RESUMEN
Inositol polyphosphate multikinase (IPMK) is a conserved protein that initiates the production of inositol phosphate intracellular messengers and is critical for regulating a variety of cellular processes. Here, we report that the C. elegans IPMK-1, which is homologous to the mammalian inositol polyphosphate multikinase, plays a crucial role in regulating rhythmic behavior and development. The deletion mutant ipmk-1(tm2687) displays a long defecation cycle period and retarded postembryonic growth. The expression of functional ipmk-1::GFP was detected in the pharyngeal muscles, amphid sheath cells, the intestine, excretory (canal) cells, proximal gonad, and spermatheca. The expression of IPMK-1 in the intestine was sufficient for the wild-type phenotype. The IP3-kinase activity of IPMK-1 is required for defecation rhythms and postembryonic development. The defective phenotypes of ipmk-1(tm2687) could be rescued by a loss-of-function mutation in type I inositol 5-phosphatase homolog (IPP-5) and improved by a supplemental Ca2+ in the medium. Our work demonstrates that IPMK-1 and the signaling molecule inositol triphosphate (IP3) pathway modulate rhythmic behaviors and development by dynamically regulating the concentration of intracellular Ca2+ in C. elegans. Advances in understanding the molecular regulation of Ca2+ homeostasis and regulation of organism development may lead to therapeutic strategies that modulate Ca2+ signaling to enhance function and counteract disease processes. Unraveling the physiological role of IPMK and the underlying functional mechanism in C. elegans would contribute to understanding the role of IPMK in other species, especially in mammals, and benefit further research on the involvement of IPMK in disease.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , Caenorhabditis elegans/enzimología , Señalización del Calcio , Desarrollo Embrionario , Inositol 1,4,5-Trifosfato/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Defecación , Eliminación de Gen , Espacio Intracelular/metabolismo , Mutación/genética , Especificidad de Órganos , Fenotipo , Fosfotransferasas (Aceptor de Grupo Alcohol)/químicaRESUMEN
Seipin, an evolutionary conserved protein, plays pivotal roles during lipid droplet (LD) biogenesis and is associated with various human diseases with unclear mechanisms. Here, we analyzed Caenorhabditis elegans mutants deleted of the sole SEIPIN gene, seip-1 Homozygous seip-1 mutants displayed penetrant embryonic lethality, which is caused by the disruption of the lipid-rich permeability barrier, the innermost layer of the C. elegans embryonic eggshell. In C. elegans oocytes and embryos, SEIP-1 is associated with LDs and is crucial for controlling LD size and lipid homeostasis. The seip-1 deletion mutants reduced the ratio of polyunsaturated fatty acids (PUFAs) in their embryonic fatty acid pool. Interestingly, dietary supplementation of selected n-6 PUFAs rescued the embryonic lethality and defective permeability barrier. Accordingly, we propose that SEIP-1 may maternally regulate LD biogenesis and lipid homeostasis to orchestrate the formation of the permeability barrier for eggshell synthesis during embryogenesis. A lipodystrophy allele of seip-1 resulted in embryonic lethality as well and could be rescued by PUFA supplementation. These experiments support a great potential for using C. elegans to model SEIPIN-associated human diseases.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Cáscara de Huevo/embriología , Genes de Helminto , Proteínas de la Membrana/genética , Animales , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/ultraestructura , Proteínas de Caenorhabditis elegans/metabolismo , Suplementos Dietéticos , Modelos Animales de Enfermedad , Cáscara de Huevo/efectos de los fármacos , Cáscara de Huevo/ultraestructura , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Embrión no Mamífero/ultraestructura , Ácidos Grasos Insaturados/farmacología , Fertilización , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Gotas Lipídicas/metabolismo , Gotas Lipídicas/ultraestructura , Lipidómica , Proteínas de la Membrana/metabolismo , Mutación/genética , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Oocitos/ultraestructura , Ovulación/efectos de los fármacos , Permeabilidad , Saccharomyces cerevisiae/genéticaRESUMEN
Chemicals that are highly prevalent in our environment, such as phthalates and pesticides, have been linked to problems associated with reproductive health. However, rapid assessment of their impact on reproductive health and understanding how they cause such deleterious effects, remain challenging due to their fast-growing numbers and the limitations of various current toxicity assessment model systems. Here, we performed a high-throughput screen in C. elegans to identify chemicals inducing aneuploidy as a result of impaired germline function. We screened 46 chemicals that are widely present in our environment, but for which effects in the germline remain poorly understood. These included pesticides, phthalates, and chemicals used in hydraulic fracturing and crude oil processing. Of the 46 chemicals tested, 41% exhibited levels of aneuploidy higher than those detected for bisphenol A (BPA), an endocrine disruptor shown to affect meiosis, at concentrations correlating well with mammalian reproductive endpoints. We further examined three candidates eliciting aneuploidy: dibutyl phthalate (DBP), a likely endocrine disruptor and frequently used plasticizer, and the pesticides 2-(thiocyanomethylthio) benzothiazole (TCMTB) and permethrin. Exposure to these chemicals resulted in increased embryonic lethality, elevated DNA double-strand break (DSB) formation, activation of p53/CEP-1-dependent germ cell apoptosis, chromosomal abnormalities in oocytes at diakinesis, impaired chromosome segregation during early embryogenesis, and germline-specific alterations in gene expression. This study indicates that this high-throughput screening system is highly reliable for the identification of environmental chemicals inducing aneuploidy, and provides new insights into the impact of exposure to three widely used chemicals on meiosis and germline function.
Asunto(s)
Caenorhabditis elegans/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Contaminantes Ambientales/toxicidad , Células Germinativas/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Aneugénicos/toxicidad , Aneuploidia , Animales , Animales Modificados Genéticamente , Benzotiazoles/toxicidad , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Roturas del ADN de Doble Cadena , Dibutil Ftalato/toxicidad , Exposición a Riesgos Ambientales , Insecticidas/toxicidad , Meiosis/efectos de los fármacos , Permetrina/toxicidad , Plastificantes/toxicidad , Tiocianatos/toxicidadRESUMEN
The tricarboxylic acid (TCA) cycle (or citric acid cycle) is responsible for the complete oxidation of acetyl-CoA and formation of intermediates required for ATP production and other anabolic pathways, such as amino acid synthesis. Here, we uncovered an additional mechanism that may help explain the essential role of the TCA cycle in the early embryogenesis of Caenorhabditis elegans. We found that knockdown of citrate synthase (cts-1), the initial and rate-limiting enzyme of the TCA cycle, results in early embryonic arrest, but that this phenotype is not because of ATP and amino acid depletions. As a possible alternative mechanism explaining this developmental deficiency, we observed that cts-1 RNAi embryos had elevated levels of intracellular acetyl-CoA, the starting metabolite of the TCA cycle. Of note, we further discovered that these embryos exhibit hyperacetylation of mitochondrial proteins. We found that supplementation with acetylase-inhibiting polyamines, including spermidine and putrescine, counteracted the protein hyperacetylation and developmental arrest in the cts-1 RNAi embryos. Contrary to the hypothesis that spermidine acts as an acetyl sink for elevated acetyl-CoA, the levels of three forms of acetylspermidine, N1-acetylspermidine, N8-acetylspermidine, and N1,N8-diacetylspermidine, were not significantly increased in embryos treated with exogenous spermidine. Instead, we demonstrated that the mitochondrial deacetylase sirtuin 4 (encoded by the sir-2.2 gene) is required for spermidine's suppression of protein hyperacetylation and developmental arrest in the cts-1 RNAi embryos. Taken together, these results suggest the possibility that during early embryogenesis, acetyl-CoA consumption by the TCA cycle in C. elegans prevents protein hyperacetylation and thereby protects mitochondrial function.
Asunto(s)
Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Ciclo del Ácido Cítrico , Desarrollo Embrionario , Proteínas Mitocondriales/metabolismo , Acetilación , Adenosina Trifosfato/metabolismo , Animales , Ácido Aspártico/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Citrato (si)-Sintasa/deficiencia , Citrato (si)-Sintasa/genética , Ácido Cítrico/metabolismo , Ácido Glutámico/metabolismo , Espacio Intracelular/metabolismo , Factores de TiempoRESUMEN
Neural tube defects are common and serious birth defects in which the brain and/or spinal cord are exposed outside the body. Supplementation of foods with folic acid, an essential vitamin, is linked to a lower risk of neural tube defects; however, the mechanisms by which folic acid influence neural tube defect risk are unclear. Our research seeks to identify the basic cellular roles of known folic acid metabolism genes during morphogenesis using the roundworm Caenorhabditis elegans (C. elegans) as a simple model system. Here, we used live imaging to characterize defects in embryonic development when mel-32 is depleted. mel-32 is an essential folic acid metabolism gene in C. elegans and a homolog to the mammalian enzyme serine hydroxymethyltransferase (Shmt). Disruption of mel-32 resulted in a doubling or tripling of cell cycle lengths and a lack of directed cell movement during embryogenesis. However, the order of cell divisions, as determined by lineage analysis, is unchanged compared to wild type embryos. These results suggest that mel-32/Shmt is required for normal cell cycle lengths in C. elegans.
Asunto(s)
Caenorhabditis elegans/fisiología , Ciclo Celular , Embrión no Mamífero/fisiología , Desarrollo Embrionario , Ácido Fólico/metabolismo , Glicina Hidroximetiltransferasa/metabolismo , Defectos del Tubo Neural/fisiopatología , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/enzimología , Embrión no Mamífero/citología , Glicina Hidroximetiltransferasa/genética , MorfogénesisRESUMEN
Dual-view inverted selective plane illumination microscopy (diSPIM) enables high-speed, long-term, four-dimensional (4D) imaging with isotropic spatial resolution. It is also compatible with conventional sample mounting on glass coverslips. However, broadening of the light sheet at distances far from the beam waist and sample-induced scattering degrades diSPIM contrast and optical sectioning. We describe two simple improvements that address both issues and entail no additional hardware modifications to the base diSPIM. First, we demonstrate improved diSPIM sectioning by keeping the light sheet and detection optics stationary, and scanning the sample through the stationary light sheet (rather than scanning the broadening light sheet and detection plane through the stationary sample, as in conventional diSPIM). This stage-scanning approach allows a thinner sheet to be used when imaging laterally extended samples, such as fixed microtubules or motile mitochondria in cell monolayers, and produces finer contrast than does conventional diSPIM. We also used stage-scanning diSPIM to obtain high-quality, 4D nuclear datasets derived from an uncompressed nematode embryo, and performed lineaging analysis to track 97% of cells until twitching. Second, we describe the improvement of contrast in thick, scattering specimens by synchronizing light-sheet synthesis with the rolling, electronic shutter of our scientific complementary metal-oxide-semiconductor (sCMOS) detector. This maneuver forms a virtual confocal slit in the detection path, partially removing out-of-focus light. We demonstrate the applicability of our combined stage- and slit-scanning- methods by imaging pollen grains and nuclear and neuronal structures in live nematode embryos. All acquisition and analysis code is freely available online.
Asunto(s)
Microscopía/instrumentación , Microscopía/métodos , Animales , Antígenos de Plantas/ultraestructura , Caenorhabditis elegans/embriología , Línea Celular Tumoral , Rastreo Celular/métodos , Humanos , Imagenología Tridimensional/métodos , Microtúbulos/ultraestructura , Mitocondrias/fisiología , Mitocondrias/ultraestructura , Extractos VegetalesRESUMEN
The Caenorhabditis elegans left and right AWC olfactory neurons communicate to establish stochastic asymmetric identities, AWC(ON) and AWC(OFF), by inhibiting a calcium-mediated signaling pathway in the future AWC(ON) cell. NSY-4/claudin-like protein and NSY-5/innexin gap junction protein are the two parallel signals that antagonize the calcium signaling pathway to induce the AWC(ON) fate. However, it is not known how the calcium signaling pathway is downregulated by nsy-4 and nsy-5 in the AWC(ON) cell. Here we identify a microRNA, mir-71, that represses the TIR-1/Sarm1 adaptor protein in the calcium signaling pathway to promote the AWC(ON) identity. Similar to tir-1 loss-of-function mutants, overexpression of mir-71 generates two AWC(ON) neurons. tir-1 expression is downregulated through its 3' UTR in AWC(ON), in which mir-71 is expressed at a higher level than in AWC(OFF). In addition, mir-71 is sufficient to inhibit tir-1 expression in AWC through the mir-71 complementary site in the tir-1 3' UTR. Our genetic studies suggest that mir-71 acts downstream of nsy-4 and nsy-5 to promote the AWC(ON) identity in a cell autonomous manner. Furthermore, the stability of mature mir-71 is dependent on nsy-4 and nsy-5. Together, these results provide insight into the mechanism by which nsy-4 and nsy-5 inhibit calcium signaling to establish stochastic asymmetric AWC differentiation.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Señalización del Calcio/genética , Proteínas del Citoesqueleto/genética , MicroARNs/genética , Neuronas Receptoras Olfatorias/metabolismo , Regiones no Traducidas 3' , Animales , Tipificación del Cuerpo/genética , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Comunicación Celular , Diferenciación Celular , Conexinas/genética , Conexinas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes , MicroARNs/metabolismo , Neuronas Receptoras Olfatorias/citología , Neuronas Receptoras Olfatorias/embriología , Estabilidad del ARN , Receptores Acoplados a Proteínas GRESUMEN
Small-molecule inhibitors of protein function are powerful tools for biological analysis and can lead to the development of new drugs. However, a major bottleneck in generating useful small-molecule tools is target identification. Here we show that Caenorhabditis elegans can provide a platform for both the discovery of new bioactive compounds and target identification. We screened 14,100 small molecules for bioactivity in wild-type worms and identified 308 compounds that induce a variety of phenotypes. One compound that we named nemadipine-A induces marked defects in morphology and egg-laying. Nemadipine-A resembles a class of widely prescribed anti-hypertension drugs called the 1,4-dihydropyridines (DHPs) that antagonize the alpha1-subunit of L-type calcium channels. Through a genetic suppressor screen, we identified egl-19 as the sole candidate target of nemadipine-A, a conclusion that is supported by several additional lines of evidence. egl-19 encodes the only L-type calcium channel alpha1-subunit in the C. elegans genome. We show that nemadipine-A can also antagonize vertebrate L-type calcium channels, demonstrating that worms and vertebrates share the orthologous protein target. Conversely, FDA-approved DHPs fail to elicit robust phenotypes, making nemadipine-A a unique tool to screen for genetic interactions with this important class of drugs. Finally, we demonstrate the utility of nemadipine-A by using it to reveal redundancy among three calcium channels in the egg-laying circuit. Our study demonstrates that C. elegans enables rapid identification of new small-molecule tools and their targets.
Asunto(s)
Caenorhabditis elegans/efectos de los fármacos , Bloqueadores de los Canales de Calcio/aislamiento & purificación , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Piridinas/farmacología , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Bloqueadores de los Canales de Calcio/química , Bloqueadores de los Canales de Calcio/farmacocinética , Felodipino/aislamiento & purificación , Felodipino/farmacocinética , Felodipino/farmacología , Oviposición/efectos de los fármacos , Fenotipo , Piridinas/química , Piridinas/aislamiento & purificaciónRESUMEN
Mutations in MCOLN1, which encodes the protein h-mucolipin-1, result in the lysosomal storage disease Mucolipidosis Type IV. Studies on CUP-5, the human orthologue of h-mucolipin-1 in Caenorhabditis elegans, have shown that these proteins are required for lysosome biogenesis. We show here that the lethality in cup-5 mutant worms is due to two defects, starvation of embryonic cells and general developmental defects. Starvation leads to apoptosis through a CED-3-mediated pathway. We also show that providing worms with a lipid-soluble metabolite partially rescues the embryonic lethality but has no effect on the developmental defects, the major cause of the lethality. These results indicate that supplementing the metabolic deficiency of Mucolipidosis Type IV patients mat not be sufficient to alleviate the symptoms due to tissue degeneration.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Genes de Helminto , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Mucolipidosis/genética , Adenosina Trifosfato/metabolismo , Alelos , Animales , Animales Modificados Genéticamente , Autofagia , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Genes Letales , Humanos , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Mutación , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Notch , Canales Catiónicos TRPM/genética , Canales de Potencial de Receptor TransitorioRESUMEN
Monomethyl branched-chain fatty acids (mmBCFAs) are commonly found in many organisms from bacteria to mammals. In humans, they have been detected in skin, brain, blood, and cancer cells. Despite a broad distribution, mmBCFAs remain exotic in eukaryotes, where their origin and physiological roles are not understood. Here we report our study of the function and regulation of mmBCFAs in Caenorhabditis elegans, combining genetics, gas chromatography, and DNA microarray analysis. We show that C. elegans synthesizes mmBCFAs de novo and utilizes the long-chain fatty acid elongation enzymes ELO-5 and ELO-6 to produce two mmBCFAs, C15ISO and C17ISO. These mmBCFAs are essential for C. elegans growth and development, as suppression of their biosynthesis results in a growth arrest at the first larval stage. The arrest is reversible and can be overcome by feeding the arrested animals with mmBCFA supplements. We show not only that the levels of C15ISO and C17ISO affect the expression of several genes, but also that the activities of some of these genes affect biosynthesis of mmBCFAs, suggesting a potential feedback regulation. One of the genes, lpd-1, encodes a homolog of a mammalian sterol regulatory element-binding protein (SREBP 1c). We present results suggesting that elo-5 and elo-6 may be transcriptional targets of LPD-1. This study exposes unexpected and crucial physiological functions of C15ISO and C17ISO in C. elegans and suggests a potentially important role for mmBCFAs in other eukaryotes.
Asunto(s)
Acetiltransferasas/genética , Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/embriología , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Unión al ADN/genética , Ácidos Grasos/fisiología , Regulación del Desarrollo de la Expresión Génica , Acetiltransferasas/fisiología , Alelos , Animales , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Cromatografía de Gases , Proteínas de Unión al ADN/química , Escherichia coli/metabolismo , Retroalimentación Fisiológica , Genes Reporteros , Técnicas Genéticas , Variación Genética , Proteínas Fluorescentes Verdes/metabolismo , Modelos Biológicos , Modelos Químicos , Neuronas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Interferencia de ARN , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Factores de Tiempo , Factores de TranscripciónRESUMEN
Inositol 1,4,5-trisphosphate (IP(3)) is an important second messenger in animal cells and is central to a wide range of cellular responses. The major intracellular activity of IP(3) is to regulate release of Ca(2+) from intracellular stores through IP(3) receptors (IP(3)Rs). We describe a system for the transient disruption of IP(3) signaling in the model organism Caenorhabditis elegans. The IP(3) binding domain of the C. elegans IP(3)R, ITR-1, was expressed from heat shock-induced promoters in live animals. This results in a dominant-negative effect caused by the overexpressed IP(3) binding domain acting as an IP(3) "sponge." Disruption of IP(3) signaling resulted in disrupted defecation, a phenotype predicted by previous genetic studies. This approach also identified two new IP(3)-mediated processes. First, the up-regulation of pharyngeal pumping in response to food is dependent on IP(3) signaling. RNA-mediated interference studies and analysis of itr-1 mutants show that this process is also IP(3)R dependent. Second, the tissue-specific expression of the dominant-negative construct enabled us to circumvent the sterility associated with loss of IP(3) signaling through the IP(3)R and thus determine that IP(3)-mediated signaling is required for multiple steps in embryogenesis, including cytokinesis and gastrulation.
Asunto(s)
Caenorhabditis elegans/enzimología , Caenorhabditis elegans/fisiología , Inositol 1,4,5-Trifosfato/metabolismo , Transducción de Señal , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/embriología , División Celular , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Gástrula , Genes Dominantes , Calor , Microscopía Fluorescente , Faringe/embriología , Fenotipo , ARN/metabolismoRESUMEN
In wild-type Caenorhabditis elegans, six cells develop as receptors for gentle touch. In egl-44 and egl-46 mutants, two other neurons, the FLP cells, express touch receptor-like features. egl-44 and egl-46 also affect the differentiation of other neurons including the HSN neurons, two cells needed for egg laying. egl-44 encodes a member of the transcription enhancer factor family. The product of the egl-46 gene, two Drosophila proteins, and two proteins in human and mice define a new family of zinc finger proteins. Both egl-44 and egl-46 are expressed in FLP and HSN neurons (and other cells); expression of egl-46 is dependent on egl-44 in the FLP cells but not in the HSN cells. Wild-type touch cells express egl-46 but not egl-44. Moreover, ectopic expression of egl-44 in the touch cells prevents touch cell differentiation in an egl-46-dependent manner. The sequences of these genes and their nuclear location as seen with GFP fusions indicate that they repress transcription of touch cell characteristics in the FLP cells.
Asunto(s)
Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Proteínas de Drosophila , Proteínas de Insectos/genética , Mutación , Tacto/genética , Alelos , Secuencia de Aminoácidos , Animales , Linaje de la Célula , Clonación Molecular , ADN Complementario/metabolismo , Drosophila , Elementos de Facilitación Genéticos , Biblioteca de Genes , Genes Reporteros , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/metabolismo , Ratones , Datos de Secuencia Molecular , Neuronas/metabolismo , Fenotipo , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
BACKGROUND: Asymmetric cell division in the Caenorhabditis elegans embryos requires products of par (partitioning defective) genes 1-6 and atypical protein kinase C (aPKC), whereas Cdc42 and Rac, members of the Rho family GTPases, play an essential role in cell polarity establishment in yeast and mammalian cells. However, little is known about a link between PAR proteins and the GTPases in cell polarization. RESULTS: Here we have cloned cDNAs for three human homologues of PAR6, designated PAR6alpha, beta and gamma, comprising 345, 372 and 376 amino acids, respectively. The PAR6 proteins harbour a PDZ domain and a CRIB-like motif, and directly interact with GTP-bound Rac and Cdc42 via this motif and with the aPKC isoforms PKCiota/lambda and PKCzeta via the N-terminal head-to-head association. These interactions are not mutually exclusive, thereby allowing the PAR6 proteins to form a ternary complex with the GTPases and aPKC, both in vitro and in vivo. When PAR6 and aPKC are expressed with a constitutively active form of Rac in HeLa or COS-7 cells, these proteins co-localize to membrane ruffles, which are known to occur at the leading edge of polarized cells during cell movement. CONCLUSION: Human PAR6 homologues most likely play an important role in the cell polarization of mammalian cells, by functioning as an adaptor protein that links activated Rac and Cdc42 to aPKC signalling.
Asunto(s)
Caenorhabditis elegans/embriología , Proteínas de Ciclo Celular/metabolismo , Polaridad Celular , Proteína Quinasa C/metabolismo , Proteínas/fisiología , Proteínas de Saccharomyces cerevisiae , Proteínas de Unión al GTP rac/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans , Cartilla de ADN , ADN Complementario , Células HeLa , Humanos , Datos de Secuencia Molecular , Unión Proteica , Proteínas/química , Proteínas/genética , ARN Mensajero/genética , Homología de Secuencia de AminoácidoRESUMEN
A genome project for the species Caenorhabditis elegans has demonstrated the presence of eight cDNAs belonging to the major intrinsic protein (MIP) family. We previously characterized one of these cDNAs known as C01G6.1. C01G6.1 was confirmed to be a water channel and newly designated as AQP-CE1 [Am. J. Physiol. 275 (1998) C1459-C1464]. In this paper, we examined the function of another MIP protein encoded by F40F9.9. This cDNA encodes a 274-amino acid protein showing a high sequence identity with mammalian aquaporin-8 (AQP8) water channel (35%) and d-TIP (34%), an AQP of Arabidopsis. The expression of F40F9.9 in Xenopus oocytes increased the osmotic water permeability (P(f)) 10.4-fold, and the activation energy for P(f) from Arrhenius plot was 4.7 kcal/mol, suggesting that F40F9.9 is a water channel (AQP-CE2). AQP-CE2 was not permeable to glycerol or urea. Oocyte P(f) was reversibly inhibited by 58% after an incubation with 0.3 mM HgCl(2). To identify the mercury-sensitive site, four individual cysteine residues in AQP-CE2 (at positions 47, 132, 149, 259) were altered to serine by site-directed mutagenesis. Of these mutants, only C132S had a P(f) similar to that of the wild-type together with an acquired mercury resistance, suggesting that Cys-132 is the mercury-sensitive site. Similar results were obtained by the mutation of Cys-132 to alanine (C132A). Replacement of Cys-132 with tryptophan decreased P(f) by 64%, but P(f) was still 2.5 times higher than that of the control. Cys-132 is located in the transmembrane helix 3, close to the transition to the extracellular loop C. These results suggest that the transmembrane helix 3, including Cys-132, might participate in the aqueous pore formation, or, alternatively, that Cys-132 might contribute to the construction of the AQP protein.
Asunto(s)
Acuaporinas/fisiología , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/fisiología , Secuencia de Aminoácidos , Animales , Acuaporina 1 , Acuaporinas/química , Acuaporinas/genética , Sitios de Unión , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Cisteína/química , Cloruro de Mercurio/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Oocitos/metabolismo , ARN Complementario , Xenopus laevisRESUMEN
The CCCH finger protein PIE-1 is a regulator of germ cell fate that segregates with the germ lineage in early embryos. At each asymmetric division, PIE-1 is inherited preferentially by the germline daughter and is excluded from the somatic daughter. We show that this asymmetry is regulated at the protein level by two complementary mechanisms. The first acts before cell division to enrich PIE-1 in the cytoplasm destined for the germline daughter. The second acts after cell division to eliminate any PIE-1 left in the somatic daughter. The latter mechanism depends on PIE-1's first CCCH finger (ZF1), which targets PIE-1 for degradation in somatic blastomeres. ZF1s in two other germline proteins, POS-1 and MEX-1, are also degraded in somatic blastomeres, suggesting that localized degradation also acts on these proteins to exclude them from somatic lineages.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/embriología , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Actinas/fisiología , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , División Celular/efectos de los fármacos , Citocalasina D/farmacología , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Proteínas Fluorescentes Verdes , Proteínas del Helminto/química , Proteínas del Helminto/metabolismo , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Microtúbulos/fisiología , Nocodazol/farmacología , Dedos de Zinc , Cigoto/citología , Cigoto/fisiologíaRESUMEN
Null mutations in the C. elegans heterochronic gene lin-41 cause precocious expression of adult fates at larval stages. Increased lin-41 activity causes the opposite phenotype, reiteration of larval fates. let-7 mutations cause similar reiterated heterochronic phenotypes that are suppressed by lin-41 mutations, showing that lin-41 is negatively regulated by let-7. lin-41 negatively regulates the timing of LIN-29 adult specification transcription factor expression. lin-41 encodes an RBCC protein, and two elements in the lin-413'UTR are complementary to the 21 nucleotide let-7 regulatory RNA. A lin-41::GFP fusion gene is downregulated in the tissues affected by lin-41 at the time that the let-7 regulatory RNA is upregulated. We suggest that late larval activation of let-7 RNA expression downregulates LIN-41 to relieve inhibition of lin-29.
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Tipificación del Cuerpo/genética , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/embriología , Proteínas de Unión al ADN/metabolismo , Genes de Helminto , ARN de Helminto/metabolismo , Factores de Transcripción/metabolismo , Regiones no Traducidas 3' , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/genética , Regulación hacia Abajo , Regulación del Desarrollo de la Expresión Génica , Proteínas del Helminto/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Factores de Tiempo , Distribución Tisular , Factores de Transcripción/genética , Dedos de ZincRESUMEN
The daf-12 gene acts at the convergence of pathways regulating larval diapause, developmental age, and adult longevity in Caenorhabditis elegans. It encodes a nuclear receptor most closely related to two C. elegans receptors, NHR-8 and NHR-48, Drosophila DHR96, and vertebrate vitamin D and pregnane-X receptors. daf-12 has three predicted protein isoforms, two of which contain DNA- and ligand-binding domains, and one of which contains the ligand-binding domain only. Mutations cluster in DNA- and ligand-binding domains, but correspond to distinct phenotypic classes. DAF-12 is expressed widely in target tissues from embryo to adult, but is upregulated during midlarval stages. In the adult, expression persists in nervous system and somatic gonad, two tissues that regulate adult longevity. We propose that DAF-12 integrates hormonal signals in cellular targets to coordinate major life history traits.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/genética , Núcleo Celular/genética , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/fisiología , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/embriología , Cósmidos , ADN/metabolismo , ADN Complementario/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ligandos , Datos de Secuencia Molecular , Mutagénesis , Mutación , Fenotipo , Filogenia , Mapeo Físico de Cromosoma , Receptor X de Pregnano , Isoformas de Proteínas , Estructura Terciaria de Proteína , Receptores de Calcitriol/química , Receptores Citoplasmáticos y Nucleares/química , Receptores de Esteroides/química , Factores de Tiempo , Distribución TisularRESUMEN
The vertebrate CRMP (collapsin-response-mediator protein) gene family comprises at least four members. These CRMPs exhibit about 60% amino acid identity with vertebrate dihydropyrimidinase (DHP), an amidohydrolase involved in the pyrimidine degradation pathway. CRMP is also referred to as DRP (DHP-related protein), TOAD-64 (turned on after division, 64 kDa) and Ulip (Unc-33-like phosphoprotein). These vertebrate CRMPs are expressed mainly in early neuronal differentiation, which suggests that they play a role in neuronal development. In this study we isolated two cDNA clones from nematode C. elegans based on their sequence homology to vertebrate CRMPs and DHP. These two molecules, termed CeCRMP/DHP-1 and -2, turned out to be Ulip-B and -A, respectively, which were previously identified in the C. elegans genomic database by Byk et al. (1998). These newly isolated molecules were believed to represent a common ancestral state before the gene duplication between CRMPs and DHP. CeCRMP/DHP-1 and -2 protein retained all putative zinc-binding residues thought to be essential for the amidohydrolase activity of DHP and exhibited a weak amidohydrolase activity when 5-bromo-dihydrouracil was used as a substrate. Whole-mount in situ hybridization and expression analysis using GFP fusions revealed that CeCRMP/DHP-1 was transiently expressed in the hypodermis of C. elegans during the early larva stage. CeCRMP/DHP-1 was also expressed in a single nerve cell between the pharynx and ring neuropil. On the other hand, expression of CeCRMP/DHP-2 was observed in the body wall muscle throughout the lifespan of C. elegans. These results indicate that a major site of CeCRMP/DHP-1 and -2 expression is non-neuronal. Targeted gene disruption of CeCRMP/DHP-2 caused no particular difference in appearance or movement phenotype.
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Caenorhabditis elegans/genética , Proteínas del Tejido Nervioso/genética , Fosfoproteínas/genética , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Caenorhabditis elegans/embriología , Caenorhabditis elegans/crecimiento & desarrollo , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Mutación , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/metabolismo , Filogenia , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Mutations in the C. elegans vab-1 gene disrupt the coordinated movements of cells during two periods of embryogenesis. vab-1 mutants are defective in the movement of neuroblasts during closure of the ventral gastrulation cleft and in the movements of epidermal cells during ventral enclosure of the embryo by the epidermis. We show that vab-1 encodes a receptor tyrosine kinase of the Eph family. Disruption of the kinase domain of VAB-1 causes weak mutant phenotypes, indicating that VAB-1 may have both kinase-dependent and kinase-independent activities. VAB-1 is expressed in neurons during epidermal enclosure and is required in these cells for normal epidermal morphogenesis, demonstrating that cell-cell interactions are required between neurons and epidermal cells for epidermal morphogenesis.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/embriología , Proteínas de Ciclo Celular/fisiología , Epidermis/embriología , Gástrula/citología , Proteínas del Helminto/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/enzimología , Proteínas de Ciclo Celular/genética , Movimiento Celular , Clonación Molecular , ADN Complementario/genética , ADN de Helmintos/genética , Células Epidérmicas , Células Epiteliales , Proteínas del Helminto/genética , Datos de Secuencia Molecular , Morfogénesis , Mosaicismo , Mutación , Sistema Nervioso/embriología , Neuronas/citología , Fenotipo , Proteínas Tirosina Quinasas Receptoras/genética , Análisis de Secuencia de ADNRESUMEN
Little is known about differential expression, functions, regulation, and targeting of "atypical" protein kinase C (aPKC) isoenzymes in vivo. We have cloned and characterized a novel cDNA that encodes a Caenorhabditis elegans aPKC (PKC3) composed of 597 amino acids. In post-embryonic animals, a 647-base pair segment of promoter/enhancer DNA directs transcription of the 3.6-kilobase pair pkc-3 gene and coordinates accumulation of PKC3 protein in approximately 85 muscle, epithelial, and hypodermal cells. These cells are incorporated into tissues involved in feeding, digestion, excretion, and reproduction. Mammalian aPKCs promote mitogenesis and survival of cultured cells. In contrast, C. elegans PKC3 accumulates in non-dividing, terminally differentiated cells that will not undergo apoptosis. Thus, aPKCs may control cell functions that are independent of cell cycle progression and programmed cell death. PKC3 is also expressed during embryogenesis. Ablation of PKC3 function by microinjection of antisense RNA into oocytes yields disorganized, developmentally arrested embryos. Thus, PKC3 is essential for viability. PKC3 is enriched in particulate fractions of disrupted embryos and larvae. Immunofluorescence microscopy revealed that PKC3 accumulates near cortical actin cytoskeleton/plasma membrane at the apical surface of intestinal cells and in embryonic cells. A candidate anchoring/targeting protein, which binds PKC3 in vitro, has been identified.