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1.
J Cell Sci ; 136(6)2023 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-36762651

RESUMO

The advance of CRISPR/Cas9 technology has enabled us easily to generate gene knockout cell lines by introducing insertion-deletion mutations (indels) at the target site via the error-prone non-homologous end joining repair system. Frameshift-promoting indels can disrupt gene functions by generation of a premature stop codon. However, there is growing evidence that targeted genes are not always knocked out by the indel-based gene disruption. Here, we established a pipeline of CRISPR-del, which induces a large chromosomal deletion by cutting two different target sites, to perform 'complete' gene knockout efficiently in human diploid cells. Quantitative analyses show that the frequency of gene deletion with this approach is much higher than that of conventional CRISPR-del methods. The lengths of the deleted genomic regions demonstrated in this study are longer than those of 95% of the human protein-coding genes. Furthermore, the pipeline enabled the generation of a model cell line having a bi-allelic cancer-associated chromosomal deletion. Overall, these data lead us to propose that the CRISPR-del pipeline is an efficient and practical approach for producing 'complete' gene knockout cell lines in human diploid cells.


Assuntos
Sistemas CRISPR-Cas , Diploide , Humanos , Técnicas de Inativação de Genes , Sistemas CRISPR-Cas/genética , Mutação INDEL/genética , Linhagem Celular , Edição de Genes/métodos
2.
PLoS Genet ; 17(4): e1009457, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33872306

RESUMO

Spatiotemporal restriction of signaling plays a critical role in animal development and tissue homeostasis. All stem and progenitor cells in newly hatched C. elegans larvae are quiescent and capable of suspending their development until sufficient food is supplied. Here, we show that ptr-18, which encodes the evolutionarily conserved patched-related (PTR)/patched domain-containing (PTCHD) protein, temporally restricts the availability of extracellular hedgehog-related protein to establish the capacity of progenitor cells to maintain quiescence. We found that neural progenitor cells exit from quiescence in ptr-18 mutant larvae even when hatched under starved conditions. This unwanted reactivation depended on the activity of a specific set of hedgehog-related grl genes including grl-7. Unexpectedly, neither PTR-18 nor GRL-7 were expressed in newly hatched wild-type larvae. Instead, at the late embryonic stage, both PTR-18 and GRL-7 proteins were first localized around the apical membrane of hypodermal and neural progenitor cells and subsequently targeted for lysosomal degradation before hatching. Loss of ptr-18 caused a significant delay in GRL-7 clearance, causing this protein to be retained in the extracellular space in newly hatched ptr-18 mutant larvae. Furthermore, the putative transporter activity of PTR-18 was shown to be required for the appropriate function of the protein. These findings not only uncover a previously undescribed role of PTR/PTCHD in the clearance of extracellular hedgehog-related proteins via endocytosis-mediated degradation but also illustrate that failure to temporally restrict intercellular signaling during embryogenesis can subsequently compromise post-embryonic progenitor cell function.


Assuntos
Caenorhabditis elegans/genética , Endocitose/genética , Proteínas Hedgehog/genética , Receptores Patched/genética , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Membrana Celular/genética , Larva/genética , Larva/crescimento & desenvolvimento , Mutação/genética , Células-Tronco Neurais/metabolismo , Transdução de Sinais/genética
3.
BMC Genomics ; 24(1): 289, 2023 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-37248464

RESUMO

BACKGROUND: Recent advances in CRISPR technology have enabled us to perform gene knock-in in various species and cell lines. CRISPR-mediated knock-in requires donor DNA which serves as a template for homology-directed repair (HDR). For knock-in of short sequences or base substitutions, ssDNA donors are frequently used among various other forms of HDR donors, such as linear dsDNA. However, partly due to the complexity of long ssDNA preparation, it remains unclear whether ssDNA is the optimal type of HDR donors for insertion of long transgenes such as fluorescent reporters in human cells. RESULTS: In this study, we established a nuclease-based simple method for the preparation of long ssDNA with high yield and purity, and comprehensively compared the performance of ssDNA and dsDNA donors with 90 bases of homology arms for endogenous gene tagging with long transgenes in human diploid RPE1 and HCT116 cells. Quantification using flow cytometry revealed lower efficiency of endogenous fluorescent tagging with ssDNA donors than with dsDNA. By analyzing knock-in outcomes using long-read amplicon sequencing and a classification framework, a variety of mis-integration events were detected regardless of the donor type. Importantly, the ratio of precise insertion was lower with ssDNA donors than with dsDNA. Moreover, in off-target integration analyses using donors without homology arms, ssDNA and dsDNA were comparably prone to non-homologous integration. CONCLUSIONS: These results indicate that ssDNA is not superior to dsDNA as long HDR donors with relatively short homology arms for gene knock-in in human RPE1 and HCT116 cells.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Células HCT116 , Diploide , DNA/metabolismo , DNA de Cadeia Simples/genética , Técnicas de Introdução de Genes , Edição de Genes/métodos
4.
Biochem Biophys Res Commun ; 520(3): 532-537, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31615656

RESUMO

The animal body contains various types of stem and progenitor cells. These undifferentiated cells coordinate the balance between quiescence and proliferation with dynamics of various physiological conditions such as the developmental stage, food availability, and injury. Although regulation of such coordination plays a critical role in maintaining tissue homeostasis, controlling the growth rate and regeneration, much of its mechanism remains elusive. Newly hatched Caenorhabditis elegans larvae possess quiescent stem and progenitor cells in several tissues, and these cells are reactivated by the insulin/insulin-like growth factor (IGF) signaling (IIS) pathway only when sufficient food is supplied. Maintenance of the quiescence of neuronal and mesodermal progenitor cells requires microRNA (miRNA), miR-235, which is upregulated under the starvation. On the other hand, feeding ample food downregulates the miRNA via the activity of the IIS pathway. As miR-235 in the hypodermis can non-autonomously regulate quiescence of neuronal and mesodermal progenitor cells, a cell-cell signaling pathway has been hypothesized to act downstream of the miRNA. Here, we provide evidence that two hedgehog-related (hh-r) genes, grl-5 and grl-7, are targets of miR-235 that promote reactivation of quiescent neuroblasts. These grl genes possess an miR-235 binding site on 3'UTRs of their transcripts, and are upregulated in starved mir-235 mutant larvae. grl-5 and grl-7 promoters can continuously drive the expression of GFP-pest reporter protein in the hypodermis under the fed condition. However, expression of these reporters is strikingly downregulated under the starvation condition after hatching. We found that miR-235 can repress expression of reporter genes via the predicted miR-235 binding sites on the grl-5 and grl-7 3'UTRs. Furthermore, activity of grl-5 and grl-7 genes are required for reactivation of neural progenitor cells in starved mir-235 mutant larvae. These findings suggest that the IIS pathway-miR-235 signaling in the hypodermis non-autonomously regulates quiescence of neural progenitor cells, partly via grl-5 and grl-7.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Genes de Helmintos , Proteínas Hedgehog/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Regiões 3' não Traduzidas , Animais , Animais Geneticamente Modificados , Sequência de Bases , Sítios de Ligação/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Hedgehog/metabolismo , Larva/citologia , Larva/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Mutação , Fase de Repouso do Ciclo Celular/genética , Transdução de Sinais/genética , Somatomedinas/metabolismo
5.
Nature ; 497(7450): 503-6, 2013 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-23644454

RESUMO

The coordination of stem- and blast-cell behaviours, such as self-renewal, differentiation and quiescence, with physiological changes underlies growth, regeneration and tissue homeostasis. Germline stem and somatic blast cells in newly hatched Caenorhabditis elegans larvae can suspend postembryonic development, which consists of diverse cellular events such as migration, proliferation and differentiation, until the nutritional state becomes favourable (termed L1 diapause). Although previous studies showed that the insulin/insulin-like growth factor (IGF) signalling (IIS) pathway regulates this developmental quiescence, the detailed mechanism by which the IIS pathway enables these multipotent cells to respond to nutrient availability is unknown. Here we show in C. elegans that the microRNA (miRNA) miR-235, a sole orthologue of mammalian miR-92 from the oncogenic miR-17-92 cluster, acts in the hypodermis and glial cells to arrest postembryonic developmental events in both neuroblasts and mesoblasts. Expression of mir-235 persists during L1 diapause, and decreases upon feeding in a manner dependent on the IIS pathway. Upregulation of one of the miR-235 targets, nhr-91, which encodes an orthologue of mammalian germ cell nuclear factor, is responsible for defects caused by loss of the miRNA. Our findings establish a novel role of a miR-92 orthologue in coupling blast-cell behaviours to the nutritional state.


Assuntos
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Ativação Linfocitária/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Estado Nutricional , Animais , Sequência de Bases , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/imunologia , Regulação para Baixo , Embrião não Mamífero/metabolismo , Privação de Alimentos , Humanos , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Larva/citologia , Larva/metabolismo , Ativação Linfocitária/fisiologia , Dados de Sequência Molecular , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neuroglia/metabolismo , Estado Nutricional/genética , RNA Longo não Codificante , Transdução de Sinais , Tela Subcutânea/metabolismo
6.
Nat Commun ; 15(1): 5793, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38987256

RESUMO

Temperature is a critical environmental cue that controls the development and lifespan of many animal species; however, mechanisms underlying low-temperature adaptation are poorly understood. Here, we describe cold-inducible diapause (CID), another type of diapause induced by low temperatures in Caenorhabditis elegans. A premature stop codon in heat shock factor 1 (hsf-1) triggers entry into CID at 9 °C, whereas wild-type animals enter CID at 4 °C. Furthermore, both wild-type and hsf-1(sy441) mutant animals undergoing CID can survive for weeks, and resume growth at 20 °C. Using epistasis analysis, we demonstrate that neural signalling pathways, namely tyraminergic and neuromedin U signalling, regulate entry into CID of the hsf-1 mutant. Overexpression of anti-ageing genes, such as hsf-1, XBP1/xbp-1, FOXO/daf-16, Nrf2/skn-1, and TFEB/hlh-30, also inhibits CID entry of the hsf-1 mutant. Based on these findings, we hypothesise that regulators of the hsf-1 mutant CID may impact longevity, and successfully isolate 16 long-lived mutants among 49 non-CID mutants via genetic screening. Furthermore, we demonstrate that the nonsense mutation of MED23/sur-2 prevents CID entry of the hsf-1(sy441) mutant and extends lifespan. Thus, CID is a powerful model to investigate neural networks involving cold acclimation and to explore new ageing mechanisms.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Temperatura Baixa , Proteínas de Ligação a DNA , Diapausa , Longevidade , Fatores de Transcrição , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Diapausa/genética , Diapausa/fisiologia , Longevidade/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Mutação , Transdução de Sinais , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Códon sem Sentido/genética , Neuropeptídeos/metabolismo , Neuropeptídeos/genética , Proteínas de Transporte , Fatores de Transcrição Hélice-Alça-Hélice Básicos
7.
Nat Commun ; 15(1): 2216, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38519454

RESUMO

The triplet microtubule, a core structure of centrioles crucial for the organization of centrosomes, cilia, and flagella, consists of unclosed incomplete microtubules. The mechanisms of its assembly represent a fundamental open question in biology. Here, we discover that the ciliopathy protein HYLS1 and the ß-tubulin isotype TUBB promote centriole triplet microtubule assembly. HYLS1 or a C-terminal tail truncated version of TUBB generates tubulin-based superstructures composed of centriole-like incomplete microtubule chains when overexpressed in human cells. AlphaFold-based structural models and mutagenesis analyses further suggest that the ciliopathy-related residue D211 of HYLS1 physically traps the wobbling C-terminal tail of TUBB, thereby suppressing its inhibitory role in the initiation of the incomplete microtubule assembly. Overall, our findings provide molecular insights into the biogenesis of atypical microtubule architectures conserved for over a billion years.


Assuntos
Centríolos , Ciliopatias , Humanos , Centríolos/metabolismo , Tubulina (Proteína)/metabolismo , Microtúbulos/metabolismo , Centrossomo/metabolismo , Ciliopatias/metabolismo , Cílios/metabolismo , Proteínas/metabolismo
8.
Genes Cells ; 17(9): 778-89, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22897658

RESUMO

Ras-family GTPases regulate a wide variety of cellular functions including cell growth and differentiation. Di-Ras, which belongs to a distinct subfamily of Ras-family GTPases, is expressed predominantly in brain, but the role of Di-Ras in nervous systems remains totally unknown. Here, we report that the Caenorhabditis elegans Di-Ras homologue drn-1 is expressed specifically in neuronal cells and involved in synaptic function at neuromuscular junctions. Loss of function of drn-1 conferred resistance to the acetylcholinesterase inhibitor aldicarb and partially suppressed the aldicarb-hypersensitive phenotypes of heterotrimeric G-protein mutants, in which acetylcholine release is up-regulated. drn-1 mutants displayed no apparent defects in the axonal distribution of the membrane-bound second messenger diacylglycerol (DAG), which is a key stimulator of acetylcholine release. Finally, we have identified EPAC-1, a C. elegans Epac homologue, as a binding partner for DRN-1. Deletion mutants of epac-1 displayed an aldicarb-resistant phenotype as drn-1 mutants. Genetic analysis of drn-1 and epac-1 showed that they acted in the same pathway to control acetylcholine release. Furthermore, DRN-1 and EPAC-1 were co-immunoprecipitated. These findings suggest that DRN-1 may function cooperatively with EPAC-1 to modulate synaptic activity in C. elegans.


Assuntos
Caenorhabditis elegans/enzimologia , Neurônios Colinérgicos/enzimologia , GTP Fosfo-Hidrolases/metabolismo , Transmissão Sináptica , Acetilcolina/genética , Acetilcolina/metabolismo , Aldicarb/farmacologia , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Neurônios Colinérgicos/efeitos dos fármacos , Neurônios Colinérgicos/fisiologia , Inibidores da Colinesterase/farmacologia , Resistência a Medicamentos , GTP Fosfo-Hidrolases/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Imunoprecipitação/métodos , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/genética , Junção Neuromuscular/metabolismo , Fenótipo , Mapeamento de Interação de Proteínas , Deleção de Sequência
9.
Commun Biol ; 6(1): 1107, 2023 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-37914777

RESUMO

Phototoxicity is an important issue in fluorescence live imaging of light-sensitive cellular processes such as mitosis. Among several approaches to reduce phototoxicity, the addition of antioxidants to the media has been used as a simple method. Here, we analyzed the impact of phototoxicity on the mitotic progression in fluorescence live imaging of human cells and performed a screen to identify the most efficient antioxidative agents that reduce it. Quantitative analysis shows that high amounts of light illumination cause various mitotic defects such as prolonged mitosis and delays of chromosome alignment and centrosome separation. Among several antioxidants, our screen reveals that ascorbic acid significantly alleviates these phototoxic effects in mitosis. Furthermore, we demonstrate that adding ascorbic acid to the media enables fluorescence imaging of mitotic events at very high temporal resolution without obvious photodamage. Thus, this study provides an optimal method to effectively reduce the phototoxic effects in fluorescence live cell imaging.


Assuntos
Antioxidantes , Ácido Ascórbico , Humanos , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Mitose , Ciclo Celular , Cromossomos
10.
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
12.
Curr Biol ; 16(8): 773-9, 2006 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-16631584

RESUMO

The molecular pathways that link nutritional cues to developmental programs are poorly understood. Caenorhabditis elegans hatchlings arrest in a dormant state termed "L1 diapause" until food is supplied. However, little is known about what signal transduction pathways mediate nutritional status to control arrest and initiation of postembryonic development. We report that C. elegans embryonic germline precursors undergo G2 arrest with condensed chromosomes and remain arrested throughout L1 diapause. Loss of the DAF-18/PTEN tumor suppressor bypasses this arrest, resulting in inappropriate germline growth dependent on the AGE-1/PI-3 and AKT-1/PKB kinases. DAF-18 also regulates an insulin/IGF-like pathway essential for longevity and dauer larva formation. However, DAF-16/FoxO, which is repressed by this pathway, is not required for germline arrest in L1 diapause. Thus, these findings indicate that quiescence of germline development during L1 diapause is not a passive consequence of nutrient deprivation, but rather is actively maintained by DAF-18 through a pathway distinct from that which regulates longevity and dauer formation.


Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/crescimento & desenvolvimento , Células Germinativas/fisiologia , Animais , Fase G2/fisiologia , Larva/fisiologia , Longevidade , Necessidades Nutricionais
13.
Curr Biol ; 25(9): 1241-8, 2015 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-25891400

RESUMO

The nutritional status of an organism can greatly impact the function and behavior of stem and progenitor cells [1]. However, the regulatory circuits that inform these cells about the dietary environment remain to be elucidated. Newly hatched C. elegans larvae (L1s) halt development in "L1 arrest" or "L1 diapause" until ample food is encountered and triggers stem and progenitor cells to exit from quiescence [2]. The insulin/insulin-like growth factor signaling (IIS) pathway plays a key role in this reactivation [3, 4], but its site(s) of action have not been elucidated nor have the nutrient molecule(s) that stimulate the pathway been identified. By tissue-specifically modulating the activity of its components, we demonstrate that the IIS pathway acts in the hypodermis to regulate nutrition-responsive reactivation of neural and mesodermal progenitor cells. We identify ethanol, a likely component of the natural Caenorhabditis habitat, and amino acids as nutrients that synergistically reactivate somatic progenitor cells and upregulate expression of insulin-like genes in starved L1 larvae. The hypodermis likely senses the availability of amino acids because forced activation of the amino-acid-responsive Rag-TORC1 (target of rapamycin complex 1) pathway in this tissue can also release somatic progenitor cell quiescence in the presence of ethanol. Finally, there appears to be crosstalk between the IIS and Rag-TORC1 pathways because constitutive activation of the IIS pathway requires Rag to promote reactivation. This work demonstrates that ethanol and amino acids act as dietary cues via the IIS and Rag-TORC1 pathways in the hypodermis to coordinately control progenitor cell behavior.


Assuntos
Aminoácidos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Etanol/farmacologia , Células-Tronco/efeitos dos fármacos , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Movimento Celular , Dieta , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/metabolismo , Células-Tronco/metabolismo , Tela Subcutânea/metabolismo
14.
Mol Biol Cell ; 24(10): 1584-92, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23485564

RESUMO

Efficient clearance of apoptotic cells by phagocytes is important for development, tissue homeostasis, and the prevention of autoimmune responses. Phagosomes containing apoptotic cells undergo acidification and mature from Rab5-positive early to Rab7-positive late stages. Phagosomes finally fuse with lysosomes to form phagolysosomes, which degrade apoptotic cells; however, the molecular mechanism underlying phagosome-lysosome fusion is not fully understood. Here we show that the Caenorhabditis elegans Arf-like small GTPase Arl8 (ARL-8) is involved in phagolysosome formation and is required for the efficient removal of apoptotic cells. Loss of function of arl-8 results in the accumulation of apoptotic germ cells. Both the engulfment of the apoptotic cells by surrounding somatic sheath cells and the phagosomal maturation from RAB-5- to RAB-7-positive stages occur in arl-8 mutants. However, the phagosomes fail to fuse with lysosomes in the arl-8 mutants, leading to the accumulation of RAB-7-positive phagosomes and the delayed degradation of apoptotic cells. ARL-8 localizes primarily to lysosomes and physically interacts with the homotypic fusion and protein sorting complex component VPS-41. Collectively our findings reveal that ARL-8 facilitates apoptotic cell removal in vivo by mediating phagosome-lysosome fusion during phagocytosis.


Assuntos
Apoptose , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/enzimologia , GTP Fosfo-Hidrolases/fisiologia , Fagossomos/enzimologia , Animais , Caenorhabditis elegans/citologia , Células Germinativas/fisiologia , Gônadas/citologia , Gônadas/enzimologia , Lisossomos/enzimologia , Transporte Proteico , Imagem com Lapso de Tempo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
15.
Biol Open ; 1(10): 929-36, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-23213370

RESUMO

Mechanisms controlling development, growth, and metabolism are coordinated in response to changes in environmental conditions, enhancing the likelihood of survival to reproductive maturity. Much remains to be learned about the molecular basis underlying environmental influences on these processes. C. elegans larvae enter a developmentally dormant state called L1 diapause when hatched into nutrient-poor conditions. The nematode pten homologue daf-18 is essential for maintenance of survival and germline stem cell quiescence during this period (Fukuyama et al., 2006; Sigmond et al., 2008), but the details of the signaling network(s) in which it functions remain to be elucidated. Here, we report that animals lacking both aak-1 and aak-2, which encode the two catalytic α subunits of AMP-activated protein kinase (AMPK), show reduced viability and failure to maintain mitotic quiescence in germline stem cells during L1 diapause. Furthermore, failure to arrest germline proliferation has a long term consequence; aak double mutants that have experienced L1 diapause develop into sterile adults when returned to food, whereas their continuously fed siblings are fertile. Both aak and daf-18 appear to maintain germline quiescence by inhibiting activity of the common downstream target, TORC1 (TOR Complex 1). In contrast, rescue of the lethality phenotype indicates that aak-2 acts not only in the intestine, as does daf-18, but also in neurons, likely promoting survival by preventing energy deprivation during L1 diapause. These results not only provide evidence that AMPK contributes to survival during L1 diapause in a manner distinct from that by which it controls dauer diapause, but they also suggest that AMPK suppresses TORC1 activity to maintain stem cell quiescence.

16.
Mol Biol Cell ; 21(14): 2434-42, 2010 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-20484575

RESUMO

Late endocytic organelles including lysosomes are highly dynamic acidic organelles. Late endosomes and lysosomes directly fuse for content mixing to form hybrid organelles, from which lysosomes are reformed. It is not fully understood how these processes are regulated and maintained. Here we show that the Caenorhabditis elegans ARL-8 GTPase is localized primarily to lysosomes and involved in late endosome-lysosome fusion in the macrophage-like coelomocytes. Loss of arl-8 results in an increase in the number of late endosomal/lysosomal compartments, which are smaller than wild type. In arl-8 mutants, late endosomal compartments containing endocytosed macromolecules fail to fuse with lysosomal compartments enriched in the aspartic protease ASP-1. Furthermore, loss of arl-8 strongly suppresses formation of enlarged late endosome-lysosome hybrid organelles caused by mutations of cup-5, which is the orthologue of human mucolipin-1. These findings suggest that ARL-8 mediates delivery of endocytosed macromolecules to lysosomes by facilitating late endosome-lysosome fusion.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Endocitose , GTP Fosfo-Hidrolases/metabolismo , Lisossomos/metabolismo , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Compartimento Celular , Desenvolvimento Embrionário , Endossomos/enzimologia , Membranas Intracelulares/enzimologia , Mutação/genética , Transporte Proteico , Soroalbumina Bovina/metabolismo , Frações Subcelulares/metabolismo , Fatores de Tempo
17.
Dev Biol ; 260(1): 273-86, 2003 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-12885569

RESUMO

Following a phase of rapid proliferation, cells in developing embryos must decide when to cease division and then whether to survive and differentiate or instead undergo programmed death. In screens for genes that regulate embryonic patterning of the endoderm in Caenorhabditis elegans, we identified overlapping chromosomal deletions that define a gene required for these decisions. These deletions result in embryonic hyperplasia in multiple somatic tissues, excessive numbers of cell corpses, and profound defects in morphogenesis and differentiation. However, cell-cycle arrest of the germline is unaffected. Cell lineage analysis of these mutants revealed that cells that normally stop dividing earlier than their close relatives instead undergo an extra round of division. These deletions define a genomic region that includes cki-1 and cki-2, adjacent genes encoding members of the Cip/Kip family of cyclin-dependent kinase inhibitors. cki-1 alone can rescue the cell proliferation, programmed cell death, and differentiation and morphogenesis defects observed in these mutants. In contrast, cki-2 is not capable of significantly rescuing these phenotypes. RNA interference of cki-1 leads to embryonic lethality with phenotypes similar to, or more severe than, the deletion mutants. cki-1 and -2 gene reporters show distinct expression patterns; while both are expressed at around the time that embryonic cells exit the cell cycle, cki-2 also shows marked expression starting early in embryogenesis, when rapid cell division occurs. Our findings demonstrate that cki-1 activity plays an essential role in embryonic cell cycle arrest, differentiation and morphogenesis, and suggest that it may be required to suppress programmed cell death or engulfment of cell corpses.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Animais , Apoptose/genética , Caenorhabditis elegans/genética , Diferenciação Celular , Divisão Celular , Linhagem da Célula , Deleção Cromossômica , Proteínas Inibidoras de Quinase Dependente de Ciclina , Embrião não Mamífero/citologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Ligação Genética , Morfogênese , Mutagênese , Mutação , RNA Interferente Pequeno/metabolismo
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