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1.
Nat Commun ; 12(1): 4898, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385431

RESUMO

Hedgehog (Hh) signaling is essential during development and in organ physiology. In the canonical pathway, Hh binding to Patched (PTCH) relieves the inhibition of Smoothened (SMO). Yet, PTCH may also perform SMO-independent functions. While the PTCH homolog PTC-3 is essential in C. elegans, worms lack SMO, providing an excellent model to probe non-canonical PTCH function. Here, we show that PTC-3 is a cholesterol transporter. ptc-3(RNAi) leads to accumulation of intracellular cholesterol and defects in ER structure and lipid droplet formation. These phenotypes were accompanied by a reduction in acyl chain (FA) length and desaturation. ptc-3(RNAi)-induced lethality, fat content and ER morphology defects were rescued by reducing dietary cholesterol. We provide evidence that cholesterol accumulation modulates the function of nuclear hormone receptors such as of the PPARα homolog NHR-49 and NHR-181, and affects FA composition. Our data uncover a role for PTCH in organelle structure maintenance and fat metabolism.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Colesterol/metabolismo , Homeostase/genética , Metabolismo dos Lipídeos/genética , Receptor Patched-1/genética , Animais , Western Blotting , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestrutura , Proteínas de Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica , Microscopia Eletrônica de Transmissão , Receptor Patched-1/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa
2.
Nat Commun ; 12(1): 4912, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389721

RESUMO

Polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) hybrid systems typically use complex protein-protein interactions to facilitate direct transfer of intermediates between these multimodular megaenzymes. In the canal-associated neurons (CANs) of Caenorhabditis elegans, PKS-1 and NRPS-1 produce the nemamides, the only known hybrid polyketide-nonribosomal peptides biosynthesized by animals, through a poorly understood mechanism. Here, we use genome editing and mass spectrometry to map the roles of individual PKS-1 and NRPS-1 enzymatic domains in nemamide biosynthesis. Furthermore, we show that nemamide biosynthesis requires at least five additional enzymes expressed in the CANs that are encoded by genes distributed across the worm genome. We identify the roles of these enzymes and discover a mechanism for trafficking intermediates between a PKS and an NRPS. Specifically, the enzyme PKAL-1 activates an advanced polyketide intermediate as an adenylate and directly loads it onto a carrier protein in NRPS-1. This trafficking mechanism provides a means by which a PKS-NRPS system can expand its biosynthetic potential and is likely important for the regulation of nemamide biosynthesis.


Assuntos
Vias Biossintéticas/genética , Proteínas de Caenorhabditis elegans/genética , Peptídeo Sintases/genética , Peptídeos/metabolismo , Policetídeo Sintases/genética , Policetídeos/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Cromatografia Líquida/métodos , Enzimas/genética , Enzimas/metabolismo , Expressão Gênica , Espectrometria de Massas/métodos , Estrutura Molecular , Mutação , Neurônios/metabolismo , Peptídeo Sintases/metabolismo , Peptídeos/química , Policetídeo Sintases/metabolismo , Policetídeos/química
3.
BMC Genomics ; 22(1): 586, 2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344317

RESUMO

BACKGROUND: Circular RNAs (circRNAs) play diverse roles in different biological and physiological environments and are always expressed in a tissue-specific manner. Especially, circRNAs are enriched in the brain tissues of almost all investigated species, including humans, mice, Drosophila, etc. Although circRNAs were found in C. elegans, the neuron-specific circRNA data is not available yet. Exon-skipping is found to be correlated to circRNA formation, but the mechanisms that link them together are not clear. RESULTS: Here, through large-scale neuron isolation from the first larval (L1) stage of C. elegans followed by RNA sequencing with ribosomal RNA depletion, the neuronal circRNA data in C. elegans were obtained. Hundreds of novel circRNAs were annotated with high accuracy. circRNAs were highly expressed in the neurons of C. elegans and were positively correlated to the levels of their cognate linear mRNAs. Disruption of reverse complementary match (RCM) sequences in circRNA flanking introns effectively abolished circRNA formation. In the zip-2 gene, deletion of either upstream or downstream RCMs almost eliminated the production of both the circular and the skipped transcript. Interestingly, the 13-nt RCM in zip-2 is highly conserved across five nematode ortholog genes, which show conserved exon-skipping patterns. Finally, through in vivo one-by-one mutagenesis of all the splicing sites and branch points required for exon-skipping and back-splicing in the zip-2 gene, I showed that back-splicing still happened without exon-skipping, and vice versa. CONCLUSIONS: Through protocol optimization, total RNA obtained from sorted neurons is increased to hundreds of nanograms. circRNAs highly expressed in the neurons of C. elegans are more likely to be derived from genes also highly expressed in the neurons. RCMs are abundant in circRNA flanking introns, and RCM-deletion is an efficient way to knockout circRNAs. More importantly, these RCMs are not only required for back-splicing but also promote the skipping of exon(s) to be circularized. Finally, RCMs in circRNA flanking introns can directly promote both exon-skipping and back-splicing, providing a new explanation for the correlation between them.


Assuntos
Caenorhabditis elegans , RNA , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Éxons , Camundongos , RNA/metabolismo , Splicing de RNA , RNA Circular
4.
Nucleic Acids Res ; 49(15): 8836-8865, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34329465

RESUMO

The Caenorhabditis elegans genome encodes nineteen functional Argonaute proteins that use 22G-RNAs, 26G-RNAs, miRNAs or piRNAs to regulate target transcripts. Only one Argonaute is essential under normal laboratory conditions: CSR-1. While CSR-1 has been studied widely, nearly all studies have overlooked the fact that the csr-1 locus encodes two isoforms. These isoforms differ by an additional 163 amino acids present in the N-terminus of CSR-1a. Using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG into the long (CSR-1a) and short (CSR-1b) isoforms, we found that CSR-1a is expressed during spermatogenesis and in several somatic tissues, including the intestine. CSR-1b is expressed constitutively in the germline. small RNA sequencing of CSR-1 complexes shows that they interact with partly overlapping sets of 22G-RNAs. Phenotypic analyses reveal that the essential functions of csr-1 described in the literature coincide with CSR-1b, while CSR-1a plays tissue specific functions. During spermatogenesis, CSR-1a integrates into an sRNA regulatory network including ALG-3, ALG-4 and WAGO-10 that is necessary for fertility at 25°C. In the intestine, CSR-1a silences immunity and pathogen-responsive genes, and its loss results in improved survival from the pathogen Pseudomonas aeruginosa. Our findings functionally distinguish the CSR-1 isoforms and highlight the importance of studying each AGO isoform independently.


Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/genética , Espermatogênese/genética , Alelos , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Feminino , Fertilidade , Expressão Gênica , Masculino , Mutação , Oócitos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Pequeno RNA não Traduzido/metabolismo , Espermatozoides/metabolismo
5.
Nature ; 596(7871): 281-284, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34290409

RESUMO

The mTOR complex 1 (mTORC1) controls cell growth in response to amino acid levels1. Here we report SAR1B as a leucine sensor that regulates mTORC1 signalling in response to intracellular levels of leucine. Under conditions of leucine deficiency, SAR1B inhibits mTORC1 by physically targeting its activator GATOR2. In conditions of leucine sufficiency, SAR1B binds to leucine, undergoes a conformational change and dissociates from GATOR2, which results in mTORC1 activation. SAR1B-GATOR2-mTORC1 signalling is conserved in nematodes and has a role in the regulation of lifespan. Bioinformatic analysis reveals that SAR1B deficiency correlates with the development of lung cancer. The silencing of SAR1B and its paralogue SAR1A promotes mTORC1-dependent growth of lung tumours in mice. Our results reveal that SAR1B is a conserved leucine sensor that has a potential role in the development of lung cancer.


Assuntos
Leucina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Transdução de Sinais , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sequência Conservada , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células HEK293 , Humanos , Leucina/deficiência , Longevidade/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/agonistas , Camundongos , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/deficiência , Proteínas Monoméricas de Ligação ao GTP/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Nature ; 596(7871): 285-290, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34321666

RESUMO

Ageing is driven by a loss of cellular integrity1. Given the major role of ubiquitin modifications in cell function2, here we assess the link between ubiquitination and ageing by quantifying whole-proteome ubiquitin signatures in Caenorhabditis elegans. We find a remodelling of the ubiquitinated proteome during ageing, which is ameliorated by longevity paradigms such as dietary restriction and reduced insulin signalling. Notably, ageing causes a global loss of ubiquitination that is triggered by increased deubiquitinase activity. Because ubiquitination can tag proteins for recognition by the proteasome3, a fundamental question is whether deficits in targeted degradation influence longevity. By integrating data from worms with a defective proteasome, we identify proteasomal targets that accumulate with age owing to decreased ubiquitination and subsequent degradation. Lowering the levels of age-dysregulated proteasome targets prolongs longevity, whereas preventing their degradation shortens lifespan. Among the proteasomal targets, we find the IFB-2 intermediate filament4 and the EPS-8 modulator of RAC signalling5. While increased levels of IFB-2 promote the loss of intestinal integrity and bacterial colonization, upregulation of EPS-8 hyperactivates RAC in muscle and neurons, and leads to alterations in the actin cytoskeleton and protein kinase JNK. In summary, age-related changes in targeted degradation of structural and regulatory proteins across tissues determine longevity.


Assuntos
Envelhecimento/metabolismo , Caenorhabditis elegans/metabolismo , Proteoma/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Citoesqueleto de Actina/metabolismo , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/microbiologia , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas do Citoesqueleto/metabolismo , Intestinos/microbiologia , Longevidade , Músculos/metabolismo , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteoma/química , Proteínas rac de Ligação ao GTP/metabolismo
7.
Cell Calcium ; 98: 102446, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34303264

RESUMO

Transient receptor potential (TRP) channels are expressed in many nonneural tissues where their functions are not well known. Using C. elegans as a model, a new study demonstrated that colonization of the Gram-positive pathogenic bacteria E. faecalis in the intestine causes intestinal distention. Two TRPM channels sense such intestinal distension to trigger fast pathogen avoidance behavior, thereby limiting pathogen infection. This work signifies the novel role of TRP channels in gut physiology and pathogen defense.


Assuntos
Proteínas de Caenorhabditis elegans , Canais de Potencial de Receptor Transitório , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo
8.
Commun Biol ; 4(1): 843, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234268

RESUMO

Age-related changes in cellular metabolism can affect brain homeostasis, creating conditions that are permissive to the onset and progression of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Although the roles of metabolites have been extensively studied with regard to cellular signaling pathways, their effects on protein aggregation remain relatively unexplored. By computationally analysing the Human Metabolome Database, we identified two endogenous metabolites, carnosine and kynurenic acid, that inhibit the aggregation of the amyloid beta peptide (Aß) and rescue a C. elegans model of Alzheimer's disease. We found that these metabolites act by triggering a cytosolic unfolded protein response through the transcription factor HSF-1 and downstream chaperones HSP40/J-proteins DNJ-12 and DNJ-19. These results help rationalise previous observations regarding the possible anti-ageing benefits of these metabolites by providing a mechanism for their action. Taken together, our findings provide a link between metabolite homeostasis and protein homeostasis, which could inspire preventative interventions against neurodegenerative disorders.


Assuntos
Doença de Alzheimer/metabolismo , Caenorhabditis elegans/metabolismo , Carnosina/metabolismo , Modelos Animais de Doenças , Ácido Cinurênico/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Doença de Alzheimer/prevenção & controle , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Carnosina/farmacologia , Citosol/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Ácido Cinurênico/farmacologia , Agregados Proteicos , Agregação Patológica de Proteínas/prevenção & controle , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos
9.
Ecotoxicol Environ Saf ; 222: 112523, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34273852

RESUMO

Micro- and nano- polystyrene particles have been widely detected in environment, posing potential threats to human health. This study was designed to evaluate the neurodevelopmental toxicity of polystyrene nanoparticles (NPs) in Caenorhabditis elegans (C. elegans), to screen crucial genes and investigate the underlying mechanism. In wild-type C. elegans, polystyrene NPs (diameter 50 nm) could concentration-dependently induce significant inhibition in body length, survival rate, head thrashes, and body bending, accompanying with increase of reactive oxygen species (ROS) production, lipofuscin accumulation, and apoptosis and decrease of dopamine (DA) contents. Moreover, pink-1 mutant was demonstrated to alleviate the locomotion disorders and oxidative damage induced by polystyrene NPs, indicating involvement of pink-1 in the polystyrene NPs-induced neurotoxicity. RNA sequencing results revealed 89 up-regulated and 56 down-regulated differently expressed genes (DEGs) response to polystyrene NPs (100 µg/L) exposure. Gene Ontology (GO) enrichment analysis revealed that predominant enriched DEGs were correlated with biological function of cuticle development and molting cycle. Furthermore, mutant strains test showed that the neurodevelopmental toxicity and oxidative stress responses induced by 50 nm polystyrene NPs were regulated by dpy-5 and rol-6. In general, polystyrene NPs induced obvious neurodevelopmental toxicity in C. elegans through oxidative damage and dopamine reduction. Crucial genes dpy-5 and rol-6 might participate in polystyrene NPs-induced neurodevelopmental toxicity through regulation on synthesis and deposition of cuticle collagen.


Assuntos
Proteínas de Caenorhabditis elegans , Nanopartículas , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Colágeno , Humanos , Nanopartículas/toxicidade , Estresse Oxidativo , Poliestirenos , Espécies Reativas de Oxigênio
10.
J Cell Sci ; 134(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34308970

RESUMO

Aberrant centrosome numbers are associated with human cancers. The levels of centrosome regulators positively correlate with centrosome number. Thus, tight control of centrosome protein levels is critical. In Caenorhabditis elegans, the anaphase-promoting complex/cyclosome and its co-activator FZR-1 (APC/CFZR-1), a ubiquitin ligase, negatively regulates centrosome assembly through SAS-5 degradation. In this study, we report the C. elegans ZYG-1 (Plk4 in humans) as a potential substrate of APC/CFZR-1. Inhibiting APC/CFZR-1 or mutating a ZYG-1 destruction (D)-box leads to elevated ZYG-1 levels at centrosomes, restoring bipolar spindles and embryonic viability to zyg-1 mutants, suggesting that APC/CFZR-1 influences centrosomal ZYG-1 via the D-box motif. We also show the Slimb/ßTrCP-binding (SB) motif is critical for ZYG-1 degradation, substantiating a conserved mechanism by which ZYG-1/Plk4 stability is regulated by the SKP1-CUL1-F-box (Slimb/ßTrCP)-protein complex (SCFSlimb/ßTrCP)-dependent proteolysis via the conserved SB motif in C. elegans. Furthermore, we show that co-mutating ZYG-1 SB and D-box motifs stabilizes ZYG-1 in an additive manner, suggesting that the APC/CFZR-1 and SCFSlimb/ßTrCP ubiquitin ligases function cooperatively for timely ZYG-1 destruction in C. elegans embryos where ZYG-1 activity remains at threshold level to ensure normal centrosome number.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Centrossomo , Ciclossomo-Complexo Promotor de Anáfase/genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Humanos , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Contendo Repetições de beta-Transducina
11.
Small ; 17(30): e2102145, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34196492

RESUMO

Significant non-genetic stochastic factors affect aging, causing lifespan differences among individuals, even those sharing the same genetic and environmental background. In Caenorhabditis elegans, differences in heat-shock response (HSR) are predictive of lifespan. However, factors contributing to the heterogeneity of HSR are still not fully elucidated. Here, the authors characterized HSR dynamics in isogenic C. elegans expressing GFP reporter for hsp-16.2 for identifying the key contributors of HSR heterogeneity. Specifically, microfluidic devices that enable cross-sectional and longitudinal measurements of HSR dynamics in C. elegans at different scales are developed: in populations, within individuals, and in embryos. The authors adapted a mathematical model of HSR to single C. elegans and identified model parameters associated with proteostasis-maintenance of protein homeostasis-more specifically, protein turnover, as the major drivers of heterogeneity in HSR dynamics. It is verified that individuals with enhanced proteostasis fidelity in early adulthood live longer. The model-based comparative analysis of protein turnover in day-1 and day-2 adult C. elegans revealed a stochastic-onset of age-related proteostasis decline that increases the heterogeneity of HSR capacity. Finally, the analysis of C. elegans embryos showed higher HSR and proteostasis capacity than young adults and established transgenerational contribution to HSR heterogeneity that depends on maternal age.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Adulto , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Estudos Transversais , Resposta ao Choque Térmico , Humanos , Proteostase
12.
Nat Commun ; 12(1): 4527, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34312384

RESUMO

Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. Here we report BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets.


Assuntos
Membrana Celular/fisiologia , Opsinas/metabolismo , Optogenética/métodos , Células Piramidais/fisiologia , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Células Cultivadas , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Furões/genética , Furões/metabolismo , Células HEK293 , Hipocampo/citologia , Humanos , Masculino , Potenciais da Membrana/fisiologia , Camundongos Transgênicos , Opsinas/genética , Técnicas de Patch-Clamp/métodos , Células Piramidais/citologia , Células Piramidais/metabolismo , Ratos Wistar , Reprodutibilidade dos Testes
13.
Molecules ; 26(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203560

RESUMO

A number of plants used in folk medicine in Thailand and Eastern Asia are attracting interest due to the high bioactivities of their extracts. The aim of this study was to screen the edible leaf extracts of 20 plants found in Thailand and investigate the potential neuroprotective effects of the most bioactive sample. The total phenol and flavonoid content and 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity were determined for all 20 leaf extracts. Based on these assays, Glochidion littorale leaf extract (GLE), which showed a high value in all tested parameters, was used in further experiments to evaluate its effects on neurodegeneration in Caenorhabditis elegans. GLE treatment ameliorated H2O2-induced oxidative stress by attenuating the accumulation of reactive oxygen species and protected the worms against 1-methyl-4-phenylpyridinium-induced neurodegeneration. The neuroprotective effects observed may be associated with the activation of the transcription factor DAF-16. The characterization of this extract by LC-MS identified several phenolic compounds, including myricetin, coumestrin, chlorogenic acid, and hesperidin, which may play a key role in neuroprotection. This study reports the novel neuroprotective activity of GLE, which may be used to develop treatments for neurodegenerative diseases such as Parkinson's syndrome.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fármacos Neuroprotetores , Estresse Oxidativo/efeitos dos fármacos , Phyllanthus/química , Extratos Vegetais , Animais , Peróxido de Hidrogênio/farmacologia , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia
14.
Methods Mol Biol ; 2276: 397-407, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060057

RESUMO

Caenorhabditis elegans is a highly versatile model system, intensively used for functional, genetic, cytometric, and integrative studies. Due to its simplicity and large muscle cell number, C. elegans has frequently been used to study mitochondrial deficiencies caused by disease or drug toxicity. Here we describe a robust and efficient method to visualize and quantify mitochondrial morphology in vivo. This method has many practical and technical advantages above traditional (manual) methods and provides a comprehensive analysis of mitochondrial morphology.


Assuntos
Caenorhabditis elegans/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Microscopia Intravital/métodos , Microscopia Confocal/métodos , Mitocôndrias/ultraestrutura , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Mitocôndrias/metabolismo
15.
Development ; 148(11)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34100067

RESUMO

Cells of the same type can be generated by distinct cellular lineages that originate in different parts of the developing embryo ('lineage convergence'). Several Caenorhabditis elegans neuron classes composed of left/right or radially symmetric class members display such lineage convergence. We show here that the C. elegans Atonal homolog lin-32 is differentially expressed in neuronal lineages that give rise to left/right or radially symmetric class members. Loss of lin-32 results in the selective loss of the expression of pan-neuronal markers and terminal selector-type transcription factors that confer neuron class-specific features. Another basic helix-loop-helix (bHLH) gene, the Achaete-Scute homolog hlh-14, is expressed in a mirror image pattern relative to lin-32 and is required to induce neuronal identity and terminal selector expression on the contralateral side of the animal. These findings demonstrate that distinct lineage histories converge via different bHLH factors at the level of induction of terminal selector identity determinants, which thus serve as integrators of distinct lineage histories. We also describe neuron-to-neuron identity transformations in lin-32 mutants, which we propose to also be the result of misregulation of terminal selector gene expression.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Linhagem da Célula/fisiologia , Neurônios/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Diferenciação Celular , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição
16.
Methods Mol Biol ; 2326: 33-46, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34097259

RESUMO

Environmental pollutants inevitably exert adverse effects on humans and other species. Quick identification and in-depth characterization of the pollutants are requisite objectives for clinicians and environmental health scientists. The nematode Caenorhabditis elegans has been utilized as a model organism for toxicity evaluation of environmental pollutants, due to its transparency, short lifespan, entire genome sequencing, and economical characteristics. However, few researchers have systematically addressed mitochondrial toxicity in response to toxicants, despite the critical role mitochondria play in energy production and respiration, as well as the generation of reactive oxygen species. Mitochondria are vulnerable to environmental pollutants, and their dysfunction contributes to cellular damage and toxicity in plethora of diseases. Here, we describe methods in step-by-step for mitochondrial toxicity evaluation in response to pollutants, including exposure of C. elegans to toxicants, mitochondrial ROS detection, mitochondrial morphology analysis, mitochondrial function analysis, such as ATP production and oxygen consumption, and gene expression studies, with the application of corresponding genetically modified strains.


Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Mitocôndrias/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Expressão Gênica/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Consumo de Oxigênio/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Testes de Toxicidade/métodos
18.
Chem Pharm Bull (Tokyo) ; 69(6): 557-563, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34078802

RESUMO

Sperm activation is an essential process by which the male gametes become capable of fertilization. Because the process in Caenorhabditis elegans is readily reproducible in vitro, this organism serves as an excellent model to investigate it. C. elegans sperm activation in vivo occurs during spermiogenesis. Membranous organelles (MOs) contained within spermatids fuse with the plasma membrane, resulting in extracellular release of their contents and relocation of some proteins indispensable for fertilization from the MO membrane onto the sperm surface. Intriguingly, these cytological alternations are exhibited similarly in mouse spermatozoa during the acrosome reaction, which also represents a form of sperm activation, prompting us to hypothesize that C. elegans and mice share a common mechanism for sperm activation. To explore this, we first screened a chemical library to identify compounds that activate C. elegans spermatozoa. Because a quinolinol analog named DDI-6 seemed to be a candidate sperm activator, we synthesized it to use for further analyses. This involved direct dechlorination and hydrogenolysis of commercially available 5-chloro-8-quinolinol, both of which are key steps to yield 1,2,3,4-tetrahydro-8-quinolinol, and we subsequently introduced the sulfonamide group to the compound. When C. elegans spermatids were stimulated with solvent alone or the newly synthesized DDI-6, approx. 3% and approx. 28% of spermatids became MO-fused spermatozoa, respectively. Moreover, DDI-6 triggered the acrosome reaction in approx. 20% of mouse spermatozoa, while approx. 12% became acrosome-reacted after mock stimulation. Thus, DDI-6 serves as a moderately effective activator for both C. elegans and mouse spermatozoa.


Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Hidroxiquinolinas/farmacologia , Espermatozoides/efeitos dos fármacos , Animais , Caenorhabditis elegans/metabolismo , Hidroxiquinolinas/síntese química , Hidroxiquinolinas/química , Masculino , Camundongos , Camundongos Endogâmicos ICR , Estrutura Molecular , Espermatozoides/metabolismo
19.
Development ; 148(11)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34109380

RESUMO

The adult nervous system has a limited capacity to regenerate after accidental damage. Post-injury functional restoration requires proper targeting of the injured axon to its postsynaptic cell. Although the initial response to axonal injury has been studied in great detail, it is rather unclear what controls the re-establishment of a functional connection. Using the posterior lateral microtubule neuron in Caenorhabditis elegans, we found that after axotomy, the regrowth from the proximal stump towards the ventral side and accumulation of presynaptic machinery along the ventral nerve cord correlated to the functional recovery. We found that the loss of insulin receptor DAF-2 promoted 'ventral targeting' in a DAF-16-dependent manner. We further showed that coordinated activities of DAF-16 in neuron and muscle promoted 'ventral targeting'. In response to axotomy, expression of the Netrin receptor UNC-40 was upregulated in the injured neuron in a DAF-16-dependent manner. In contrast, the DAF-2-DAF-16 axis contributed to the age-related decline in Netrin expression in muscle. Therefore, our study revealed an important role for insulin signaling in regulating the axon guidance molecules during the functional rewiring process.


Assuntos
Axônios/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Moléculas de Adesão Celular/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Netrinas/metabolismo , Animais , Orientação de Axônios , Proteínas de Caenorhabditis elegans/genética , Moléculas de Adesão Celular/genética , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Microtúbulos/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Netrina/metabolismo , Netrinas/genética , Neurônios/metabolismo , Transdução de Sinais
20.
Nat Commun ; 12(1): 3492, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108460

RESUMO

In the Caenorhabditis elegans germline, thousands of mRNAs are concomitantly expressed with antisense 22G-RNAs, which are loaded into the Argonaute CSR-1. Despite their essential functions for animal fertility and embryonic development, how CSR-1 22G-RNAs are produced remains unknown. Here, we show that CSR-1 slicer activity is primarily involved in triggering the synthesis of small RNAs on the coding sequences of germline mRNAs and post-transcriptionally regulates a fraction of targets. CSR-1-cleaved mRNAs prime the RNA-dependent RNA polymerase, EGO-1, to synthesize 22G-RNAs in phase with translating ribosomes, in contrast to other 22G-RNAs mostly synthesized in germ granules. Moreover, codon optimality and efficient translation antagonize CSR-1 slicing and 22G-RNAs biogenesis. We propose that codon usage differences encoded into mRNA sequences might be a conserved strategy in eukaryotes to regulate small RNA biogenesis and Argonaute targeting.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Uso do Códon , Biossíntese de Proteínas , RNA Interferente Pequeno/biossíntese , Animais , Proteínas Argonauta/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Catálise , Citosol/metabolismo , Mutação , Oogônios/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Ribossomos/metabolismo
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