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1.
Nat Commun ; 14(1): 7564, 2023 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-37985670

RESUMO

Even slight imbalance between the growth rate of different organs can accumulate to a large deviation from their appropriate size during development. Here, we use live imaging of the pharynx of C. elegans to ask if and how organ size scaling nevertheless remains uniform among individuals. Growth trajectories of hundreds of individuals reveal that pharynxes grow by a near constant volume per larval stage that is independent of their initial size, such that undersized pharynxes catch-up in size during development. Tissue-specific depletion of RAGA-1, an activator of mTOR and growth, shows that maintaining correct pharynx-to-body size proportions involves a bi-directional coupling between pharynx size and body growth. In simulations, this coupling cannot be explained by limitation of food uptake alone, and genetic experiments reveal an involvement of the mechanotransducing transcriptional co-regulator yap-1. Our data suggests that mechanotransduction coordinates pharynx growth with other tissues, ensuring body plan uniformity among individuals.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Humanos , Animais , Caenorhabditis elegans/genética , Faringe/metabolismo , Mecanotransdução Celular , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Sinalização YAP
2.
PLoS Genet ; 19(9): e1010938, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37721956

RESUMO

mTORC1 (mechanistic target of rapamycin complex 1) is a metabolic sensor that promotes growth when nutrients are abundant. Ubiquitous inhibition of mTORC1 extends lifespan in multiple organisms but also disrupts several anabolic processes resulting in stunted growth, slowed development, reduced fertility, and disrupted metabolism. However, it is unclear if these pleiotropic effects of mTORC1 inhibition can be uncoupled from longevity. Here, we utilize the auxin-inducible degradation (AID) system to restrict mTORC1 inhibition to C. elegans neurons. We find that neuron-specific degradation of RAGA-1, an upstream activator of mTORC1, or LET-363, the ortholog of mammalian mTOR, is sufficient to extend lifespan in C. elegans. Unlike raga-1 loss of function genetic mutations or somatic AID of RAGA-1, neuronal AID of RAGA-1 robustly extends lifespan without impairing body size, developmental rate, brood size, or neuronal function. Moreover, while degradation of RAGA-1 in all somatic tissues alters the expression of thousands of genes, demonstrating the widespread effects of mTORC1 inhibition, degradation of RAGA-1 in neurons only results in around 200 differentially expressed genes with a specific enrichment in metabolism and stress response. Notably, our work demonstrates that targeting mTORC1 specifically in the nervous system in C. elegans uncouples longevity from growth and reproductive impairments, and that many canonical effects of low mTORC1 activity are not required to promote healthy aging. These data challenge previously held ideas about the mechanisms of mTORC1 lifespan extension and underscore the potential of promoting longevity by neuron-specific mTORC1 modulation.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Caenorhabditis elegans/metabolismo , Longevidade/genética , Complexos Multiproteicos/genética , Reprodução/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Mamíferos/metabolismo
3.
Cell Rep Methods ; 3(3): 100433, 2023 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-37056370

RESUMO

Here, we introduce a single-copy knockin translating ribosome immunoprecipitation (SKI TRIP) toolkit, a collection of Caenorhabditis elegans strains engineered by CRISPR in which tissue-specific expression of FLAG-tagged ribosomal subunit protein RPL-22 is driven by cassettes present in single copy from defined sites in the genome. Through in-depth characterization of the effects of the FLAG tag in animals in which endogenous RPL-22 has been tagged, we show that it incorporates into actively translating ribosomes and efficiently and cleanly pulls down cell-type-specific transcripts. Importantly, the presence of the tag does not impact overall mRNA translation, create bias in transcript use, or cause changes to fitness of the animal. We propose SKI TRIP use for the study of tissue-specific differences in translation and for investigating processes that are acutely sensitive to changes in translation like development or aging.


Assuntos
Caenorhabditis elegans , Biossíntese de Proteínas , Animais , Caenorhabditis elegans/genética , RNA Mensageiro/genética , Biossíntese de Proteínas/genética , Ribossomos/genética , Proteínas Ribossômicas/genética , Imunoprecipitação
4.
Elife ; 82019 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-31411562

RESUMO

Target of rapamycin complex 1 (TORC1) and AMP-activated protein kinase (AMPK) antagonistically modulate metabolism and aging. However, how they coordinate to determine longevity and if they act via separable mechanisms is unclear. Here, we show that neuronal AMPK is essential for lifespan extension from TORC1 inhibition, and that TORC1 suppression increases lifespan cell non autonomously via distinct mechanisms from global AMPK activation. Lifespan extension by null mutations in genes encoding raga-1 (RagA) or rsks-1 (S6K) is fully suppressed by neuronal-specific rescues. Loss of RAGA-1 increases lifespan via maintaining mitochondrial fusion. Neuronal RAGA-1 abrogation of raga-1 mutant longevity requires UNC-64/syntaxin, and promotes mitochondrial fission cell nonautonomously. Finally, deleting the mitochondrial fission factor DRP-1 renders the animal refractory to the pro-aging effects of neuronal RAGA-1. Our results highlight a new role for neuronal TORC1 in cell nonautonomous regulation of longevity, and suggest TORC1 in the central nervous system might be targeted to promote healthy aging.


Assuntos
Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/fisiologia , Longevidade , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Dinâmica Mitocondrial , Proteínas Quinases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Animais
5.
G3 (Bethesda) ; 9(7): 2195-2198, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31064766

RESUMO

We have generated a single-copy knock-in loci for defined gene expression (SKI LODGE) system to insert any DNA by CRISPR/Cas9 at defined safe harbors in the Caenorhabditis elegans genome. Utilizing a single crRNA guide, which also acts as a Co-CRISPR enrichment marker, any DNA sequence can be introduced as a single copy, regulated by different tissue-specific promoters. The SKI LODGE system provides a fast, economical, and effective approach for generating single-copy ectopic transgenes in C. elegans.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Dosagem de Genes , Regulação da Expressão Gênica , Loci Gênicos , Animais , Sistemas CRISPR-Cas , Técnicas de Introdução de Genes , Marcação de Genes
6.
Elife ; 62017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29231812

RESUMO

The neural control of social behaviors in rodents requires the encoding of pheromonal cues by the vomeronasal system. Here we show that the typical preference of male mice for females is eliminated in mutants lacking oxytocin, a neuropeptide modulating social behaviors in many species. Ablation of the oxytocin receptor in aromatase-expressing neurons of the medial amygdala (MeA) fully recapitulates the elimination of female preference in males. Further, single-unit recording in the MeA uncovered significant changes in the sensory representation of conspecific cues in the absence of oxytocin signaling. Finally, acute manipulation of oxytocin signaling in adults is sufficient to alter social interaction preferences in males as well as responses of MeA neurons to chemosensory cues. These results uncover the critical role of oxytocin signaling in a molecularly defined neuronal population in order to modulate the behavioral and physiological responses of male mice to females on a moment-to-moment basis.


Assuntos
Tonsila do Cerebelo/fisiologia , Ocitocina/farmacologia , Receptores de Ocitocina/fisiologia , Células Receptoras Sensoriais/fisiologia , Comportamento Sexual Animal/efeitos dos fármacos , Comportamento Social , Tonsila do Cerebelo/citologia , Tonsila do Cerebelo/efeitos dos fármacos , Animais , Células Cultivadas , Sinais (Psicologia) , Discriminação Psicológica , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ocitócicos/farmacologia , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/efeitos dos fármacos
8.
Nature ; 541(7635): 102-106, 2017 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-27919065

RESUMO

Ageing is driven by a loss of transcriptional and protein homeostasis and is the key risk factor for multiple chronic diseases. Interventions that attenuate or reverse systemic dysfunction associated with age therefore have the potential to reduce overall disease risk in the elderly. Precursor mRNA (pre-mRNA) splicing is a fundamental link between gene expression and the proteome, and deregulation of the splicing machinery is linked to several age-related chronic illnesses. However, the role of splicing homeostasis in healthy ageing remains unclear. Here we demonstrate that pre-mRNA splicing homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans. Using transcriptomics and in-depth splicing analysis in young and old animals fed ad libitum or subjected to dietary restriction, we find defects in global pre-mRNA splicing with age that are reduced by dietary restriction via splicing factor 1 (SFA-1; the C. elegans homologue of SF1, also known as branchpoint binding protein, BBP). We show that SFA-1 is specifically required for lifespan extension by dietary restriction and by modulation of the TORC1 pathway components AMPK, RAGA-1 and RSKS-1/S6 kinase. We also demonstrate that overexpression of SFA-1 is sufficient to extend lifespan. Together, these data demonstrate a role for RNA splicing homeostasis in dietary restriction longevity and suggest that modulation of specific spliceosome components may prolong healthy ageing.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Restrição Calórica , Longevidade/genética , Longevidade/fisiologia , Complexos Multiproteicos/metabolismo , Fatores de Processamento de RNA/metabolismo , Splicing de RNA , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Envelhecimento/genética , Animais , Proteínas de Caenorhabditis elegans/genética , Genoma/genética , Homeostase , Alvo Mecanístico do Complexo 1 de Rapamicina , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transcriptoma
9.
Nat Neurosci ; 16(7): 949-57, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23685720

RESUMO

Odor stimulation evokes complex spatiotemporal activity in the olfactory bulb, suggesting that both the identity of activated neurons and the timing of their activity convey information about odors. However, whether and how downstream neurons decipher these temporal patterns remains unknown. We addressed this question by measuring the spiking activity of downstream neurons while optogenetically stimulating two foci in the olfactory bulb with varying relative timing in mice. We found that the overall spike rates of piriform cortex neurons (PCNs) were sensitive to the relative timing of activation. Posterior PCNs showed higher sensitivity to relative input times than neurons in the anterior piriform cortex. In contrast, olfactory bulb neurons rarely showed such sensitivity. Thus, the brain can transform a relative time code in the periphery into a firing rate-based representation in central brain areas, providing evidence for the relevance of a relative time-based code in the olfactory bulb.


Assuntos
Neurônios/fisiologia , Bulbo Olfatório/fisiologia , Condutos Olfatórios/citologia , Condutos Olfatórios/fisiologia , Olfato/fisiologia , Potenciais de Ação/fisiologia , Animais , Mapeamento Encefálico , Channelrhodopsins , Discriminação Psicológica/fisiologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibição Neural/fisiologia , Odorantes , Proteína de Marcador Olfatório/genética , Proteína de Marcador Olfatório/metabolismo , Optogenética , Técnicas de Patch-Clamp , Proteínas com Domínio T/genética , Fatores de Tempo
10.
EMBO J ; 26(12): 2966-80, 2007 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-17510633

RESUMO

Individual cell types can elaborate morphologically diverse cilia. Cilia are assembled via intraflagellar transport (IFT) of ciliary precursors; however, the mechanisms that generate ciliary diversity are unknown. Here, we examine IFT in the structurally distinct cilia of the ASH/ASI and the AWB chemosensory neurons in Caenorhabditis elegans, enabling us to compare IFT in specific cilia types. We show that unlike in the ASH/ASI cilia, the OSM-3 kinesin moves independently of the kinesin-II motor in the AWB cilia. Although OSM-3 is essential to extend the distal segments of the ASH/ASI cilia, it is not required to build the AWB distal segments. Mutations in the fkh-2 forkhead domain gene result in AWB-specific defects in ciliary morphology, and FKH-2 regulates kinesin-II subunit gene expression specifically in AWB. Our results suggest that cell-specific regulation of IFT contributes to the generation of ciliary diversity, and provide insights into the networks coupling the acquisition of ciliary specializations with other aspects of cell fate.


Assuntos
Caenorhabditis elegans/citologia , Cílios/ultraestrutura , Animais , Animais Geneticamente Modificados , Transporte Biológico , Proteínas de Caenorhabditis elegans/fisiologia , Cinesinas/fisiologia , Microscopia Eletrônica , Mutação , Neurônios Receptores Olfatórios/ultraestrutura
11.
Dev Biol ; 290(1): 139-51, 2006 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-16376329

RESUMO

Members of the HES subfamily of bHLH proteins play crucial roles in neural patterning via repression of neurogenesis. In C. elegans, loss-of-function mutations in ref-1, a distant nematode-specific member of this subfamily, were previously shown to cause ectopic neurogenesis from postembryonic lineages. However, while the vast majority of the nervous system in C. elegans is generated embryonically, the role of REF-1 in regulating these neural lineage decisions is unknown. Here, we show that mutations in ref-1 result in the generation of multiple ectopic neuron types derived from an embryonic neuroblast. In wild-type animals, neurons derived from this sublineage are present in a left/right symmetrical manner. However, in ref-1 mutants, while the ectopically generated neurons exhibit gene expression profiles characteristic of neurons on the left, they are present only on the right side. REF-1 functions in a Notch-independent manner to regulate this ectopic lineage decision. We also demonstrate that loss of REF-1 function results in defective differentiation of an embryonically generated serotonergic neuron type. These results indicate that REF-1 functions in both Notch-dependent and independent pathways to regulate multiple developmental decisions in different neuronal sublineages.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Padronização Corporal , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Linhagem da Célula , Neurônios/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Mutação , Neurônios/metabolismo , Receptores Notch/metabolismo , Fatores de Transcrição/metabolismo
12.
Curr Biol ; 14(24): 2245-51, 2004 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-15620651

RESUMO

Most C. elegans sensory neuron types consist of a single bilateral pair of neurons, and respond to a unique set of sensory stimuli. Although genes required for the development and function of individual sensory neuron types have been identified in forward genetic screens, these approaches are unlikely to identify genes that when mutated result in subtle or pleiotropic phenotypes. Here, we describe a complementary approach to identify sensory neuron type-specific genes via microarray analysis using RNA from sorted AWB olfactory and AFD thermosensory neurons. The expression patterns of subsets of these genes were further verified in vivo. Genes identified by this analysis encode 7-transmembrane receptors, kinases, and nuclear factors including dac-1, which encodes a homolog of the highly conserved Dachshund protein. dac-1 is expressed in a subset of sensory neurons including the AFD neurons and is regulated by the TTX-1 OTX homeodomain protein. On thermal gradients, dac-1 mutants fail to suppress a cryophilic drive but continue to track isotherms at the cultivation temperature, representing the first genetic separation of these AFD-mediated behaviors. Expression profiling of single neuron types provides a rapid, powerful, and unbiased method for identifying neuron-specific genes whose functions can then be investigated in vivo.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Perfilação da Expressão Gênica , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Nervo Olfatório/metabolismo , Sensação Térmica/genética , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Células Cultivadas , Citometria de Fluxo , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência
13.
Curr Opin Neurobiol ; 14(1): 22-30, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15018934

RESUMO

Cellular diversity in the nervous system arises from the presence of multiple neuronal subtypes, each of which is specialized to perform a unique function. Work in Caenorhabditis elegans has begun to reveal the pathways that are essential for the specification of identities of neuronal subtypes in its chemosensory system. The functions of each chemosensory neuron subtype are specified by distinct developmental cascades, using molecules from well-conserved transcription factor families. Additional cellular complexity is generated by novel mechanisms that further diversify the identities of the left and right members of a bilateral sensory neuron pair.


Assuntos
Caenorhabditis elegans/citologia , Células Quimiorreceptoras/citologia , Neurônios Aferentes/citologia , Animais , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/fisiologia , Células Quimiorreceptoras/fisiologia , Humanos , Neurônios Motores/citologia , Neurônios Motores/fisiologia , Neurônios Aferentes/fisiologia , Fatores de Transcrição/fisiologia
14.
Dev Cell ; 5(4): 621-33, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14536063

RESUMO

The mechanisms by which the diverse functional identities of neurons are generated are poorly understood. C. elegans responds to thermal and chemical stimuli using 12 types of sensory neurons. The Otx/otd homolog ttx-1 specifies the identities of the AFD thermosensory neurons. We show here that ceh-36 and ceh-37, the remaining two Otx-like genes in the C. elegans genome, specify the identities of AWC, ASE, and AWB chemosensory neurons, defining a role for this gene family in sensory neuron specification. All C. elegans Otx genes and rat Otx1 can substitute for ceh-37 and ceh-36, but only ceh-37 functionally substitutes for ttx-1. Functional substitution in the AWB neurons is mediated by activation of the same downstream target lim-4 by different Otx genes. Misexpression experiments indicate that although the specific identity adopted upon expression of an Otx gene may be constrained by the cellular context, individual Otx genes preferentially promote distinct neuronal identities.


Assuntos
Caenorhabditis elegans/genética , Genes Homeobox/fisiologia , Proteínas de Homeodomínio/genética , Neurônios Aferentes/metabolismo , Alelos , Animais , Animais Geneticamente Modificados , Comportamento Animal , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Drosophila , Reação de Fuga/efeitos dos fármacos , Regulação da Expressão Gênica , Genes de Helmintos , Genes Homeobox/genética , Marcadores Genéticos , Estruturas Genéticas , Genótipo , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/fisiologia , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Cetonas/farmacologia , Dados de Sequência Molecular , Mutação , Neurônios Aferentes/classificação , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/fisiologia , Odorantes , Fatores de Transcrição Otx , Alinhamento de Sequência
15.
Neuron ; 33(3): 369-81, 2002 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-11832225

RESUMO

Sensory signals regulate multiple developmental and behavioral circuits in C. elegans, providing a genetically tractable system in which to investigate the mechanisms underlying the acquisition and integration of sensory information. kin-29 mutants are defective in the expression of a set of chemoreceptor genes, and exhibit characteristics associated with altered sensory signaling, including increased lifespan, decreased body size, and deregulated entry into the dauer developmental stage. kin-29 encodes a Ser/Thr kinase with similarity to the MARK and AMPK/SNF1 family of kinases. We show that KIN-29 acts cell-autonomously and non-cell-autonomously in sensory neurons to regulate chemoreceptor expression, body size, and the dauer decision, suggesting that kin-29 function is essential for the correct acquisition and transduction of sensory information.


Assuntos
Caenorhabditis elegans/fisiologia , Células Quimiorreceptoras/fisiologia , Regulação da Expressão Gênica , Neurônios Aferentes/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Transporte Ativo do Núcleo Celular/fisiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas de Caenorhabditis elegans , Clonagem Molecular , Genes Reporter , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Temperatura Alta/efeitos adversos , Humanos , Expectativa de Vida , Dados de Sequência Molecular , Mutação , Feromônios/metabolismo , Filogenia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/classificação , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência
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