RESUMO
Spindle assembly requires the coordinated action of multiple cellular structures to nucleate and organize microtubules in a precise spatiotemporal manner. Among them, the contributions of centrosomes, chromosomes, and microtubules have been well studied, yet the involvement of membrane-bound organelles remains largely elusive. Here, we provide mechanistic evidence for a membrane-based, Golgi-derived microtubule assembly pathway in mitosis. Upon mitotic entry, the Golgi matrix protein GM130 interacts with importin α via a classical nuclear localization signal that recruits importin α to the Golgi membranes. Sequestration of importin α by GM130 liberates the spindle assembly factor TPX2, which activates Aurora-A kinase and stimulates local microtubule nucleation. Upon filament assembly, nascent microtubules are further captured by GM130, thus linking Golgi membranes to the spindle. Our results reveal an active role for the Golgi in regulating spindle formation to ensure faithful organelle inheritance.
Assuntos
Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Animais , Aurora Quinase A/metabolismo , Células HeLa , Humanos , Carioferinas/metabolismo , Camundongos , Microtúbulos/metabolismo , Mitose , Fosfoproteínas/metabolismo , Fuso Acromático , Xenopus/metabolismo , Proteínas de Xenopus/metabolismoRESUMO
Secreted Wnt morphogens are signaling molecules essential for embryogenesis, pathogenesis, and regeneration and require distinct modifications for secretion, gradient formation, and activity. Whether Wnt proteins can be posttranslationally inactivated during development and homeostasis is unknown. Here we identify, through functional cDNA screening, a transmembrane protein Tiki1 that is expressed specifically in the dorsal Spemann-Mangold Organizer and is required for anterior development during Xenopus embryogenesis. Tiki1 antagonizes Wnt function in embryos and human cells via a TIKI homology domain that is conserved from bacteria to mammals and acts likely as a protease to cleave eight amino-terminal residues of a Wnt protein, resulting in oxidized Wnt oligomers that exhibit normal secretion but minimized receptor-binding capability. Our findings identify a Wnt-specific protease that controls head formation, reveal a mechanism for morphogen inactivation through proteolysis-induced oxidation-oligomerization, and suggest a role of the Wnt amino terminus in evasion of oxidizing inactivation. TIKI proteins may represent potential therapeutic targets.
Assuntos
Padronização Corporal , Cabeça/embriologia , Proteínas de Membrana/metabolismo , Metaloproteases/metabolismo , Via de Sinalização Wnt , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Sequência de Aminoácidos , Animais , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/genética , Metaloproteases/genética , Dados de Sequência Molecular , Organizadores Embrionários/metabolismo , Alinhamento de Sequência , Xenopus/metabolismo , Proteínas de Xenopus/genéticaRESUMO
The eukaryotic replicative DNA helicase, CMG, unwinds DNA by an unknown mechanism. In some models, CMG encircles and translocates along one strand of DNA while excluding the other strand. In others, CMG encircles and translocates along duplex DNA. To distinguish between these models, replisomes were confronted with strand-specific DNA roadblocks in Xenopus egg extracts. An ssDNA translocase should stall at an obstruction on the translocation strand but not the excluded strand, whereas a dsDNA translocase should stall at obstructions on either strand. We found that replisomes bypass large roadblocks on the lagging strand template much more readily than on the leading strand template. Our results indicate that CMG is a 3' to 5' ssDNA translocase, consistent with unwinding via "steric exclusion." Given that MCM2-7 encircles dsDNA in G1, the data imply that formation of CMG in S phase involves remodeling of MCM2-7 from a dsDNA to a ssDNA binding mode.
Assuntos
DNA Helicases/metabolismo , Replicação do DNA , DNA/metabolismo , Xenopus/metabolismo , Animais , DNA de Cadeia Simples/metabolismo , Modelos Biológicos , Fase SRESUMO
Neural crest cells (NCCs) are migratory, multipotent embryonic cells that are unique to vertebrates and form an array of clade-defining adult features. The evolution of NCCs has been linked to various genomic events, including the evolution of new gene-regulatory networks1,2, the de novo evolution of genes3 and the proliferation of paralogous genes during genome-wide duplication events4. However, conclusive functional evidence linking new and/or duplicated genes to NCC evolution is lacking. Endothelin ligands (Edns) and endothelin receptors (Ednrs) are unique to vertebrates3,5,6, and regulate multiple aspects of NCC development in jawed vertebrates7-10. Here, to test whether the evolution of Edn signalling was a driver of NCC evolution, we used CRISPR-Cas9 mutagenesis11 to disrupt edn, ednr and dlx genes in the sea lamprey, Petromyzon marinus. Lampreys are jawless fishes that last shared a common ancestor with modern jawed vertebrates around 500 million years ago12. Thus, comparisons between lampreys and gnathostomes can identify deeply conserved and evolutionarily flexible features of vertebrate development. Using the frog Xenopus laevis to expand gnathostome phylogenetic representation and facilitate side-by-side analyses, we identify ancient and lineage-specific roles for Edn signalling. These findings suggest that Edn signalling was activated in NCCs before duplication of the vertebrate genome. Then, after one or more genome-wide duplications in the vertebrate stem, paralogous Edn pathways functionally diverged, resulting in NCC subpopulations with different Edn signalling requirements. We posit that this new developmental modularity facilitated the independent evolution of NCC derivatives in stem vertebrates. Consistent with this, differences in Edn pathway targets are associated with differences in the oropharyngeal skeleton and autonomic nervous system of lampreys and modern gnathostomes. In summary, our work provides functional genetic evidence linking the origin and duplication of new vertebrate genes with the stepwise evolution of a defining vertebrate novelty.
Assuntos
Endotelinas/metabolismo , Evolução Molecular , Crista Neural/citologia , Petromyzon/metabolismo , Transdução de Sinais , Xenopus/metabolismo , Animais , Desenvolvimento Ósseo , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Linhagem da Célula , Endotelinas/genética , Feminino , Cabeça/crescimento & desenvolvimento , Coração/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Ligantes , Masculino , Petromyzon/genética , Petromyzon/crescimento & desenvolvimento , Receptores de Endotelina/deficiência , Receptores de Endotelina/genética , Receptores de Endotelina/metabolismo , Xenopus/genética , Xenopus/crescimento & desenvolvimentoRESUMO
Hybrid incompatibility as a kind of reproductive isolation contributes to speciation. The nucleocytoplasmic incompatibility between Xenopus tropicalis eggs and Xenopus laevis sperm (te×ls) leads to specific loss of paternal chromosomes 3L and 4L. The hybrids die before gastrulation, of which the lethal causes remain largely unclear. Here, we show that the activation of the tumor suppressor protein P53 at late blastula stage contributes to this early lethality. We find that in stage 9 embryos, P53-binding motif is the most enriched one in the up-regulated Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) peaks between te×ls and wild-type X. tropicalis controls, which correlates with an abrupt stabilization of P53 protein in te×ls hybrids at stage 9. Inhibition of P53 activity via either tp53 knockout or overexpression of a dominant-negative P53 mutant or Murine double minute 2 proto-oncogene (Mdm2), a negative regulator of P53, by mRNA injection can rescue the te×ls early lethality. Our results suggest a causal function of P53 on hybrid lethality prior to gastrulation.
Assuntos
Sêmen , Proteína Supressora de Tumor p53 , Animais , Masculino , Camundongos , Cromossomos/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Sêmen/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
Sister chromatid cohesion is essential for chromosome segregation and is mediated by cohesin bound to DNA. Cohesin-DNA interactions can be reversed by the cohesion-associated protein Wapl, whereas a stably DNA-bound form of cohesin is thought to mediate cohesion. In vertebrates, Sororin is essential for cohesion and stable cohesin-DNA interactions, but how Sororin performs these functions is unknown. We show that DNA replication and cohesin acetylation promote binding of Sororin to cohesin, and that Sororin displaces Wapl from its binding partner Pds5. In the absence of Wapl, Sororin becomes dispensable for cohesion. We propose that Sororin maintains cohesion by inhibiting Wapl's ability to dissociate cohesin from DNA. Sororin has only been identified in vertebrates, but we show that many invertebrate species contain Sororin-related proteins, and that one of these, Dalmatian, is essential for cohesion in Drosophila. The mechanism we describe here may therefore be widely conserved among different species.
Assuntos
Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/química , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/química , Humanos , Fase S , Xenopus/metabolismo , CoesinasRESUMO
The size of the nucleus varies among different cell types, species, and disease states, but mechanisms of nuclear size regulation are poorly understood. We investigated nuclear scaling in the pseudotetraploid frog Xenopus laevis and its smaller diploid relative Xenopus tropicalis, which contains smaller cells and nuclei. Nuclear scaling was recapitulated in vitro using egg extracts, demonstrating that titratable cytoplasmic factors determine nuclear size to a greater extent than DNA content. Nuclear import rates correlated with nuclear size, and varying the concentrations of two transport factors, importin α and Ntf2, was sufficient to account for nuclear scaling between the two species. Both factors modulated lamin B3 import, with importin α increasing overall import rates and Ntf2 reducing import based on cargo size. Importin α also contributes to nuclear size changes during early X. laevis development. Thus, nuclear transport mechanisms are physiological regulators of both interspecies and developmental nuclear scaling.
Assuntos
Núcleo Celular , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Xenopus/metabolismo , alfa Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Lamina Tipo B/metabolismo , Xenopus/embriologia , Xenopus laevis/embriologiaRESUMO
The FET family of atypical RNA-binding proteins includes Fused in sarcoma (FUS), Ewing's sarcoma (EWS) and the TATA-binding protein-associate factor 15 (TAF15). FET proteins are highly conserved, suggesting specialized requirements for each protein. Fus regulates splicing of transcripts required for mesoderm differentiation and cell adhesion in Xenopus, but the roles of Ews and Taf15 remain unknown. Here, we analyze the roles of maternally deposited and zygotically transcribed Taf15, which is essential for the correct development of dorsoanterior neural tissues. By measuring changes in exon usage and transcript abundance from Taf15-depleted embryos, we found that Taf15 may regulate dorsoanterior neural development through fgfr4 and ventx2.1. Taf15 uses distinct mechanisms to downregulate Fgfr4 expression, namely retention of a single intron within fgfr4 when maternal and zygotic Taf15 is depleted, and reduction in the total fgfr4 transcript when zygotic Taf15 alone is depleted. The two mechanisms of gene regulation (post-transcriptional versus transcriptional) suggest that Taf15-mediated gene regulation is target and co-factor dependent, contingent on the milieu of factors that are present at different stages of development.
Assuntos
Encéfalo/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Xenopus/metabolismo , Animais , Encéfalo/fisiologia , Diferenciação Celular/fisiologia , Éxons/fisiologia , Feminino , Masculino , Neurônios/fisiologia , Xenopus/fisiologiaRESUMO
Mutations in the RNA helicase DDX3 have emerged as a frequent cause of intellectual disability in humans. Because many individuals carrying DDX3 mutations have additional defects in craniofacial structures and other tissues containing neural crest (NC)-derived cells, we hypothesized that DDX3 is also important for NC development. Using Xenopus tropicalis as a model, we show that DDX3 is required for normal NC induction and craniofacial morphogenesis by regulating AKT kinase activity. Depletion of DDX3 decreases AKT activity and AKT-dependent inhibitory phosphorylation of GSK3ß, leading to reduced levels of ß-catenin and Snai1: two GSK3ß substrates that are crucial for NC induction. DDX3 function in regulating these downstream signaling events during NC induction is likely mediated by RAC1, a small GTPase whose translation depends on the RNA helicase activity of DDX3. These results suggest an evolutionarily conserved role of DDX3 in NC development by promoting AKT activity, and provide a potential mechanism for the NC-related birth defects displayed by individuals harboring mutations in DDX3 and its downstream effectors in this signaling cascade.
Assuntos
RNA Helicases DEAD-box/metabolismo , Crista Neural/embriologia , Crista Neural/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Xenopus/metabolismo , Animais , Cartilagem/embriologia , Cartilagem/metabolismo , Embrião não Mamífero/metabolismo , Face/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Glicogênio Sintase Quinase 3 beta/metabolismo , Morfogênese/genética , Fosforilação , Estabilidade Proteica , Crânio/embriologia , Crânio/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Via de Sinalização Wnt , Xenopus/genética , beta Catenina/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Hybridization of eggs and sperm from closely related species can give rise to genetic diversity, or can lead to embryo inviability owing to incompatibility. Although central to evolution, the cellular and molecular mechanisms underlying post-zygotic barriers that drive reproductive isolation and speciation remain largely unknown. Species of the African clawed frog Xenopus provide an ideal system to study hybridization and genome evolution. Xenopus laevis is an allotetraploid with 36 chromosomes that arose through interspecific hybridization of diploid progenitors, whereas Xenopus tropicalis is a diploid with 20 chromosomes that diverged from a common ancestor approximately 48 million years ago. Differences in genome size between the two species are accompanied by organism size differences, and size scaling of the egg and subcellular structures such as nuclei and spindles formed in egg extracts. Nevertheless, early development transcriptional programs, gene expression patterns, and protein sequences are generally conserved. Whereas the hybrid produced when X. laevis eggs are fertilized by X. tropicalis sperm is viable, the reverse hybrid dies before gastrulation. Here we apply cell biological tools and high-throughput methods to study the mechanisms underlying hybrid inviability. We reveal that two specific X. laevis chromosomes are incompatible with the X. tropicalis cytoplasm and are mis-segregated during mitosis, leading to unbalanced gene expression at the maternal to zygotic transition, followed by cell-autonomous catastrophic embryo death. These results reveal a cellular mechanism underlying hybrid incompatibility that is driven by genome evolution and contributes to the process by which biological populations become distinct species.
Assuntos
Cromossomos/genética , Hibridização Genética , Herança Paterna/genética , Xenopus/genética , Xenopus/metabolismo , Animais , Segregação de Cromossomos , Cromossomos/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Perda do Embrião/veterinária , Evolução Molecular , Feminino , Especiação Genética , Masculino , Mitose , Xenopus laevis/genéticaRESUMO
Heart development is a delicate and complex process regulated by coordination of various signaling pathways. In this study, we investigated the role of sox18 in heart development by modulating Wnt/ß-Catenin signaling pathways. Our spatiotemporal expression analysis revealed that sox18 is mainly expressed in the heart, branchial arch, pharyngeal arch, spinal cord, and intersegmental vessels at the tailbud stage of Xenopus tropicalis embryo. Overexpression of sox18 in the X. tropicalis embryos causes heart edema, while loss-of-function of sox18 can change the signal of developmental heart marker gata4 at different stages, suggesting that sox18 plays an essential role in the development of the heart. Knockdown of SOX18 in human umbilical vein endothelial cells suggests a link between Sox18 and ß-CATENIN, a key regulator of the Wnt signaling pathway. Sox18 negatively regulates islet1 and tbx3, the downstream factors of Wnt/ß-Catenin signaling, during the linear heart tube formation and the heart looping stage. Taken together, our findings highlight the crucial role of Sox18 in the development of the heart via inhibiting Wnt/ß-Catenin signaling.
Assuntos
Fatores de Transcrição SOXF , Proteínas de Xenopus , beta Catenina , Animais , Humanos , beta Catenina/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismo , Via de Sinalização Wnt , Xenopus/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
Early life exposure to endocrine disrupting chemicals (EDCs) has been suggested to adversely affect reproductive health in humans and wildlife. Here, we characterize endocrine and adverse effects on the reproductive system after juvenile exposure to propiconazole (PROP) or imazalil (IMZ), two common azole fungicides with complex endocrine modes of action. Using the frog Xenopus tropicalis, two short-term (2-weeks) studies were conducted. I: Juveniles (2 weeks post metamorphosis (PM)) were exposed to 0, 17 or 178 µg PROP/L. II: Juveniles (6 weeks PM) were exposed to 0, 1, 12 or 154 µg IMZ/L. Histological analysis of the gonads revealed an increase in the number of dark spermatogonial stem cells (SSCs)/testis area, and in the ratio secondary spermatogonia: dark SSCs were increased in all IMZ groups compared to control. Key genes in gametogenesis, retinoic acid and sex steroid pathways were also analysed in the gonads. Testicular levels of 3ß-hsd, ddx4 were increased and cyp19 and id4 levels were decreased in the IMZ groups. In PROP exposed males, increased testicular aldh1a2 levels were detected, but no histological effects observed. Although no effects on ovarian histology were detected, ovarian levels of esr1, rsbn1 were increased in PROP groups, and esr1 levels were decreased in IMZ groups. In conclusion, juvenile azole exposure disrupted testicular expression of key genes in retinoic acid (PROP) and sex steroid pathways and in gametogenesis (IMZ). Our results further show that exposure to environmental concentrations of IMZ disrupted spermatogenesis in the juvenile testis, which is a cause for concern as it may lead to impaired fertility. Testicular levels of id4, ddx4 and the id4:ddx4 ratio were associated with the number of dark SSCs and secondary spermatogonia suggesting that they may serve as a molecular markers for disrupted spermatogenesis.
Assuntos
Fungicidas Industriais , Humanos , Masculino , Feminino , Animais , Fungicidas Industriais/metabolismo , Xenopus laevis , Azóis/toxicidade , Xenopus/metabolismo , Testículo , Espermatogênese , Hormônios Esteroides Gonadais/metabolismo , Tretinoína , Esteroides/metabolismo , Família Aldeído Desidrogenase 1/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/farmacologia , Retinal Desidrogenase/metabolismoRESUMO
The endoplasmic reticulum (ER) membrane protein complex (EMC) is essential for the insertion of a wide variety of transmembrane proteins into the plasma membrane across cell types. Each EMC is composed of Emc1-7, Emc10, and either Emc8 or Emc9. Recent human genetics studies have implicated variants in EMC genes as the basis for a group of human congenital diseases. The patient phenotypes are varied but appear to affect a subset of tissues more prominently than others. Namely, craniofacial development seems to be commonly affected. We previously developed an array of assays in Xenopus tropicalis to assess the effects of emc1 depletion on the neural crest, craniofacial cartilage, and neuromuscular function. We sought to extend this approach to additional EMC components identified in patients with congenital malformations. Through this approach, we determine that EMC9 and EMC10 are important for neural crest development and the development of craniofacial structures. The phenotypes observed in patients and our Xenopus model phenotypes similar to EMC1 loss of function likely due to a similar mechanism of dysfunction in transmembrane protein topogenesis.
Assuntos
Retículo Endoplasmático , Crista Neural , Animais , Humanos , Crista Neural/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Xenopus/genética , Xenopus/metabolismoRESUMO
Regeneration of complex tissues is initiated by an injury-induced stress response, eventually leading to activation of developmental signaling pathways such as Wnt signaling. How early injury cues are interpreted and coupled to activation of these developmental signals and their targets is not well understood. Here, we show that Hif1α, a stress induced transcription factor, is required for tail regeneration in Xenopus tropicalis. We find that Hif1α is required for regeneration of differentiated axial tissues, including axons and muscle. Using RNA-sequencing, we find that Hif1α and Wnt converge on a broad set of genes required for posterior specification and differentiation, including the posterior hox genes. We further show that Hif1α is required for transcription via a Wnt-responsive element, a function that is conserved in both regeneration and early neural patterning. Our findings indicate that Hif1α has regulatory roles in Wnt target gene expression across multiple tissue contexts.
Assuntos
Padronização Corporal/genética , Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Cauda/metabolismo , Proteínas Wnt/genética , Via de Sinalização Wnt/genética , Proteínas de Xenopus/genética , Xenopus/genética , Animais , Axônios/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Larva/genética , Músculos/metabolismo , Regeneração/genética , Proteínas Wnt/metabolismo , Xenopus/metabolismo , Proteínas de Xenopus/metabolismoRESUMO
Since CRISPR-based genome editing technology works effectively in the diploid frog Xenopus tropicalis, a growing number of studies have successfully modeled human genetic diseases in this species. However, most of their targets were limited to non-syndromic diseases that exhibit abnormalities in a small fraction of tissues or organs in the body. This is likely because of the complexity of interpreting the phenotypic variations resulting from somatic mosaic mutations generated in the founder animals (crispants). In this study, we attempted to model the syndromic disease campomelic dysplasia (CD) by generating sox9 crispants in X. tropicalis. The resulting crispants failed to form neural crest cells at neurula stages and exhibited various combinations of jaw, gill, ear, heart, and gut defects at tadpole stages, recapitulating part of the syndromic phenotype of CD patients. Genotyping of the crispants with a variety of allelic series of mutations suggested that the heart and gut defects depend primarily on frame-shift mutations expected to be null, whereas the jaw, gill, and ear defects could be induced not only by such mutations but also by in-frame deletion mutations expected to delete part of the jawed vertebrate-specific domain from the encoded Sox9 protein. These results demonstrate that Xenopus crispants are useful for investigating the phenotype-genotype relationships behind syndromic diseases and examining the tissue-specific role of each functional domain within a single protein, providing novel insights into vertebrate jaw evolution.
Assuntos
Displasia Campomélica , Animais , Humanos , Xenopus laevis/metabolismo , Displasia Campomélica/genética , Xenopus/genética , Xenopus/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Fenótipo , GenótipoRESUMO
Cryptochromes (CRYs) are multifunctional molecules that act as a circadian clock oscillating factor, a blue-light sensor, and a light-driven magnetoreceptor. Cry genes are classified into several groups based on the evolutionary relationships. Cryptochrome 6 gene (Cry6) is present in invertebrates and lower vertebrates such as amphibians and fishes. Here we identified a Cry6 ortholog in Xenopus tropicalis (XtCry6). XtCRY6 retains a conserved long N-terminal extension (termed CRY N-terminal extension; CNE) that is not found in any CRY in the other groups. A structural prediction suggested that CNE contained unique structures; a tetrahelical fold structure topologically related to KaiA/RbsU domain, overlapping nuclear- and nucleolar-localizing signals (NLS/NoLS), and a novel motif (termed DI-UIM) overlapping a double-sided ubiquitin-interacting motif (DUIM) and an inverted ubiquitin-interacting motif (IUIM). Potential activities of the NLS/NoLS and DI-UIM were examined to infer the molecular function of XtCRY6. GFP-NLS/NoLS fusion protein exogenously expressed in HEK293 cells was mostly observed in the nucleolus, while GFP-XtCRY6 was observed in the cytoplasm. A glutathione S-transferase (GST) pull-down assay suggested that the DI-UIM physically interacts with polyubiquitin. Consistently, protein docking simulations implied that XtCRY6 DI-UIM binds two ubiquitin molecules in a relationship of a twofold rotational symmetry with the symmetry axis parallel or perpendicular to the DI-UIM helix. These results strongly suggested that XtCRY6 does not function as a circadian transcriptional repressor and that it might have another function such as photoreceptive molecule regulating light-dependent protein degradation or gene expression through a CNE-mediated interaction with ubiquitinated proteins in the cytoplasm and/or nucleolus.
Assuntos
Criptocromos , Ubiquitina , Animais , Humanos , Criptocromos/genética , Criptocromos/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Xenopus/genética , Xenopus/metabolismo , Células HEK293 , Fatores de TranscriçãoRESUMO
Thyroid hormone (T3) is essential for normal development and metabolism, especially during postembryonic development, a period around birth in mammals when plasma T3 levels reach their peak. T3 functions through two T3 receptors, TRα and TRß. However, little is known about the tissue-specific functions of TRs during postembryonic development because of maternal influence and difficulty in manipulation of mammalian models. We have studied Xenopus tropicalis metamorphosis as a model for human postembryonic development. By using TRα knockout (Xtr·thratmshi ) tadpoles, we have previously shown that TRα is important for T3-dependent intestinal remodeling and hindlimb development but not tail resorption during metamorphosis. Here, we have identified genes bound by TR in premetamorphic wild-type and Xtr·thratmshi tails with or without T3 treatment by using chromatin immunoprecipitation-sequencing and compared them with those in the intestine and hindlimb. Compared to other organs, the tail has much fewer genes bound by TR or affected by TRα knockout. Bioinformatic analyses revealed that among the genes bound by TR in wild-type but not Xtr·thratmshi organs, fewer gene ontology (GO) terms or biological pathways related to metamorphosis were enriched in the tail compared to those in the intestine and hindlimb. This difference likely underlies the drastic effects of TRα knockout on the metamorphosis of the intestine and hindlimb but not the tail. Thus, TRα has tissue-specific roles in regulating T3-dependent anuran metamorphosis by directly targeting the pathways and GO terms important for metamorphosis.
Assuntos
Receptores alfa dos Hormônios Tireóideos , Proteínas de Xenopus , Xenopus , Animais , Humanos , Regulação da Expressão Gênica no Desenvolvimento/genética , Mamíferos/metabolismo , Metamorfose Biológica/genética , Receptores alfa dos Hormônios Tireóideos/genética , Receptores alfa dos Hormônios Tireóideos/metabolismo , Tri-Iodotironina/genética , Tri-Iodotironina/metabolismo , Tri-Iodotironina/farmacologia , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
Among its multiple functions, p53 is a critical regulator of TGF-beta responses. Sasai et al. (2008) now identify a new p53 inhibitory protein, XFDL156. During embryonic development, this factor is expressed in the ectoderm germ layer and maintains the pluripotency of ectodermal cells by inhibiting TGF-beta target genes that promote mesoderm specification.
Assuntos
Diferenciação Celular , Fator de Crescimento Transformador beta/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Xenopus/embriologia , Animais , Ectoderma/citologia , Ectoderma/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Xenopus/metabolismoRESUMO
Ghrelin is a gut-derived peptide with several physiological functions, including feeding, gastrointestinal motility, and hormonal secretion. Recently, a host defense peptide, liver-expressed antimicrobial peptide-2 (LEAP2), was reported as an endogenous antagonist of growth hormone secretagogue receptor (GHS-R). The physiological relevance of the molecular LEAP2-GHS-R interaction in mammals has been explored; however, studies on non-mammals are limited. Here, we report the identification and functional characterization of ghrelin and its related molecules in Western clawed frog (Xenopus tropicalis), a known model organism. We first identified cDNA encoding X. tropicalis ghrelin and GHS-R. RT-qPCR revealed that ghrelin mRNA expression was most abundant in the stomach. GHS-R mRNA was widely distributed in the brain and peripheral tissues, and a relatively strong signal was observed in the stomach and intestine. In addition, LEAP2 was mainly expressed in intestinal tissues at higher levels than in the liver. In functional analysis, X. tropicalis ghrelin and human ghrelin induced intracellular Ca2+ mobilization with EC50 values in the low nanomolar range in CHO-K1 cells expressing X. tropicalis GHS-R. Furthermore, ghrelin-induced GHS-R activation was antagonized with IC50 values in the nanomolar range by heterologous human LEAP2. We also validated the expression of ghrelin and feeding-related factors under fasting conditions. After 2 days of fasting, no changes in ghrelin mRNA levels were observed in the stomach, but GHS-R mRNA levels were significantly increased, associated with significant downregulation of nucb2. In addition, LEAP2 upregulation was observed in the duodenum. These results provide the first evidence that LEAP2 functions as an antagonist of GHS-R in the anuran amphibian X. tropicalis. It has also been suggested that the ghrelin/GHS-R/LEAP2 system may be involved in energy homeostasis in X. tropicalis.
Assuntos
Grelina , Receptores de Grelina , Animais , Cricetinae , Humanos , Grelina/genética , Grelina/metabolismo , Xenopus/metabolismo , Receptores de Grelina/metabolismo , Cricetulus , Clonagem Molecular , RNA MensageiroRESUMO
We identified the bullfrog Rana catesbeiana sulfotransferase 1 (SULT1) family from the BLAST search tool of the public databases based on the SULT1 families of Nanorana parkeri, Xenopus laevis, and Xenopus tropicalis as queries, revealing the characteristics of the anuran SULT1 family. The results showed that the anuran SULT1 family comprises six subfamilies, four of which were related to the mammalian SULT1 subfamily. Additionally, the bullfrog has two SULT1Cc subfamily members that are consistent with the characteristics of the expanded Xenopus SULT1C subfamily. Several members of the bullfrog SULT1 family were suggested to play important roles in sulfation during metamorphosis. Among these, cDNAs encoding SULT1Cc1 and SULT1Y1 were cloned, and the sulfation activity was analyzed using recombinant proteins. The affinity for 2-naphthol and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and the enzymatic reaction rate were higher in SULT1Cc1 than in SULT1Y1. Both the enzymes showed inhibitory effect of many thyroid hormones (THs) analogs on the sulfation of 2-naphthol. The potency of sulfation activities of SULT1Cc1 and SULT1Y1 against T4 indicated their possible role in the intracellular T4 clearance during metamorphosis.