RESUMO
BACKGROUND AND OBJECTIVE: Bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation has been shown to occur through the canonical Wnt/ßcatenin pathway, whereas factors promoting canonical Wnt signaling in cementoblasts inhibit cell differentiation and promote cell proliferation in vitro. The aim of this study was to investigate whether putative precursor cells of cementoblasts, dental follicle cells (murine SVF4 cells), when stimulated with BMP2, would exhibit changes in genes/proteins associated with the Wnt/ß-catenin pathway. MATERIAL AND METHODS: SVF4 cells were stimulated with BMP2, and the following assays were carried out: (i) Wnt/ß-catenin pathway activation assessed by western blotting, ß-catenin/transcription factor (TCF) reporter assays and expression of the lymphoid enhancer-binding factor-1 (Lef1), transcription factor 7 (Tcf7), Wnt inhibitor factor 1 (Wif1) and Axin2 (Axin2) genes; and (ii) cementoblast/osteoblast differentiation assessed by mineralization in vitro, and by the mRNA levels of runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), osteocalcin (Ocn) and bone sialoprotein (Bsp), determined by quantitative PCR after treatment with wingless-type MMTV integration site family, member 3A (WNT3A) and knockdown of ß-catenin. RESULTS: WNT3A induced ß-catenin nuclear translocation and up-regulated the transcriptional activity of a canonical Wnt-responsive reporter, suggesting that the Wnt/ß-catenin pathway functions in SVF4 cells. Activation of Wnt signaling with WNT3A suppressed BMP2-mediated induction of cementoblast/osteoblast maturation of SVF4 cells. However, ß-catenin knockdown showed that the BMP2-induced expression of cementoblast/osteoblast differentiation markers requires endogenous ß-catenin. WNT3A down-regulated transcripts for Runx2, Alp and Ocn in SVF4 cells compared with untreated cells. In contrast, BMP2 induction of Bsp transcripts occurred independently of Wnt/ß-catenin signaling. CONCLUSION: These data suggest that stabilization of ß-catenin by WNT3A inhibits BMP2-mediated induction of cementoblast/osteoblast differentiation in SVF4 cells, although BMP2 requires endogenous Wnt/ß-catenin signaling to promote cell maturation.
Assuntos
Proteína Morfogenética Óssea 2/fisiologia , Saco Dentário/citologia , Via de Sinalização Wnt/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Fosfatase Alcalina/análise , Animais , Proteína Axina/análise , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células , Subunidade alfa 1 de Fator de Ligação ao Core/análise , Cemento Dentário/efeitos dos fármacos , Cemento Dentário/fisiologia , Saco Dentário/efeitos dos fármacos , Proteínas da Matriz Extracelular/análise , Técnicas de Silenciamento de Genes , Fator 1-alfa Nuclear de Hepatócito , Peptídeos e Proteínas de Sinalização Intercelular/análise , Fator 1 de Ligação ao Facilitador Linfoide/análise , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Osteocalcina/análise , Osteogênese/fisiologia , Osteopontina/análise , Fator de Transcrição Sp7 , Fator 1 de Transcrição de Linfócitos T/análise , Fatores de Transcrição/análise , Transcrição Gênica/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt3A/farmacologia , Dedos de Zinco , beta Catenina/genéticaRESUMO
The Wnt proteins constitute a family of secreted glycoproteins the members of which have essential signaling roles during embryogenesis. The recent identification of several new regulators of this signal transduction pathway have revealed unexpectedly intricate levels of constraint on Wnt-dependent gene activation, and studies in developing embryos and in cell culture systems have allowed a more complete understanding of the functional and biochemical interactions between components of this evolutionarily conserved pathway.
Assuntos
Regulação para Baixo/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais/fisiologia , Proteínas de Peixe-Zebra , Animais , Regulação para Baixo/genética , Humanos , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/genética , Regulação para Cima/genética , Regulação para Cima/fisiologia , Proteínas WntRESUMO
In vertebrates, wnt8 has been implicated in the early patterning of the mesoderm. To determine directly the embryonic requirements for wnt8, we generated a chromosomal deficiency in zebrafish that removes the bicistronic wnt8 locus. We report that homozygous mutants exhibit pronounced defects in dorso-ventral mesoderm patterning and in the antero-posterior neural pattern. Despite differences in their signaling activities, either coding region of the bicistronic RNA can rescue the deficiency phenotype. Specific interference of wnt8 translation by morpholino antisense oligomers phenocopies the deficiency, and interference with wnt8 translation in ntl and spt mutants produces embryos lacking trunk and tail. These data demonstrate that the zebrafish wnt8 locus is required during gastrulation to pattern both the mesoderm and the neural ectoderm properly.
Assuntos
Padronização Corporal/fisiologia , Proteínas/genética , Transcrição Gênica/fisiologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Proteínas do Citoesqueleto , Ectoderma/fisiologia , Proteínas Fetais , Regulação da Expressão Gênica no Desenvolvimento , Cabeça/embriologia , Mesoderma/fisiologia , Dados de Sequência Molecular , Mutagênese/fisiologia , Oligonucleotídeos Antissenso , Fases de Leitura Aberta , Fenótipo , RNA Mensageiro/genética , Proteínas com Domínio T/genética , Cauda/embriologia , Proteínas WntRESUMO
In chicken embryo erythroid cells, newly synthesized vimentin first enters a Triton X-100 (TX-100)-soluble pool and subsequently assembles posttranslationally into TX-100-insoluble vimentin filaments (Blikstad I., and E. Lazarides, J. Cell Biol., 96:1803-1808). Here we show that incubation of chicken embryo erythroid cells in a medium in which arginine has been substituted by its amino acid analogue, canavanine, results in the inhibition of the posttranslational assembly of vimentin into the TX-100-insoluble filaments. Immunoprecipitation and subsequent SDS gel electrophoresis showed that the synthesis of canavanine-vimentin is not inhibited and that it accumulates in the TX-100-soluble compartment. Pulse-chase experiments with [35S]methionine demonstrated that while arginine-vimentin can be rapidly chased from the soluble to the cytoskeletal fraction, canavanine-vimentin remains in the soluble fraction, where it turns over. The effect of canavanine on the assembly of vimentin did not prevent the assembly of arginine-vimentin, as cells labeled with [35S]methionine first in the presence of canavanine and then in the presence of arginine contained labeled canavanine-vimentin only in the soluble fraction, and arginine-vimentin in both the soluble and cytoskeletal fractions. These results suggest that arginine residues play an essential role in the assembly of vimentin in vivo.
Assuntos
Canavanina/farmacologia , Citoesqueleto/metabolismo , Eritrócitos/metabolismo , Proteínas de Filamentos Intermediários/sangue , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Animais , Arginina/farmacologia , Embrião de Galinha , Cinética , VimentinaRESUMO
Ankyrin is an extrinsic membrane protein in human erythrocytes that links the alpha beta-spectrin-based extrinsic membrane skeleton to the membrane by binding simultaneously to the beta-spectrin subunit and to the transmembrane anion transporter. To analyse the temporal and spatial regulation of assembly of this membrane skeleton, we investigated the kinetics of synthesis and assembly of ankyrin ( goblin ) with respect to those of spectrin in chicken embryo erythroid cells. Electrophoretic analysis of Triton X-100 soluble and cytoskeletal fractions show that at steady state both ankyrin and spectrin are detected exclusively in the cytoskeleton. In contrast, continuous labeling of erythroid cells with [35S]methionine, and immunoprecipitation of ankyrin and alpha- and beta-spectrin, reveals that newly synthesized ankyrin and spectrin are partitioned into both the cytoskeletal and Triton X-100 soluble fractions. The soluble pools of ankyrin and beta-spectrin reach a plateau of labeling within 1 h, whereas the soluble pool of alpha-spectrin is substantially larger and reaches a plateau more slowly, reflecting an approximately 3:1 ratio of synthesis of alpha- to beta-spectrin. Ankyrin and beta-spectrin enter the cytoskeletal fraction within 10 min of labeling, and the amount assembled into the cytoskeletal fraction exceeds the amount present in their respective soluble pools within 1 h of labeling. Although alpha-spectrin enters the cytoskeletal fraction with similar kinetics to beta-spectrin and ankyrin, and in amounts equimolar to beta-spectrin, the amount of cytoskeletal alpha-spectrin does not exceed the amount of soluble alpha-spectrin even after 3 h of labeling. Pulse-chase labeling experiments reveal that ankyrin and alpha- and beta-spectrin assembled into the cytoskeleton exhibit no detectable turnover, whereas the Triton X-100 soluble polypeptides are rapidly catabolized, suggesting that stable assembly of the three polypeptides is dependent upon their association with their respective membrane receptor(s). The existence in the detergent-soluble compartment of newly synthesized ankyrin and alpha- and beta-spectrin that are catabolized, rather than assembled, suggests that ankyrin and spectrin are synthesized in excess of available respective membrane binding sites, and that the assembly of these polypeptides, while rapid, is not tightly coupled to their synthesis. We hypothesize that the availability of the high affinity receptor(s) localized on the membrane mediates posttranslationally the extent of assembly of the three cytoskeletal proteins in the correct stoichiometry, their stability, and their spatial localization.
Assuntos
Membrana Eritrocítica/ultraestrutura , Proteínas de Membrana/metabolismo , Espectrina/metabolismo , Animais , Anquirinas , Compartimento Celular , Embrião de Galinha , Substâncias Macromoleculares , Proteínas de Membrana/biossíntese , Morfogênese , Processamento de Proteína Pós-Traducional , SolubilidadeRESUMO
Affinity-purified antibodies and cDNA probes specific for the chicken erythrocyte anion transporter (also referred to as band 3) have been used to demonstrate that this protein is expressed in a highly cell-type-specific manner in the avian kidney. Indirect immunofluorescence analysis indicates that this polypeptide is present in only a small subset of total kidney cells and is predominantly localized to the proximal convoluted tubule of this organ. Chicken erythrocytes synthesize and accumulate two structurally and serologically related band 3 polypeptides. The polypeptide that accumulates in kidney membranes has an apparent molecular weight greater than either of its erythroid counterparts. This diversity is also reflected at the RNA level, as the single band 3 mRNA species detected during various stages of erythroid development is distinct in size from that found in kidney cells. Genomic DNA blot analysis suggests that both the erythroid and kidney band 3 RNAs arise from a single gene. Furthermore, of the adult tissues we have examined that are known to express ankyrin and spectrin polypeptides, only kidney accumulates detectable levels of the band 3 mRNA and polypeptide. These observations suggest that a subset of kidney cells use an anion transport mechanism analogous to that of erythrocytes and that band 3 is expressed in a noncoordinate manner with other components of the erythroid membrane skeleton in nonerythroid cells.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/genética , Proteínas de Transporte/metabolismo , Eritrócitos/fisiologia , Animais , Proteína 1 de Troca de Ânion do Eritrócito/imunologia , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Proteínas de Transporte de Ânions , Proteínas de Transporte/genética , Galinhas , Clonagem Molecular , Proteínas do Citoesqueleto/genética , DNA/genética , Regulação da Expressão Gênica , Rim/fisiologia , Fragmentos de Peptídeos/análise , RNA Mensageiro/genéticaRESUMO
In Xenopus embryos, beta-catenin has been shown to be both necessary and sufficient for the establishment of dorsal cell fates. This signaling activity is thought to depend on the binding of beta-catenin to members of the Lef/Tcf family of transcription factors and the regulation of gene expression by this complex. To test whether beta-catenin must accumulate in nuclei to establish dorsal cell fate, we constructed various localization mutants that restrict beta-catenin to either the plasma membrane, the cytosol, or the nucleus. When overexpressed in Xenopus embryos, the proteins localize as predicted, but surprisingly all forms induce an ectopic axis, indicative of inducing dorsal cell fates. Given this unexpected result, we focused on the membrane-tethered form of beta-catenin to resolve the apparent discrepancy between its membrane localization and the hypothesized role of nuclear beta-catenin in establishing dorsal cell fate. We demonstrate that overexpression of membrane-tethered beta-catenin elevates the level of free endogenous beta-catenin, which subsequently accumulates in nuclei. Consistent with the hypothesis that it is this pool of non-membrane-associated beta-catenin that signals in the presence of membrane-tethered beta-catenin, overexpression of cadherin, which binds free beta-catenin, blocks the axis-inducing activity of membrane- tethered beta-catenin. The mechanism by which ectopic membrane-tethered beta-catenin increases the level of endogenous beta-catenin likely involves competition for the adenomatous polyposis coli (APC) protein, which in other systems has been shown to play a role in degradation of beta-catenin. Consistent with this hypothesis, membrane-tethered beta-catenin coimmunoprecipitates with APC and relocalizes APC to the membrane in cells. Similar results are observed with ectopic plakoglobin, casting doubt on a normal role for plakoglobin in axis specification and indicating that ectopic proteins that interact with APC can artifactually elevate the level of endogenous beta-catenin, likely by interfering with its degradation. These results highlight the difficulty in interpreting the activity of an ectopic protein when it is assayed in a background containing the endogenous protein. We next investigated whether the ability of beta-catenin to interact with potential protein partners in the cell may normally be regulated by phosphorylation. Compared with nonphosphorylated beta-catenin, beta-catenin phosphorylated by glycogen synthase kinase-3 preferentially associates with microsomal fractions expressing the cytoplasmic region of N-cadherin. These results suggest that protein-protein interactions of beta-catenin can be influenced by its state of phosphorylation, in addition to prior evidence that this phosphorylation modulates the stability of beta-catenin.
Assuntos
Proteínas do Citoesqueleto/genética , Transdução de Sinais/genética , Transativadores , Xenopus/fisiologia , Proteína da Polipose Adenomatosa do Colo , Animais , Ligação Competitiva , Caderinas/biossíntese , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas do Citoesqueleto/biossíntese , Proteínas do Citoesqueleto/metabolismo , Desmoplaquinas , Desmossomos/metabolismo , Regulação para Baixo/genética , Embrião não Mamífero/metabolismo , Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica/fisiologia , Genes APC , Proteínas de Fluorescência Verde , Humanos , Líquido Intracelular/metabolismo , Líquido Intracelular/fisiologia , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Mutagênese , Fosforilação , Xenopus/genética , Proteínas de Xenopus , beta Catenina , gama CateninaRESUMO
Beta-catenin plays a pivotal role in the transcriptional activation of Wnt-responsive genes by binding to TCF/LEF transcription factors. Although it has been suggested that the COOH-terminal region of beta-catenin functions as an activation domain, the mechanisms of activation remain unclear. To screen for potential transcriptional coactivators that bind to the COOH-terminal region of beta-catenin, we used a novel yeast two-hybrid system, the Ras recruitment system (RRS) that detects protein-protein interactions at the inner surface of the plasma membrane. Using this system, we isolated the CREB-binding protein (CBP). Armadillo (Arm) repeat 10 to the COOH terminus of beta-catenin is involved in binding to CBP, whereas beta-catenin interacts directly with the CREB-binding domain of CBP. Beta-catenin synergizes with CBP to stimulate the activity of a synthetic reporter in vivo. Conversely, beta-catenin-dependent transcriptional activation is repressed by E1A, an antagonist of CBP function, but not by an E1A mutant that does not bind to CBP. The activation of Wnt target genes such as siamois and Xnr3 in Xenopus embryos is also sensitive to E1A. These findings suggest that CBP provides a link between beta-catenin and the transcriptional machinery, and possibly mediates the oncogenic function of beta-catenin.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Proteínas de Xenopus , Animais , Sítios de Ligação , Células COS , Proteína de Ligação a CREB , Caderinas/fisiologia , Linhagem Celular , Clonagem Molecular , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Células HeLa , Proteínas de Homeodomínio/genética , Humanos , Luciferases/análise , Saccharomyces cerevisiae , Ativação Transcricional , Transfecção , Fator de Crescimento Transformador beta/genética , Xenopus laevis/embriologia , beta CateninaRESUMO
cDNA probes for three components of the erythroid membrane skeleton, alpha spectrin, beta spectrin, and ankyrin, were obtained by using monospecific antibodies to screen a lambda gt11 expression vector library containing cDNA prepared from chicken erythroid poly(A)+ RNA. Each cDNA appears to hybridize to one gene type in the chicken genome. Qualitatively distinct RNA species in myogenic and erythroid cells are detected for beta spectrin and ankyrin, while alpha spectrin exists as a single species of transcript in all tissues examined. This tissue-specific expression of RNAs is regulated quantitatively during myogenesis in vitro, since all three accumulate only upon myoblast fusion. Furthermore, RNAs for two of the three genes do not accumulate to detectable levels in chicken embryo fibroblasts, demonstrating that their accumulation can be noncoordinate. These observations suggest that independent gene regulation and tissue-specific production of heterogeneous transcripts from the beta spectrin and ankyrin genes underlie the formation of distinct membrane skeletons in erythroid and muscle cells.
Assuntos
Proteínas de Membrana/genética , Espectrina/genética , Transcrição Gênica , Animais , Anquirinas , Células Cultivadas , Embrião de Galinha , Clonagem Molecular , DNA/metabolismo , Enzimas de Restrição do DNA , Genes , Músculos/citologia , Músculos/metabolismo , Hibridização de Ácido Nucleico , Biossíntese de ProteínasRESUMO
Fodrin (nonerythroid spectrin) and its associated proteins have been previously implicated in the establishment of specialized membrane-cytoskeletal domains in differentiating cells. Using antiserum which is monospecific for the alpha-subunit of fodrin, we demonstrate that alpha-fodrin is present in oocytes and adult tissues of Xenopus laevis. Analyses of the de novo synthesis of alpha-fodrin during embryonic development reveal that alpha-fodrin is synthesized in oocytes, but not during early development. To investigate the level of control of alpha-fodrin expression, we isolated two cDNA clones for oocyte alpha-fodrin. The oocyte cDNA clones were identified as encoding portions of alpha-fodrin based on DNA sequence analysis and on the comparison of the predicted amino acid sequence of the cDNAs with the known sequence of human erythrocyte alpha-spectrin. The Xenopus alpha-fodrin cDNAs hybridize to a transcript of approximately 9 kb on RNA blots, and probably to a single gene type on genomic DNA blots. Both RNA blot analyses and S1 nuclease protection assays with the Xenopus alpha-fodrin cDNAs demonstrate that the observed decline in the de novo synthesis of alpha-fodrin polypeptides is controlled by a dramatic decrease in the abundance of alpha-fodrin transcripts after fertilization. In contrast, levels of actin transcripts do not decrease during this period. Inasmuch as steady-state levels of alpha-fodrin transcripts rise by the neurula stage of development, these results suggest that the synthesis of alpha-fodrin polypeptides during embryonic development of Xenopus is regulated, rather than constitutive, and that the primary level of control is the steady-state abundance of mRNA.
Assuntos
Proteínas de Transporte/genética , Clonagem Molecular , DNA/metabolismo , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Oócitos/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/biossíntese , Células Cultivadas , Enzimas de Restrição do DNA , Embrião não Mamífero/metabolismo , Feminino , Proteínas dos Microfilamentos/biossíntese , Xenopus laevisRESUMO
We have examined whether the development of embryonic muscle fiber type is regulated by competing influences between Hedgehog and TGF-beta signals, as previously shown for development of neuronal cell identity in the neural tube. We found that ectopic expression of Hedgehogs or inhibition of protein kinase A in zebrafish embryos induces slow muscle precursors throughout the somite but muscle pioneer cells only in the middle of the somite. Ectopic expression in the notochord of Dorsalin-1, a member of the TGF-beta superfamily, inhibits the formation of muscle pioneer cells, demonstrating that TGF-beta signals can antagonize the induction of muscle pioneer cells by Hedgehog. We propose that a Hedgehog signal first induces the formation of slow muscle precursor cells, and subsequent Hedgehog and TGF-beta signals exert competing positive and negative influences on the development of muscle pioneer cells.
Assuntos
Regulação para Baixo/genética , Proteínas de Drosophila , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Fibras Musculares Esqueléticas/fisiologia , Fator de Crescimento Transformador beta/genética , Regulação para Cima/genética , Proteínas de Peixe-Zebra , Animais , Diferenciação Celular , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Drosophila , Embrião não Mamífero , Proteínas Hedgehog , Proteínas de Insetos/biossíntese , Proteínas de Insetos/fisiologia , Família Multigênica , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares de Contração Lenta/citologia , Fibras Musculares de Contração Lenta/fisiologia , Notocorda/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/fisiologia , Fatores de Crescimento Transformadores/biossíntese , Fatores de Crescimento Transformadores/fisiologia , Peixe-ZebraRESUMO
Eggs of Xenopus laevis undergo a postfertilization cortical rotation that specifies the position of the dorso-ventral axis and activates a transplantable dorsal-determining activity in dorsal blastomeres by the 32-cell stage. There have heretofore been no reported dorso-ventral asymmetries in endogenous signaling proteins that may be involved in this dorsal-determining activity during early cleavage stages. We focused on beta-catenin as a candidate for an asymmetrically localized dorsal-determining factor since it is both necessary and sufficient for dorsal axis formation. We report that beta-catenin displays greater cytoplasmic accumulation on the future dorsal side of the Xenopus embryo by the two-cell stage. This asymmetry persists and increases through early cleavage stages, with beta-catenin accumulating in dorsal but not ventral nuclei by the 16- to 32-cell stages. We then investigated which potential signaling factors and pathways are capable of modulating the steady-state levels of endogenous beta-catenin. Steady-state levels and nuclear accumulation of beta-catenin increased in response to ectopic Xenopus Wnt-8 (Xwnt-8) and to the inhibition of glycogen synthase kinase-3, whereas neither Xwnt-5A, BVg1, nor noggin increased beta-catenin levels before the mid-blastula stage. As greater levels and nuclear accumulation of beta-catenin on the future dorsal side of the embryo correlate with the induction of specific dorsal genes, our data suggest that early asymmetries in beta-catenin presage and may specify dorso-ventral differences in gene expression and cell fate. Our data further support the hypothesis that these dorso-ventral differences in beta-catenin arise in response to the postfertilization activation of a signaling pathway that involves Xenopus glycogen synthase kinase-3.
Assuntos
Padronização Corporal , Proteínas do Citoesqueleto/análise , Proteínas/fisiologia , Transdução de Sinais/fisiologia , Transativadores , Proteínas de Xenopus , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Proteínas de Transporte , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Glicoproteínas/fisiologia , Cloreto de Lítio/farmacologia , Proteínas Proto-Oncogênicas c-myc/genética , Fator de Crescimento Transformador beta , Raios Ultravioleta , Proteínas Wnt , Proteína Wnt-5a , Xenopus laevis/embriologia , Proteínas de Peixe-Zebra , beta CateninaRESUMO
We examined the spatial and temporal control of actin assembly in living Xenopus eggs. Within minutes of egg activation, dynamic actin-rich comet tails appeared on a subset of cytoplasmic vesicles that were enriched in protein kinase C (PKC), causing the vesicles to move through the cytoplasm. Actin comet tail formation in vivo was stimulated by the PKC activator phorbol myristate acetate (PMA), and this process could be reconstituted in a cell-free system. We used this system to define the characteristics that distinguish vesicles associated with actin comet tails from other vesicles in the extract. We found that the protein, N-WASP, was recruited to the surface of every vesicle associated with an actin comet tail, suggesting that vesicle movement results from actin assembly nucleated by the Arp2/3 complex, the immediate downstream target of N-WASP. The motile vesicles accumulated the dye acridine orange, a marker for endosomes and lysosomes. Furthermore, vesicles associated with actin comet tails had the morphological features of multivesicular endosomes as revealed by electron microscopy. Endosomes and lysosomes from mammalian cells preferentially nucleated actin assembly and moved in the Xenopus egg extract system. These results define endosomes and lysosomes as recruitment sites for the actin nucleation machinery and demonstrate that actin assembly contributes to organelle movement. Conversely, by nucleating actin assembly, intracellular membranes may contribute to the dynamic organization of the actin cytoskeleton.
Assuntos
Actinas/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Actinas/ultraestrutura , Animais , Sistema Livre de Células , Endossomos/ultraestrutura , Feminino , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Lisossomos/ultraestrutura , Masculino , Proteínas do Tecido Nervoso/ultraestrutura , Óvulo/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteína Quinase C-alfa , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich , Xenopus/metabolismoRESUMO
When overexpressed in Xenopus embryos, Xwnt-1, -3A, -8 and -8b define a functional class of Wnts (the Wnt-1 class) that promotes duplication of the embryonic axis, whereas Xwnt-5A, -4, and -11 define a distinct class (the Wnt-5A class) that alters morphogenetic movements (Du, S., S. Purcell, J. Christian, L. McGrew, and R. Moon. 1995. Mol. Cell. Biol. 15:2625-2634). Since come embryonic cells may be exposed to signals from both functional classes of Wnt during vertebrate development, this raises the question of how the signaling pathways of these classes of Wnts might interact. To address this issue, we coexpressed various Xwnts and components of the Wnt-1 class signaling pathway in developing Xenopus embryos. Members of the Xwnt-5A class antagonized the ability of ectopic Wnt-1 class to induce goosecoid expression and a secondary axis. Interestingly, the Wnt-5A class did not block goosecoid expression or axis induction in response to overexpression of cytoplasmic components of the Wnt-1 signaling pathway, beta-catenin or a kinase-dead gsk-3, or to the unrelated secreted factor, BVg1. The ability of the Wnt-5A class to block responses to the Wnt-1 class may involve decreases in cell adhesion, since ectopic expression of Xwnt-5A leads to decreased Ca2+-dependent cell adhesion and the activity of Xwnt-5A to block Wnt-1 class signals is mimicked by a dominant negative N-cadherin. These data underscore the importance of cell adhesion in modulating the responses of embryonic cells to signaling molecules and suggest that the Wnt-5A functional class of signaling factors can interact with the Wnt-1 class in an antagonistic manner.
Assuntos
Caderinas/fisiologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/fisiologia , Transativadores , Xenopus laevis/embriologia , Xenopus laevis/fisiologia , Proteínas de Peixe-Zebra , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/farmacologia , Proteínas do Citoesqueleto/farmacologia , Feminino , Quinase 3 da Glicogênio Sintase , Imuno-Histoquímica , Hibridização In Situ , Microinjeções , Sinais Direcionadores de Proteínas/antagonistas & inibidores , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia , Proteínas Proto-Oncogênicas/genética , RNA/administração & dosagem , RNA/genética , Transdução de Sinais , Proteínas Wnt , Proteína Wnt-5a , Proteína Wnt1 , Proteínas de Xenopus , Xenopus laevis/genética , beta CateninaRESUMO
Examination of the subcellular localization of Dishevelled (Dsh) in fertilized Xenopus eggs revealed that Dsh is associated with vesicle-like organelles that are enriched on the prospective dorsal side of the embryo after cortical rotation. Dorsal enrichment of Dsh is blocked by UV irradiation of the vegetal pole, a treatment that inhibits development of dorsal cell fates, linking accumulation of Dsh and specification of dorsal cell fates. Investigation of the dynamics of Dsh localization using Dsh tagged with green fluorescent protein (Dsh-GFP) demonstrated that Dsh-GFP associates with small vesicle-like organelles that are directionally transported along the parallel array of microtubules towards the prospective dorsal side of the embryo during cortical rotation. Perturbing the assembly of the microtubule array with D(2)O, a treatment that promotes the random assembly of the array and the dorsalization of embryos, randomizes translocation of Dsh-GFP. Conversely, UV irradiation of the vegetal pole abolishes movement of Dsh-GFP. Finally, we demonstrate that overexpression of Dsh can stabilize beta-catenin in Xenopus. These data suggest that the directional translocation of Dsh along microtubules during cortical rotation and its subsequent enrichment on the prospective dorsal side of the embryo play a role in locally activating a maternal Wnt pathway responsible for establishing dorsal cell fates in Xenopus.
Assuntos
Padronização Corporal , Polaridade Celular , Desenvolvimento Embrionário , Fosfoproteínas/metabolismo , Transativadores , Proteínas de Xenopus , Proteínas Adaptadoras de Transdução de Sinal , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/efeitos da radiação , Blastocisto/citologia , Blastocisto/metabolismo , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/efeitos da radiação , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos da radiação , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/efeitos da radiação , Proteínas do Citoesqueleto/metabolismo , Óxido de Deutério/farmacologia , Proteínas Desgrenhadas , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Embrião não Mamífero/efeitos da radiação , Receptores Frizzled , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Modelos Biológicos , Nocodazol/farmacologia , Organelas/efeitos dos fármacos , Organelas/metabolismo , Fosfoproteínas/genética , Ratos , Receptores Acoplados a Proteínas G , Receptores de Neurotransmissores/genética , Receptores de Neurotransmissores/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Raios Ultravioleta , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Zigoto/citologia , Zigoto/efeitos dos fármacos , Zigoto/metabolismo , Zigoto/efeitos da radiação , beta CateninaRESUMO
The proto-oncogene wnt-1 (previously referred to as int-1) is thought to be important in embryonic pattern formation although its mechanisms of action are unknown. Premature and increased expression of the Wnt-1 protein, achieved by injection of synthetic wnt-1 RNA into fertilized Xenopus eggs, enhanced gap junctional communication between ventral cells of the developing embryo. This result is consistent with the hypothesis that Wnt proteins activate a receptor-mediated signal transduction pathway and that gap junctional communication can be a target of this pathway. The effects of two Wnt-1-related proteins on gap junctional communication were also investigated: overexpression of Xwnt-8 increased gap junctional coupling in a manner similar to Wnt-1, whereas Xwnt-5A did not. These findings are consistent with the existence of multiple receptors for Wnt proteins.
Assuntos
Blastômeros/fisiologia , Comunicação Celular , Embrião não Mamífero/fisiologia , Junções Intercelulares/fisiologia , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Proteínas de Peixe-Zebra , Animais , Blastômeros/citologia , Embrião não Mamífero/citologia , Feminino , Masculino , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais , Interações Espermatozoide-Óvulo , Proteínas Wnt , Proteína Wnt1 , Xenopus , Proteínas de XenopusRESUMO
The frizzled receptors, which mediate development and display seven hydrophobic, membrane-spanning segments, are cell membrane-localized. We constructed a chimeric receptor with the ligand-binding and transmembrane segments from the beta2-adrenergic receptor (beta2AR) and the cytoplasmic domains from rat Frizzled-1 (Rfz1). Stimulation of mouse F9 clones expressing the chimera (beta2AR-Rfz1) with the beta-adrenergic agonist isoproterenol stimulated stabilization of beta-catenin, activation of a beta-catenin-sensitive promoter, and formation of primitive endoderm. The response was blocked by inactivation of pertussis toxin-sensitive, heterotrimeric guanine nucleotide-binding proteins (G proteins) and by depletion of Galphaq and Galphao. Thus, G proteins are elements of Wnt/Frizzled-1 signaling to the beta-catenin-lymphoid-enhancer factor (LEF)-T cell factor (Tcf) pathway.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores de Neurotransmissores/metabolismo , Transdução de Sinais , Transativadores , Fatores de Transcrição/metabolismo , Proteínas de Xenopus , Proteínas de Peixe-Zebra , Sequência de Aminoácidos , Animais , Clonagem Molecular , Embrião não Mamífero/metabolismo , Endoderma/fisiologia , Receptores Frizzled , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Guanosina Trifosfato/metabolismo , Isoproterenol/metabolismo , Isoproterenol/farmacologia , Camundongos , Dados de Sequência Molecular , Toxina Pertussis , Propranolol/metabolismo , Propranolol/farmacologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Receptores Acoplados a Proteínas G , Receptores de Neurotransmissores/química , Receptores de Neurotransmissores/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Células Tumorais Cultivadas , Fatores de Virulência de Bordetella/farmacologia , Proteínas Wnt , Xenopus , beta CateninaRESUMO
Dysregulation of Wnt-beta-catenin signaling disrupts axis formation in vertebrate embryos and underlies multiple human malignancies. The adenomatous polyposis coli (APC) protein, axin, and glycogen synthase kinase 3beta form a Wnt-regulated signaling complex that mediates the phosphorylation-dependent degradation of beta-catenin. A protein phosphatase 2A (PP2A) regulatory subunit, B56, interacted with APC in the yeast two-hybrid system. Expression of B56 reduced the abundance of beta-catenin and inhibited transcription of beta-catenin target genes in mammalian cells and Xenopus embryo explants. The B56-dependent decrease in beta-catenin was blocked by oncogenic mutations in beta-catenin or APC, and by proteasome inhibitors. B56 may direct PP2A to dephosphorylate specific components of the APC-dependent signaling complex and thereby inhibit Wnt signaling.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Transativadores , Proteínas de Peixe-Zebra , Proteína da Polipose Adenomatosa do Colo , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Proteínas do Citoesqueleto/genética , Regulação para Baixo , Genes Reporter , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Humanos , Leupeptinas/farmacologia , Complexos Multienzimáticos/metabolismo , Mutação , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Fosforilação , Complexo de Endopeptidases do Proteassoma , Proteína Fosfatase 2 , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas , Proteínas Wnt , Xenopus , Proteínas de Xenopus , beta CateninaRESUMO
To reveal how cells exit human pluripotency, we designed a CRISPR-Cas9 screen exploiting the metabolic and epigenetic differences between naïve and primed pluripotent cells. We identify the tumor suppressor, Folliculin(FLCN) as a critical gene required for the exit from human pluripotency. Here we show that FLCN Knock-out (KO) hESCs maintain the naïve pluripotent state but cannot exit the state since the critical transcription factor TFE3 remains active in the nucleus. TFE3 targets up-regulated in FLCN KO exit assay are members of Wnt pathway and ESRRB. Treatment of FLCN KO hESC with a Wnt inhibitor, but not ESRRB/FLCN double mutant, rescues the cells, allowing the exit from the naïve state. Using co-immunoprecipitation and mass spectrometry analysis we identify unique FLCN binding partners. The interactions of FLCN with components of the mTOR pathway (mTORC1 and mTORC2) reveal a mechanism of FLCN function during exit from naïve pluripotency.
Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Via de Sinalização Wnt/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiologia , Linhagem Celular , Estrona/genética , Estrona/metabolismo , Humanos , Imunoprecipitação , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Proteômica , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Via de Sinalização Wnt/genéticaRESUMO
In order to function properly, the brain must be wired correctly during critical periods in early development. Mistakes in this process are hypothesized to occur in disorders like autism and schizophrenia. Later in life, signaling pathways are essential in maintaining proper communication between neuronal and non-neuronal cells, and disrupting this balance may result in disorders like Alzheimer's disease. The Wnt/beta-catenin pathway has a well-established role in cancer. Here, we review recent evidence showing the involvement of Wnt/beta-catenin signaling in neurodevelopment as well as in neurodegenerative diseases. We suggest that the onset/development of such pathological conditions may involve the additive effect of genetic variation within Wnt signaling components and of molecules that modulate the activity of this signaling cascade.