in PABA synthesis, reversing the ability of this mutant to increase C. elegans lifespan. CONCLUSIONS: Folic acid supplementation in C. elegans occurs chiefly indirectly via bacterial uptake of breakdown products via E. coli AbgT, and can impact C. elegans development and longevity. Examining how folic acid supplementation affects bacterial folate synthesis in the human gut may help us to better understand the safety of folic acid supplementation.


Assuntos
Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/microbiologia , Suplementos Nutricionais , Escherichia coli/metabolismo , Ácido Fólico/metabolismo , Interações entre Hospedeiro e Microrganismos , Animais , Deficiência de Ácido Fólico/prevenção & controle , Humanos , Longevidade
11.
Mol Cell ; 70(3): 531-544.e9, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29727621

RESUMO

While the majority of phosphatidylinositol-4, 5-bisphosphate (PI-4, 5-P2) in mammalian cells is generated by the conversion of phosphatidylinositol-4-phosphate (PI-4-P) to PI-4, 5-P2, a small fraction can be made by phosphorylating phosphatidylinositol-5-phosphate (PI-5-P). The physiological relevance of this second pathway is not clear. Here, we show that deletion of the genes encoding the two most active enzymes in this pathway, Pip4k2a and Pip4k2b, in the liver of mice causes a large enrichment in lipid droplets and in autophagic vesicles during fasting. These changes are due to a defect in the clearance of autophagosomes that halts autophagy and reduces the supply of nutrients salvaged through this pathway. Similar defects in autophagy are seen in nutrient-starved Pip4k2a-/-Pip4k2b-/- mouse embryonic fibroblasts and in C. elegans lacking the PI5P4K ortholog. These results suggest that this alternative pathway for PI-4, 5-P2 synthesis evolved, in part, to enhance the ability of multicellular organisms to survive starvation.


Assuntos
Autofagia/fisiologia , Jejum/metabolismo , Metabolismo dos Lipídeos/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Autofagossomos/metabolismo , Caenorhabditis elegans/metabolismo , Linhagem Celular , Fibroblastos/metabolismo , Células HEK293 , Humanos , Fígado/metabolismo , Camundongos , Fosfatos de Fosfatidilinositol/metabolismo , Transdução de Sinais/fisiologia
12.
Subcell Biochem ; 90: 351-371, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30779015

RESUMO

Understanding how the human gut microbiota might influence ageing is challenging. The gut microbiota is a hugely complex ecology of organisms that varies greatly with individuals and time, making age-related changes difficult to measure. However, elderly and younger populations do show differences in gut microbe composition. The key question is whether these differences only reflect age-related changes in host physiology and diet, or if microbes can drive host ageing? Model organisms allow this question to be addressed. Longitudinal analyses in the fruit fly Drosophila melanogaster show that changes in microbial composition precedes intestinal and host ageing, and antibiotic treatment increases lifespan, implicating microbes in accelerating ageing. Antibiotics also extend the lifespan of middle-aged killifish but additional transplantation of gut microbes from young killifish extends lifespan further, suggesting a positive effect of microbes associated with young animals. Microbes from old, but not young, mice induce inflammation when added to germ-free mice suggesting that microbes become more harmful to the host with age. These studies implicate broad classes of bacteria, particularly members of the phylum Proteobacteria, as drivers of ageing in a feed-forward loop with intestinal degradation and inflammation. The nematode Caenorhabditis elegans can be associated with single strains of genetically-tractable bacteria, and this simplified system has revealed specific interventions in bacterial metabolism, such as inhibition of bacterial folate synthesis, that extend animal lifespan. Transferring this understanding to the human microbiota is challenging but promises to reveal how manipulation of the gut microbiota might be a route to maintain health in old age.


Assuntos
Envelhecimento/fisiologia , Microbioma Gastrointestinal/fisiologia , Longevidade/fisiologia , Envelhecimento/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/microbiologia , Drosophila melanogaster/fisiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos , Inflamação/microbiologia , Intestinos/efeitos dos fármacos , Intestinos/microbiologia , Longevidade/efeitos dos fármacos
14.
Cell Rep ; 14(7): 1611-1620, 2016 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-26876180

RESUMO

Folates are cofactors for biosynthetic enzymes in all eukaryotic and prokaryotic cells. Animals cannot synthesize folate and must acquire it from their diet or microbiota. Previously, we showed that inhibiting E. coli folate synthesis increases C. elegans lifespan. Here, we show that restriction or supplementation of C. elegans folate does not influence lifespan. Thus, folate is required in E. coli to shorten worm lifespan. Bacterial proliferation in the intestine has been proposed as a mechanism for the life-shortening influence of E. coli. However, we found no correlation between C. elegans survival and bacterial growth in a screen of 1,000+ E. coli deletion mutants. Nine mutants increased worm lifespan robustly, suggesting specific gene regulation is required for the life-shortening activity of E. coli. Disrupting the biosynthetic folate cycle did not increase lifespan. Thus, folate acts through a growth-independent route in E. coli to accelerate animal aging.


Assuntos
Envelhecimento/metabolismo , Caenorhabditis elegans/metabolismo , Escherichia coli/metabolismo , Ácido Fólico/biossíntese , Interações Hospedeiro-Patógeno , Ácido 4-Aminobenzoico/metabolismo , Envelhecimento/genética , Animais , Antibacterianos/farmacologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/microbiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Regulação da Expressão Gênica , Glutamato Carboxipeptidase II/deficiência , Glutamato Carboxipeptidase II/genética , Leucovorina/farmacologia , Longevidade/genética , Microbiota/fisiologia , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Transdução de Sinais , Sulfametoxazol/farmacologia , Taxa de Sobrevida
15.
EBioMedicine ; 2(8): 968-84, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26425705

RESUMO

Gut microbiota has been implicated as a pivotal contributing factor in diet-related obesity; however, its role in development of disease phenotypes in human genetic obesity such as Prader-Willi syndrome (PWS) remains elusive. In this hospitalized intervention trial with PWS (n = 17) and simple obesity (n = 21) children, a diet rich in non-digestible carbohydrates induced significant weight loss and concomitant structural changes of the gut microbiota together with reduction of serum antigen load and alleviation of inflammation. Co-abundance network analysis of 161 prevalent bacterial draft genomes assembled directly from metagenomic datasets showed relative increase of functional genome groups for acetate production from carbohydrates fermentation. NMR-based metabolomic profiling of urine showed diet-induced overall changes of host metabotypes and identified significantly reduced trimethylamine N-oxide and indoxyl sulfate, host-bacteria co-metabolites known to induce metabolic deteriorations. Specific bacterial genomes that were correlated with urine levels of these detrimental co-metabolites were found to encode enzyme genes for production of their precursors by fermentation of choline or tryptophan in the gut. When transplanted into germ-free mice, the pre-intervention gut microbiota induced higher inflammation and larger adipocytes compared with the post-intervention microbiota from the same volunteer. Our multi-omics-based systems analysis indicates a significant etiological contribution of dysbiotic gut microbiota to both genetic and simple obesity in children, implicating a potentially effective target for alleviation. RESEARCH IN CONTEXT: Poorly managed diet and genetic mutations are the two primary driving forces behind the devastating epidemic of obesity-related diseases. Lack of understanding of the molecular chain of causation between the driving forces and the disease endpoints retards progress in prevention and treatment of the diseases. We found that children genetically obese with Prader-Willi syndrome shared a similar dysbiosis in their gut microbiota with those having diet-related obesity. A diet rich in non-digestible but fermentable carbohydrates significantly promoted beneficial groups of bacteria and reduced toxin-producers, which contributes to the alleviation of metabolic deteriorations in obesity regardless of the primary driving forces.


Assuntos
Carboidratos da Dieta/administração & dosagem , Disbiose/dietoterapia , Disbiose/microbiologia , Microbioma Gastrointestinal , Síndrome de Prader-Willi/dietoterapia , Síndrome de Prader-Willi/microbiologia , Adolescente , Animais , Antígenos de Bactérias/sangue , Criança , Pré-Escolar , Disbiose/sangue , Disbiose/genética , Feminino , Humanos , Masculino , Camundongos , Síndrome de Prader-Willi/sangue , Síndrome de Prader-Willi/genética
17.
Cell ; 153(1): 228-39, 2013 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-23540700

RESUMO

The biguanide drug metformin is widely prescribed to treat type 2 diabetes and metabolic syndrome, but its mode of action remains uncertain. Metformin also increases lifespan in Caenorhabditis elegans cocultured with Escherichia coli. This bacterium exerts complex nutritional and pathogenic effects on its nematode predator/host that impact health and aging. We report that metformin increases lifespan by altering microbial folate and methionine metabolism. Alterations in metformin-induced longevity by mutation of worm methionine synthase (metr-1) and S-adenosylmethionine synthase (sams-1) imply metformin-induced methionine restriction in the host, consistent with action of this drug as a dietary restriction mimetic. Metformin increases or decreases worm lifespan, depending on E. coli strain metformin sensitivity and glucose concentration. In mammals, the intestinal microbiome influences host metabolism, including development of metabolic disease. Thus, metformin-induced alteration of microbial metabolism could contribute to therapeutic efficacy-and also to its side effects, which include folate deficiency and gastrointestinal upset.


Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/microbiologia , Ácido Fólico/metabolismo , Hipoglicemiantes/farmacologia , Longevidade/efeitos dos fármacos , Metformina/farmacologia , Metionina/metabolismo , Adenilato Quinase/metabolismo , Envelhecimento/efeitos dos fármacos , Animais , Biguanidas/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Restrição Calórica , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Escherichia coli/metabolismo , Humanos , Hipoglicemiantes/metabolismo , Metagenoma , Metformina/metabolismo , Fatores de Transcrição/metabolismo
18.
BMC Biol ; 10: 67, 2012 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-22849329

RESUMO

BACKGROUND: Gut microbes influence animal health and thus, are potential targets for interventions that slow aging. Live E. coli provides the nematode worm Caenorhabditis elegans with vital micronutrients, such as folates that cannot be synthesized by animals. However, the microbe also limits C. elegans lifespan. Understanding these interactions may shed light on how intestinal microbes influence mammalian aging. RESULTS: Serendipitously, we isolated an E. coli mutant that slows C. elegans aging. We identified the disrupted gene to be aroD, which is required to synthesize aromatic compounds in the microbe. Adding back aromatic compounds to the media revealed that the increased C. elegans lifespan was caused by decreased availability of para-aminobenzoic acid, a precursor to folate. Consistent with this result, inhibition of folate synthesis by sulfamethoxazole, a sulfonamide, led to a dose-dependent increase in C. elegans lifespan. As expected, these treatments caused a decrease in bacterial and worm folate levels, as measured by mass spectrometry of intact folates. The folate cycle is essential for cellular biosynthesis. However, bacterial proliferation and C. elegans growth and reproduction were unaffected under the conditions that increased lifespan. CONCLUSIONS: In this animal:microbe system, folates are in excess of that required for biosynthesis. This study suggests that microbial folate synthesis is a pharmacologically accessible target to slow animal aging without detrimental effects.


Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/microbiologia , Escherichia coli/crescimento & desenvolvimento , Ácido Fólico/biossíntese , Longevidade/fisiologia , Modelos Biológicos , Ácido 4-Aminobenzoico/farmacologia , Animais , Caenorhabditis elegans/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Genes Bacterianos/genética , Longevidade/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , Mutação/genética , Plasmídeos/metabolismo , Interferência de RNA/efeitos dos fármacos , Sulfametoxazol/farmacologia
19.
Dev Cell ; 15(2): 198-208, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18694560

RESUMO

Spindle positioning is an essential feature of asymmetric cell division. The conserved PAR proteins together with heterotrimeric G proteins control spindle positioning in animal cells, but how these are linked is not known. In C. elegans, PAR protein activity leads to asymmetric spindle placement through cortical asymmetry of Galpha regulators GPR-1/2. Here, we establish that the casein kinase 1 gamma CSNK-1 and a PIP(2) synthesis enzyme (PPK-1) transduce PAR polarity to asymmetric Galpha regulation. PPK-1 is posteriorly enriched in the one-celled embryo through PAR and CSNK-1 activities. Loss of CSNK-1 causes uniformly high PPK-1 levels, high symmetric cortical levels of GPR-1/2 and LIN-5, and increased spindle pulling forces. In contrast, knockdown of ppk-1 leads to low GPR-1/2 levels and decreased spindle forces. Furthermore, loss of CSNK-1 leads to increased levels of PIP(2). We propose that asymmetric generation of PIP(2) by PPK-1 directs the posterior enrichment of GPR-1/2 and LIN-5, leading to posterior spindle displacement.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Caseína Quinase I/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/enzimologia , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Núcleo Celular/enzimologia , Embrião não Mamífero/citologia , Embrião não Mamífero/enzimologia , Modelos Biológicos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Transporte Proteico , Interferência de RNA
20.
Dev Biol ; 313(1): 384-97, 2008 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18037397

RESUMO

Growth cones are dynamic membrane structures that migrate to target tissue by rearranging their cytoskeleton in response to environmental cues. The lipid phosphatidylinositol (4,5) bisphosphate (PIP(2)) resides on the plasma membrane of all eukaryotic cells and is thought to be required for actin cytoskeleton rearrangements. Thus PIP(2) is likely to play a role during neuron development, but this has never been tested in vivo. In this study, we have characterized the PIP(2) synthesizing enzyme Type I PIP kinase (ppk-1) in Caenorhabditis elegans. PPK-1 is strongly expressed in the nervous system, and can localize to the plasma membrane. We show that PPK-1 purified from C. elegans can generate PIP(2)in vitro and that overexpression of the kinase causes an increase in PIP(2) levels in vivo. In developing neurons, PPK-1 overexpression leads to growth cones that become stalled, produce ectopic membrane projections, and branched axons. Once neurons are established, PPK-1 overexpression results in progressive membrane overgrowth and degeneration during adulthood. These data suggest that overexpression of the Type I PIP kinase inhibits growth cone collapse, and that regulation of PIP(2) levels in established neurons may be important to maintain structural integrity and prevent neuronal degeneration.


Assuntos
Axônios/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Cones de Crescimento/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Membrana Celular/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética
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