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1.
Int J Mol Sci ; 22(19)2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34638618

RESUMO

Wildlife is chronically exposed to various sources of ionizing radiations, both environmental or anthropic, due to nuclear energy use, which can induce several defects in organisms. In invertebrates, reproduction, which directly impacts population dynamics, has been found to be the most radiosensitive endpoint. Understanding the underlying molecular pathways inducing this reproduction decrease can help in predicting the effects at larger scales (i.e., population). In this study, we used a life stage dependent approach in order to better understand the molecular determinants of reproduction decrease in the roundworm C. elegans. Worms were chronically exposed to 50 mGy·h-1 external gamma ionizing radiations throughout different developmental periods (namely embryogenesis, gametogenesis, and full development). Then, in addition to reproduction parameters, we performed a wide analysis of lipids (different class and fatty acid via FAMES), which are both important signaling molecules for reproduction and molecular targets of oxidative stress. Our results showed that reproductive defects are life stage dependent, that lipids are differently misregulated according to the considered exposure (e.g., upon embryogenesis and full development) and do not fully explain radiation induced reproductive defects. Finally, our results enable us to propose a conceptual model of lipid signaling after radiation stress in which both the soma and the germline participate.


Assuntos
Caenorhabditis elegans/efeitos da radiação , Metabolismo dos Lipídeos/efeitos da radiação , Reprodução/efeitos da radiação , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/fisiologia , Ácidos Graxos/metabolismo , Feminino , Raios gama/efeitos adversos , Masculino , Modelos Biológicos , Estresse Oxidativo/efeitos da radiação , Tolerância a Radiação , Reprodução/fisiologia
2.
Int J Mol Sci ; 22(19)2021 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-34639093

RESUMO

Aggregation of ß2 microglobulin (ß2m) into amyloid fibrils is associated with systemic amyloidosis, caused by the deposition of amyloid fibrils containing the wild-type protein and its truncated variant, ΔN6 ß2m, in haemo-dialysed patients. A second form of familial systemic amyloidosis caused by the ß2m variant, D76N, results in amyloid deposits in the viscera, without renal dysfunction. Although the folding and misfolding mechanisms of ß2 microglobulin have been widely studied in vitro and in vivo, we lack a comparable understanding of the molecular mechanisms underlying toxicity in a cellular and organismal environment. Here, we established transgenic C. elegans lines expressing wild-type (WT) human ß2m, or the two highly amyloidogenic naturally occurring variants, D76N ß2m and ΔN6 ß2m, in the C. elegans bodywall muscle. Nematodes expressing the D76N ß2m and ΔN6 ß2m variants exhibit increased age-dependent and cell nonautonomous proteotoxicity associated with reduced motility, delayed development and shortened lifespan. Both ß2m variants cause widespread endogenous protein aggregation contributing to the increased toxicity in aged animals. We show that expression of ß2m reduces the capacity of C. elegans to cope with heat and endoplasmic reticulum (ER) stress, correlating with a deficiency to upregulate BiP/hsp-4 transcripts in response to ER stress in young adult animals. Interestingly, protein secretion in all ß2m variants is reduced, despite the presence of the natural signal sequence, suggesting a possible link between organismal ß2m toxicity and a disrupted ER secretory metabolism.


Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Estresse do Retículo Endoplasmático , Longevidade , Mutação , Agregados Proteicos , Resposta a Proteínas não Dobradas , Microglobulina beta-2/toxicidade , Animais , Caenorhabditis elegans/genética , Resposta ao Choque Térmico , Humanos , Microglobulina beta-2/genética
3.
Elife ; 102021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34505574

RESUMO

Preferably, lifespan-extending therapies should work when applied late in life without causing undesired pathologies. Reducing insulin/insulin-like growth factor (IGF)-1 signaling (IIS) increases lifespan across species, but the effects of reduced IIS interventions in extreme geriatric ages remains unknown. Using the nematode Caenorhabditis elegans, we engineered the conditional depletion of the DAF-2/insulin/IGF-1 transmembrane receptor using an auxin-inducible degradation (AID) system. This allowed for the temporal and spatial reduction in DAF-2 protein levels at time points after which interventions such as RNAi become ineffective. Using this system, we found that AID-mediated depletion of DAF-2 protein surpasses the longevity of daf-2 mutants. Depletion of DAF-2 during early adulthood resulted in multiple adverse phenotypes, including growth retardation, germline shrinkage, egg retention, and reduced brood size. By contrast, AID-mediated depletion of DAF-2 post-reproduction, or specifically in the intestine in early adulthood, resulted in an extension of lifespan without these deleterious effects. Strikingly, at geriatric ages, when 75% of the population had died, AID-mediated depletion of DAF-2 protein resulted in a doubling in lifespan. Thus, we provide a proof-of-concept that even close to the end of an individual's lifespan, it is possible to slow aging and promote longevity.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Longevidade/genética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Mutação , Fenótipo , Receptor IGF Tipo 1/genética , Transdução de Sinais/genética
4.
Nature ; 596(7871): 257-261, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34349261

RESUMO

An animal's nervous system changes as its body grows from birth to adulthood and its behaviours mature1-8. The form and extent of circuit remodelling across the connectome is unknown3,9-15. Here we used serial-section electron microscopy to reconstruct the full brain of eight isogenic Caenorhabditis elegans individuals across postnatal stages to investigate how it changes with age. The overall geometry of the brain is preserved from birth to adulthood, but substantial changes in chemical synaptic connectivity emerge on this consistent scaffold. Comparing connectomes between individuals reveals substantial differences in connectivity that make each brain partly unique. Comparing connectomes across maturation reveals consistent wiring changes between different neurons. These changes alter the strength of existing connections and create new connections. Collective changes in the network alter information processing. During development, the central decision-making circuitry is maintained, whereas sensory and motor pathways substantially remodel. With age, the brain becomes progressively more feedforward and discernibly modular. Thus developmental connectomics reveals principles that underlie brain maturation.


Assuntos
Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Caenorhabditis elegans/citologia , Conectoma , Modelos Neurológicos , Vias Neurais , Sinapses/fisiologia , Envelhecimento/metabolismo , Animais , Encéfalo/anatomia & histologia , Encéfalo/ultraestrutura , Caenorhabditis elegans/anatomia & histologia , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/ultraestrutura , Individualidade , Interneurônios/citologia , Microscopia Eletrônica , Neurônios/citologia , Comportamento Estereotipado
5.
Cells ; 10(8)2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34440735

RESUMO

Knowledge regarding complex radiation responses in biological systems can be enhanced using genetically amenable model organisms. In this manuscript, we reviewed the use of the nematode, Caenorhabditis elegans (C. elegans), as a model organism to investigate radiation's biological effects. Diverse types of experiments were conducted on C. elegans, using acute and chronic exposure to different ionizing radiation types, and to assess various biological responses. These responses differed based on the type and dose of radiation and the chemical substances in which the worms were grown or maintained. A few studies compared responses to various radiation types and doses as well as other environmental exposures. Therefore, this paper focused on the effect of irradiation on C. elegans, based on the intensity of the radiation dose and the length of exposure and ways to decrease the effects of ionizing radiation. Moreover, we discussed several studies showing that dietary components such as vitamin A, polyunsaturated fatty acids, and polyphenol-rich food source may promote the resistance of C. elegans to ionizing radiation and increase their life span after irradiation.


Assuntos
Caenorhabditis elegans/efeitos da radiação , Radiação Ionizante , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Ácidos Graxos Insaturados/química , Ácidos Graxos Insaturados/farmacologia , Glucosídeos/farmacologia , Lignanas/farmacologia , Longevidade/efeitos da radiação , Reprodução/efeitos dos fármacos , Reprodução/efeitos da radiação , Vitamina A/química , Vitamina A/farmacologia
6.
PLoS Comput Biol ; 17(7): e1009185, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34280180

RESUMO

Complex dendritic trees are a distinctive feature of neurons. Alterations to dendritic morphology are associated with developmental, behavioral and neurodegenerative changes. The highly-arborized PVD neuron of C. elegans serves as a model to study dendritic patterning; however, quantitative, objective and automated analyses of PVD morphology are missing. Here, we present a method for neuronal feature extraction, based on deep-learning and fitting algorithms. The extracted neuronal architecture is represented by a database of structural elements for abstracted analysis. We obtain excellent automatic tracing of PVD trees and uncover that dendritic junctions are unevenly distributed. Surprisingly, these junctions are three-way-symmetrical on average, while dendritic processes are arranged orthogonally. We quantify the effect of mutation in git-1, a regulator of dendritic spine formation, on PVD morphology and discover a localized reduction in junctions. Our findings shed new light on PVD architecture, demonstrating the effectiveness of our objective analyses of dendritic morphology and suggest molecular control mechanisms.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Dendritos/metabolismo , Algoritmos , Animais , Comportamento Animal/fisiologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/genética , Biologia Computacional , Dendritos/genética , Dendritos/ultraestrutura , Modelos Neurológicos , Mutação , Redes Neurais de Computação , Neurogênese/genética , Neurogênese/fisiologia , Plasticidade Neuronal/genética , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Neurônios/ultraestrutura , Fenótipo
7.
PLoS Genet ; 17(7): e1009635, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34237064

RESUMO

The intracellular level of fatty aldehydes is tightly regulated by aldehyde dehydrogenases to minimize the formation of toxic lipid and protein adducts. Importantly, the dysregulation of aldehyde dehydrogenases has been implicated in neurologic disorder and cancer in humans. However, cellular responses to unresolved, elevated fatty aldehyde levels are poorly understood. Here, we report that ALH-4 is a C. elegans aldehyde dehydrogenase that specifically associates with the endoplasmic reticulum, mitochondria and peroxisomes. Based on lipidomic and imaging analysis, we show that the loss of ALH-4 increases fatty aldehyde levels and reduces fat storage. ALH-4 deficiency in the intestine, cell-nonautonomously induces NHR-49/NHR-79-dependent hypodermal peroxisome proliferation. This is accompanied by the upregulation of catalases and fatty acid catabolic enzymes, as indicated by RNA sequencing. Such a response is required to counteract ALH-4 deficiency since alh-4; nhr-49 double mutant animals are sterile. Our work reveals unexpected inter-tissue communication of fatty aldehyde levels and suggests pharmacological modulation of peroxisome proliferation as a therapeutic strategy to tackle pathology related to excess fatty aldehydes.


Assuntos
Aldeído Desidrogenase/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Aldeído Desidrogenase/química , Aldeído Desidrogenase/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Regulação da Expressão Gênica , Lipase/genética , Lipase/metabolismo , Gotículas Lipídicas/metabolismo , Lipólise/genética , Mutação , Peroxissomos/genética , Receptores Citoplasmáticos e Nucleares/genética
8.
PLoS One ; 16(7): e0243522, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34197476

RESUMO

Lowering the activity of the Insulin/IGF-1 Signaling (IIS) cascade results in elevated stress resistance, enhanced protein homeostasis (proteostasis) and extended lifespan of worms, flies and mice. In the nematode Caenorhabditis elegans (C. elegans), the longevity phenotype that stems from IIS reduction is entirely dependent upon the activities of a subset of transcription factors including the Forkhead factor DAF-16/FOXO (DAF-16), Heat Shock Factor-1 (HSF-1), SKiNhead/Nrf (SKN-1) and ParaQuat Methylviologen responsive (PQM-1). While DAF-16 determines lifespan exclusively during early adulthood and governs proteostasis in early adulthood and midlife, HSF-1 executes these functions foremost during development. Despite the central roles of SKN-1 as a regulator of lifespan and proteostasis, the temporal requirements of this transcription factor were unknown. Here we employed conditional knockdown techniques and discovered that in C. elegans, SKN-1 is primarily important for longevity and proteostasis during late larval development through early adulthood. Our findings indicate that events that occur during late larval developmental through early adulthood affect lifespan and proteostasis and suggest that subsequent to HSF-1, SKN-1 sets the conditions, partially overlapping temporally with DAF-16, that enable IIS reduction to promote longevity and proteostasis. Our findings raise the intriguing possibility that HSF-1, SKN-1 and DAF-16 function in a coordinated and sequential manner to promote healthy aging.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Ligação a DNA/metabolismo , Longevidade , Proteostase/fisiologia , Fatores de Transcrição/metabolismo , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Fatores de Transcrição Forkhead/metabolismo , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/metabolismo , Peptídeos/farmacologia , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/genética , Ribonuclease III/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética
9.
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
10.
Nat Commun ; 12(1): 4212, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244496

RESUMO

CSR-1 is an essential Argonaute protein that binds to a subclass of 22G-RNAs targeting most germline-expressed genes. Here we show that the two isoforms of CSR-1 have distinct expression patterns; CSR-1B is ubiquitously expressed throughout the germline and during all stages of development while CSR-1A expression is restricted to germ cells undergoing spermatogenesis. Furthermore, CSR-1A associates preferentially with 22G-RNAs mapping to spermatogenesis-specific genes whereas CSR-1B-bound small RNAs map predominantly to oogenesis-specific genes. Interestingly, the exon unique to CSR-1A contains multiple dimethylarginine modifications, which are necessary for the preferential binding of CSR-1A to spermatogenesis-specific 22G-RNAs. Thus, we have discovered a regulatory mechanism for C. elegans Argonaute proteins that allows for specificity of small RNA binding between similar Argonaute proteins with overlapping temporal and spatial localization.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Interferência de RNA , Espermatogênese/genética , Animais , Animais Geneticamente Modificados , Arginina/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Éxons/genética , Feminino , Masculino , Metilação , Oogênese/genética , Ligação Proteica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/metabolismo , Processos de Determinação Sexual/genética
11.
PLoS Biol ; 19(7): e3000968, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34228701

RESUMO

Centromere protein A (CENP-A) is a histone H3 variant that defines centromeric chromatin and is essential for centromere function. In most eukaryotes, CENP-A-containing chromatin is epigenetically maintained, and centromere identity is inherited from one cell cycle to the next. In the germ line of the holocentric nematode Caenorhabditis elegans, this inheritance cycle is disrupted. CENP-A is removed at the mitosis-to-meiosis transition and is reestablished on chromatin during diplotene of meiosis I. Here, we show that the N-terminal tail of CENP-A is required for the de novo establishment of centromeres, but then its presence becomes dispensable for centromere maintenance during development. Worms homozygous for a CENP-A tail deletion maintain functional centromeres during development but give rise to inviable offspring because they fail to reestablish centromeres in the maternal germ line. We identify the N-terminal tail of CENP-A as a critical domain for the interaction with the conserved kinetochore protein KNL-2 and argue that this interaction plays an important role in setting centromere identity in the germ line. We conclude that centromere establishment and maintenance are functionally distinct in C. elegans.


Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Proteína Centromérica A/genética , Centrômero , Impressão Genômica , Células Germinativas , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteína Centromérica A/química , Proteína Centromérica A/metabolismo , Cromatina/metabolismo , Cromossomos , Feminino , Homozigoto , Cinetocoros , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica , Domínios Proteicos
12.
Sci Rep ; 11(1): 12304, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112855

RESUMO

Small RNAs that originate from transfer RNA (tRNA) species, tRNA-derived fragments (tRFs), play diverse biological functions but little is known for their association with aging. Moreover, biochemical aspects of tRNAs limit discovery of functional tRFs by high throughput sequencing. In particular, genes encoding tRNAs exist as multiple copies throughout genome, and mature tRNAs have various modified bases, contributing to ambiguities for RNA sequencing-based analysis of tRFs. Here, we report age-dependent changes of tRFs in Caenorhabditis elegans. We first analyzed published RNA sequencing data by using a new strategy for tRNA-associated sequencing reads. Our current method used unique mature tRNAs as a reference for the sequence alignment, and properly filtered out false positive enrichment for tRFs. Our analysis successfully distinguished de novo mutation sites from differences among homologous copies, and identified potential RNA modification sites. Overall, the majority of tRFs were upregulated during aging and originated from 5'-ends, which we validated by using Northern blot analysis. Importantly, we revealed that the major source of tRFs upregulated during aging was the tRNAs with abundant gene copy numbers. Our analysis suggests that tRFs are useful biomarkers of aging particularly when they originate from abundant homologous gene copies.


Assuntos
Envelhecimento/genética , Caenorhabditis elegans/genética , RNA de Transferência/genética , Homologia de Sequência , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Sequenciamento de Nucleotídeos em Larga Escala , Alinhamento de Sequência , Análise de Sequência de RNA
13.
Sci Rep ; 11(1): 12289, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112931

RESUMO

Traditionally Caenorhabditis elegans lifespan assays are performed by manually inspecting nematodes with a dissection microscope, which involves daily counting of live/dead worms cultured in Petri plates for 21-25 days. This manual inspection requires the screening of hundreds of worms to ensure statistical robustness, and is therefore a time-consuming approach. In recent years, various automated artificial vision systems have been reported to increase the throughput, however they usually provide less accurate results than manual assays. The main problems identified when using these vision systems are the false positives and false negatives, which occur due to culture media changes, occluded zones, dirtiness or condensation of the Petri plates. In this work, we developed and described a new C. elegans monitoring machine, SiViS, which consists of a flexible and compact platform design to analyse C. elegans cultures using the standard Petri plates seeded with E. coli. Our system uses an active vision illumination technique and different image-processing pipelines for motion detection, both previously reported, providing a fully automated image processing pipeline. In addition, this study validated both these methods and the feasibility of the SiViS machine for lifespan experiments by comparing them with manual lifespan assays. Results demonstrated that the automated system yields consistent replicates (p-value log rank test 0.699), and there are no significant differences between automated system assays and traditionally manual assays (p-value 0.637). Finally, although we have focused on the use of SiViS in longevity assays, the system configuration is flexible and can, thus, be adapted to other C. elegans studies such as toxicity, mobility and behaviour.


Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Processamento de Imagem Assistida por Computador , Longevidade/fisiologia , Animais , Caenorhabditis elegans/genética , Escherichia coli
14.
J Virol ; 95(17): e0026421, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34132570

RESUMO

Uncharacterized viral genomes that encode circular replication-associated proteins of single-stranded DNA viruses have been discovered by metagenomics/metatranscriptomics approaches. Some of these novel viruses are classified in the newly formed family Genomoviridae. Here, we determined the host range of a novel genomovirus, SlaGemV-1, through the transfection of Sclerotinia sclerotiorum with infectious clones. Inoculating with the rescued virions, we further transfected Botrytis cinerea and Monilinia fructicola, two economically important members of the family Sclerotiniaceae, and Fusarium oxysporum. SlaGemV-1 causes hypovirulence in S. sclerotiorum, B. cinerea, and M. fructicola. SlaGemV-1 also replicates in Spodoptera frugiperda insect cells but not in Caenorhabditis elegans or plants. By expressing viral genes separately through site-specific integration, the replication protein alone was sufficient to cause debilitation. Our study is the first to demonstrate the reconstruction of a metagenomically discovered genomovirus without known hosts with the potential of inducing hypovirulence, and the infectious clone allows for studying mechanisms of genomovirus-host interactions that are conserved across genera. IMPORTANCE Little is known about the exact host range of widespread genomoviruses. The genome of soybean leaf-associated gemygorvirus-1 (SlaGemV-1) was originally assembled from a metagenomic/metatranscriptomic study without known hosts. Here, we rescued SlaGemV-1 and found that it could infect three important plant-pathogenic fungi and fall armyworm (S. frugiperda Sf9) insect cells but not a model nematode, C. elegans, or model plant species. Most importantly, SlaGemV-1 shows promise for inducing hypovirulence of the tested fungal species in the family Sclerotiniaceae, including Sclerotinia sclerotiorum, Botrytis cinerea, and Monilinia fructicola. The viral determinant of hypovirulence was further identified as replication initiation protein. As a proof of concept, we demonstrate that viromes discovered in plant metagenomes can be a valuable genetic resource when novel viruses are rescued and characterized for their host range.


Assuntos
Ascomicetos/virologia , Geminiviridae/isolamento & purificação , Especificidade de Hospedeiro , Metagenoma , Doenças das Plantas/prevenção & controle , Tabaco/crescimento & desenvolvimento , Virulência , Animais , Ascomicetos/genética , Ascomicetos/patogenicidade , Botrytis/genética , Botrytis/patogenicidade , Botrytis/virologia , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/microbiologia , Caenorhabditis elegans/virologia , Fusarium/genética , Fusarium/patogenicidade , Fusarium/virologia , Geminiviridae/classificação , Geminiviridae/genética , Genoma Viral , Controle Biológico de Vetores , Doenças das Plantas/microbiologia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/microbiologia , Folhas de Planta/virologia , Soja/crescimento & desenvolvimento , Soja/microbiologia , Tabaco/microbiologia , Tabaco/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion
15.
Elife ; 102021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34165430

RESUMO

The generation of the enormous diversity of neuronal cell types in a differentiating nervous system entails the activation of neuron type-specific gene batteries. To examine the regulatory logic that controls the expression of neuron type-specific gene batteries, we interrogate single cell expression profiles of all 118 neuron classes of the Caenorhabditis elegans nervous system for the presence of DNA binding motifs of 136 neuronally expressed C. elegans transcription factors. Using a phylogenetic footprinting pipeline, we identify cis-regulatory motif enrichments among neuron class-specific gene batteries and we identify cognate transcription factors for 117 of the 118 neuron classes. In addition to predicting novel regulators of neuronal identities, our nervous system-wide analysis at single cell resolution supports the hypothesis that many transcription factors directly co-regulate the cohort of effector genes that define a neuron type, thereby corroborating the concept of so-called terminal selectors of neuronal identity. Our analysis provides a blueprint for how individual components of an entire nervous system are genetically specified.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/fisiologia , Fatores de Transcrição/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Simulação por Computador , Sistema Nervoso/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo
16.
PLoS Genet ; 17(6): e1009618, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34115759

RESUMO

Coordination of neurite extension with surrounding glia development is critical for neuronal function, but the underlying molecular mechanisms remain poorly understood. Through a genome-wide mutagenesis screen in C. elegans, we identified dyf-4 and daf-6 as two mutants sharing similar defects in dendrite extension. DAF-6 encodes a glia-specific patched-related membrane protein that plays vital roles in glial morphogenesis. We cloned dyf-4 and found that DYF-4 encodes a glia-secreted protein. Further investigations revealed that DYF-4 interacts with DAF-6 and functions in a same pathway as DAF-6 to regulate sensory compartment formation. Furthermore, we demonstrated that reported glial suppressors of daf-6 could also restore dendrite elongation and ciliogenesis in both dyf-4 and daf-6 mutants. Collectively, our data reveal that DYF-4 is a regulator for DAF-6 which promotes the proper formation of the glial channel and indirectly affects neurite extension and ciliogenesis.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Genoma Helmíntico , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas do Tecido Nervoso/genética , Neurogênese/genética , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Comunicação Celular , Cílios/genética , Cílios/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutagênese , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
17.
Methods Mol Biol ; 2326: 19-32, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34097258

RESUMO

The contamination of heavy metals, a class of naturally occurring and persistent toxicants, has become a major public health concern due to increasing industrial and anthropogenic activities. The use of COPAS Biosort, a flow cytometer capable of measuring thousands of nematodes in minutes via high-throughput assays, has been widely applied in C. elegans studies for assessing toxicity of individual metals; however, such application yet to be seen for metals or other chemical mixtures. In the present protocol, we investigated toxic effects of individual metals, Cd, Pb, and Mn, as well as their binary and ternary mixtures, using nematode C. elegans. The toxic outcomes, including effects on growth, reproduction, and feeding behavior, were measured using high-throughput platform analysis (COAPS Biosort).


Assuntos
Cádmio/toxicidade , Caenorhabditis elegans/efeitos dos fármacos , Chumbo/toxicidade , Manganês/toxicidade , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/fisiologia , Comportamento Alimentar/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Reprodução/efeitos dos fármacos , Testes de Toxicidade/métodos
18.
Development ; 148(18)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34170296

RESUMO

Several microfluidic-based methods for Caenorhabditis elegans imaging have recently been introduced. Existing methods either permit imaging across multiple larval stages without maintaining a stable worm orientation, or allow for very good immobilization but are only suitable for shorter experiments. Here, we present a novel microfluidic imaging method that allows parallel live-imaging across multiple larval stages, while maintaining worm orientation and identity over time. This is achieved through an array of microfluidic trap channels carefully tuned to maintain worms in a stable orientation, while allowing growth and molting to occur. Immobilization is supported by an active hydraulic valve, which presses worms onto the cover glass during image acquisition only. In this way, excellent quality images can be acquired with minimal impact on worm viability or developmental timing. The capabilities of the devices are demonstrated by observing the hypodermal seam and P-cell divisions and, for the first time, the entire process of vulval development from induction to the end of morphogenesis. Moreover, we demonstrate feasibility of on-chip RNAi by perturbing basement membrane breaching during anchor cell invasion.


Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Microfluídica/métodos , Animais , Dispositivos Lab-On-A-Chip
19.
J Cell Biol ; 220(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34128967

RESUMO

While much is known about how transcription is controlled at individual genes, comparatively little is known about how cells regulate gene expression on a genome-wide level. Here, we identify a molecular pathway in the C. elegans germline that controls transcription globally in response to nutritional stress. We report that when embryos hatch into L1 larvae, they sense the nutritional status of their environment, and if food is unavailable, they repress gene expression via a global chromatin compaction (GCC) pathway. GCC is triggered by the energy-sensing kinase AMPK and is mediated by a novel mechanism that involves the topoisomerase II/condensin II axis acting upstream of heterochromatin assembly. When the GCC pathway is inactivated, then transcription persists during starvation. These results define a new mode of whole-genome control of transcription.


Assuntos
Caenorhabditis elegans/genética , Cromatina/química , Regulação da Expressão Gênica no Desenvolvimento , Genoma Helmíntico , Proteínas Quinases/genética , Inanição/genética , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Embrião não Mamífero , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histonas/genética , Histonas/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Masculino , Óvulo/metabolismo , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Espermatozoides/metabolismo , Inanição/metabolismo , Transcrição Genética
20.
Dev Cell ; 56(11): 1631-1645.e7, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34051143

RESUMO

Spermiogenesis in nematodes is a process whereby round and quiescent spermatids differentiate into asymmetric and crawling spermatozoa. The molecular mechanism underlying this symmetry breaking remains uncharacterized. In this study, we revealed that sperm-specific Na+/K+-ATPase (NKA) is evenly distributed on the plasma membrane (PM) of Caenorhabditis elegans spermatids but is translocated to and subsequently enters the invaginated membrane of the spermatozoa cell body during sperm activation. The polarization of NKA depends on the transport of cholesterol from the PM to membranous organelles (MOs) via membrane contact sites (MCSs). The inositol 5-phosphatase CIL-1 and the MO-localized PI4P phosphatase SAC-1 may mediate PI4P metabolism to drive cholesterol countertransport via sterol/lipid transport proteins through MCSs. Furthermore, the NKA function is required for C. elegans sperm motility and reproductive success. Our data imply that the lipid dynamics mediated by MCSs might play crucial roles in the establishment of cell polarity. eGraphical abstract.


Assuntos
Transporte Biológico/genética , Proteínas de Caenorhabditis elegans/genética , Colesterol/genética , Esterases/genética , Proteínas de Membrana/genética , ATPase Trocadora de Sódio-Potássio/genética , Espermatogênese/genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Colesterol/metabolismo , Masculino , Membranas Mitocondriais/metabolismo , Organelas/genética , Motilidade Espermática/genética , Espermátides/crescimento & desenvolvimento , Espermatozoides/crescimento & desenvolvimento
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