RESUMO
The evolutionarily conserved Toll signaling pathway controls innate immunity across phyla and embryonic patterning in insects. In the Drosophila embryo, Toll is required to establish gene expression domains along the dorsal-ventral axis. Pathway activation induces degradation of the IκB inhibitor Cactus, resulting in a ventral-to-dorsal nuclear gradient of the NFκB effector Dorsal. Here, we investigate how cactus modulates Toll signals through its effects on the Dorsal gradient and on Dorsal target genes. Quantitative analysis using a series of loss- and gain-of-function conditions shows that the ventral and lateral aspects of the Dorsal gradient can behave differently with respect to Cactus fluctuations. In lateral and dorsal embryo domains, loss of Cactus allows more Dorsal to translocate to the nucleus. Unexpectedly, cactus loss-of-function alleles decrease Dorsal nuclear localization ventrally, where Toll signals are high. Overexpression analysis suggests that this ability of Cactus to enhance Toll stems from the mobilization of a free Cactus pool induced by the Calpain A protease. These results indicate that Cactus acts to bolster Dorsal activation, in addition to its role as a NFκB inhibitor, ensuring a correct response to Toll signals.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Fosfoproteínas/metabolismo , Alelos , Animais , Calpaína/genética , Calpaína/metabolismo , Proteínas de Ligação a DNA/genética , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
A wide variety of cellular processes and signaling events are regulated by the proteolytic enzyme γ-secretase. Notch-1 is one of the substrates of γ-secretase and its role in the regulation of muscle differentiation has been well described. Importantly, besides Notch-1, a number of proteins have been identified to undergo proteolysis by γ-secretase. To date, the specific role of γ-secretase during embryonic skeletal muscle differentiation has not been studied. Therefore, we address this question through the analysis of in vitro grown chick myogenic cells during the formation of multinucleated myotubes. The γ-secretase inhibitor DAPT (N-N[-(3,5-Difluorophenacetyl-l-alanyl)]-S-328 phenylglycine-t-butyl-ester) induces muscle hypertrophy. Knockdown of Notch-1 using siRNA specific to chick shows no significant effect in myotube size, suggesting that γ-secretase-dependent effects on muscle hypertrophy in chick myogenic cells are Notch-1-independent. We also investigate the effects of γ-secretase inhibition in the whole proteomic profile of chick myogenic cells. We identified 276 differentially expressed proteins from Label-free proteomic approach. Data overview of interaction network obtained from STRING show that after γ-secretase inhibition cells exhibited imbalance in protein metabolism, cytoskeleton/adhesion, and Sonic Hedgehog signaling. The collection of these results provides new insights into the role of γ-secretase in skeletal muscle hypertrophy.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Proteínas Aviárias/metabolismo , Diaminas/toxicidade , Hipertrofia/veterinária , Proteínas Musculares/metabolismo , Doenças Musculares/veterinária , Receptores Notch/metabolismo , Tiazóis/toxicidade , Animais , Células Cultivadas , Embrião de Galinha , Hipertrofia/induzido quimicamente , Hipertrofia/fisiopatologia , Doenças Musculares/induzido quimicamente , Doenças Musculares/fisiopatologia , Mapas de Interação de Proteínas , Proteômica , Transdução de SinaisRESUMO
Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome (â¼ 702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immune-deficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods.
Assuntos
Adaptação Fisiológica/genética , Doença de Chagas , Interações Hospedeiro-Parasita/genética , Insetos Vetores , Rhodnius , Trypanosoma cruzi/fisiologia , Animais , Sequência de Bases , Transferência Genética Horizontal , Humanos , Insetos Vetores/genética , Insetos Vetores/parasitologia , Dados de Sequência Molecular , Rhodnius/genética , Rhodnius/parasitologia , Wolbachia/genéticaRESUMO
Nitric oxide (NO) contributes to myogenesis by regulating the transition between myoblast proliferation and fusion through cGMP signaling. NO can form S-nitrosothiols (RSNO), which control signaling pathways in many different cell types. However, neither the role of RSNO content nor its regulation by the denitrosylase activity of S-nitrosoglutathione reductase (GSNOR) during myogenesis is understood. Here, we used primary cultures of chick embryonic skeletal muscle cells to investigate whether changes in intracellular RSNO alter proliferation and fusion of myoblasts in the presence and absence of cGMP. Cultures were grown to fuse most of the myoblasts into myotubes, with and without S-nitrosocysteine (CysNO), 8-Br-cGMP, DETA-NO, or inhibitors for NO synthase (NOS), GSNOR, soluble guanylyl cyclase (sGC), or a combination of these, followed by analysis of GSNOR activity, protein expression, RSNO, cGMP, and cell morphology. Although the activity of GSNOR increased progressively over 72 h, inhibiting GSNOR (by GSNOR inhibitor - GSNORi - or by knocking down GSNOR with siRNA) produced an increase in RSNO and in the number of myoblasts and fibroblasts, accompanied by a decrease in myoblast fusion index. This was also detected with CysNO supplementation. Enhanced myoblast number was proportional to GSNOR inhibition. Effects of the GSNORi and GSNOR knockdown were blunted by NOS inhibition, suggesting their dependence on NO synthesis. Interestingly, GSNORi and GSNOR knockdown reversed the attenuated proliferation obtained with sGC inhibition in myoblasts, but not in fibroblasts. Hence myoblast proliferation is enhanced by increasing RSNO, and regulated by GSNOR activity, independently of cGMP production and signaling.
Assuntos
Aldeído Oxirredutases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Desenvolvimento Muscular/genética , Mioblastos/metabolismo , Óxido Nítrico/metabolismo , Aldeído Oxirredutases/antagonistas & inibidores , Aldeído Oxirredutases/genética , Animais , Diferenciação Celular , Fusão Celular , Embrião de Galinha , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Cisteína/análogos & derivados , Cisteína/metabolismo , Cisteína/farmacologia , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , S-Nitrosoglutationa/metabolismo , S-Nitrosotióis/metabolismo , S-Nitrosotióis/farmacologia , Transdução de Sinais , Guanilil Ciclase Solúvel/genética , Guanilil Ciclase Solúvel/metabolismo , Guanilil Ciclase Solúvel/farmacologia , Tionucleotídeos/farmacologia , Triazenos/farmacologiaRESUMO
Rhodnius prolixus is a blood-feeding insect that transmits Trypanosoma cruzi and Trypanosoma rangeli to vertebrate hosts. Rhodnius prolixus is also a classical model in insect physiology, and the recent availability of R. prolixus genome has opened new avenues on triatomine research. Glycogen synthase kinase 3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism, also acting as a downstream component of the Wnt pathway during embryogenesis. GSK-3 has been shown to be highly conserved among several organisms, mainly in the catalytic domain region. Meanwhile, the role of GSK-3 during R. prolixus embryogenesis or glycogen metabolism has not been investigated. Here we show that chemical inhibition of GSK-3 by alsterpaullone, an ATP-competitive inhibitor of GSK3, does not affect adult survival rate, though it alters oviposition and egg hatching. Specific GSK-3 gene silencing by dsRNA injection in adult females showed a similar phenotype. Furthermore, bright field and 4'-6-diamidino-2-phenylindole (DAPI) staining analysis revealed that ovaries and eggs from dsGSK-3 injected females exhibited specific morphological defects. We also demonstrate that glycogen content was inversely related to activity and transcription levels of GSK-3 during embryogenesis. Lastly, after GSK-3 knockdown, we observed changes in the expression of the Wingless (Wnt) downstream target ß-catenin as well as in members of other pathways such as the receptor Notch. Taken together, our results show that GSK-3 regulation is essential for R. prolixus oogenesis and embryogenesis.
Assuntos
Desenvolvimento Embrionário , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio/metabolismo , Rhodnius/embriologia , Rhodnius/enzimologia , Animais , Benzazepinas/metabolismo , Inibidores Enzimáticos/metabolismo , Perfilação da Expressão Gênica , Inativação Gênica , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Indóis/metabolismo , OogêneseRESUMO
The life cycle of the protozoan parasite Trypanosoma cruzi comprises rounds of proliferative cycles and differentiation in distinct host environments. Ras GTPases are molecular switches that play pivotal regulatory functions in cell fate. Rjl is a novel GTPase with unknown function. Herein we show that TcRjl blocks in vivo cell differentiation. The forced expression of TcRjl leads to changes in the overall tyrosine protein phosphorylation profile of parasites. TcRjl expressing parasites sustained DNA synthesis regardless the external stimuli for differentiation. Heterologous expression in the Drosophila melanogaster genetic system strongly suggests a role from TcRjl protein in RTK-dependent pathways and MAPK activation.
Assuntos
Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/enzimologia , Animais , Animais Geneticamente Modificados , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Genes de Protozoários , Sistema de Sinalização das MAP Quinases , Proteínas Monoméricas de Ligação ao GTP/genética , Fenótipo , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Chelicerates, which include spiders, ticks, mites, scorpions, and horseshoe crabs, are members of the phylum Arthropoda. In recent years, several molecular experimental studies of chelicerates have examined the embryology of spiders; however, the embryology of other groups, such as ticks (Acari: Parasitiformes), has been largely neglected. Ticks and mites are believed to constitute a monophyletic group, the Acari. Due to their blood-sucking activities, ticks are also known to be vectors of several diseases. In this study, we analyzed the embryonic development of the cattle tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). First, we developed an embryonic staging system consisting of 14 embryonic stages. Second, histological analysis and antibody staining unexpectedly revealed the presence of a population of tick cells with similar characteristics to the spider cumulus. Cumulus cell populations also exist in other chelicerates; these cells are responsible for the breaking of radial symmetry through bone morphogenetic protein signaling. Third, it was determined that the posterior (opisthosomal) embryonic region of R. microplus is segmented. Finally, we identified the presence of a transient ventral midline furrow and the formation and regression of a fourth leg pair; these features may be regarded as hallmarks of late tick embryogenesis. Importantly, most of the aforementioned features are absent from mite embryos, suggesting that mites and ticks do not constitute a monophyletic group or that mites have lost these features. Taken together, our findings provide fundamental common ground for improving knowledge regarding tick embryonic development, thereby facilitating the establishment of a new chelicerate model system.
Assuntos
Rhipicephalus/embriologia , Animais , Evolução Biológica , Bovinos , Células do Cúmulo/citologia , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário , Feminino , Modelos Animais , Filogenia , Rhipicephalus/citologiaRESUMO
Bone morphogenetic proteins (BMPs) perform a variety of functions during development. Considering a single BMP, what enables its multiple roles in tissues of varied sizes and shapes? What regulates the spatial distribution and activity patterns of the BMP in these different developmental contexts? Some BMP functions require controlling spread of the BMP morphogen, while others require formation of localized, high concentration peaks of BMP activity. Here we review work in Drosophila that describes spatial regulation of the BMP encoded by decapentaplegic (dpp) in different developmental contexts. We concentrate on extracellular modulation of BMP function and discuss the mechanisms that generate concentrated peaks of Dpp activity, subdivide territories of different activity levels or regulate spread of the Dpp morphogen from a point source. We compare these findings with data from vertebrates and non-model organisms to discuss how changes in the regulation of Dpp distribution by extracellular modulators may lead to variability in dpp function in different species.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Animais , Transporte Biológico , Proteínas Morfogenéticas Ósseas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Espaço Extracelular/metabolismo , Morfogênese , Oogênese , RNA Mensageiro/genética , Transdução de Sinais/genética , Especificidade da EspécieRESUMO
Bone morphogenetic proteins (BMPs) have important functions during epithelial development. In Drosophila, extracellular Short gastrulation (Sog) limits the action of the BMP family member Decapentaplegic (Dpp). We have shown that Integrin receptors regulate Sog activity and distribution during pupal wing development to direct placement of wing veins. Here, we show that Integrins perform a similar function in the follicular epithelium, impacting Dpp function during oogenesis and embryonic development. As reported for the wing, this effect is specific to mew, which codes for alphaPS1 integrin. Sog is subject to cleavage by metalloproteases, generating fragments with different properties. We also show that Integrins regulate the distribution of C- and N-terminal Sog fragments in both epithelia, suggesting they may regulate the quality of BMP outputs. Our data indicate that alphaPS1betaPS integrin receptors regulate the amount and type of Sog fragments available for diffusion in the extracellular space during oogenesis and pupal wing development.
Assuntos
Proteínas de Drosophila/metabolismo , Células Epiteliais/metabolismo , Integrina alfa1/metabolismo , Cadeias beta de Integrinas/metabolismo , Animais , Polaridade Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Dinaminas/genética , Dinaminas/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Epitélio/embriologia , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Hibridização In Situ , Integrina alfa1/genética , Cadeias beta de Integrinas/genética , Larva/genética , Larva/crescimento & desenvolvimento , Masculino , Microscopia Confocal , Oogênese , Folículo Ovariano/citologia , Folículo Ovariano/embriologia , Folículo Ovariano/metabolismo , Asas de Animais/embriologia , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismoRESUMO
The multifunctional protein Lmo7 has been implicated in some aspects of myogenesis in mammals. Here we studied the distribution and expression of Lmo7 and the effects of Lmo7 knockdown in primary cultures of chick skeletal muscle cells. Lmo7 was localized within the nuclei of myoblasts and at the perinuclear region of myotubes. Knockdown of Lmo7 using siRNA specific to chick reduces the number and width of myotubes and the number of MyoD positive-myoblasts. Both Wnt3a enriched medium and Bio, activators of the Wnt/beta-catenin pathway, could rescue the effects of the Lmo7 knockdown suggesting a crosstalk between the Wnt/beta-catenin and Lmo7-mediated signaling pathways. Our data shows a role of Lmo7 during the initial events of chick skeletal myogenesis, particularly in myoblast survival.
Assuntos
Proteínas Aviárias/metabolismo , Proteínas com Domínio LIM/metabolismo , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas Aviárias/antagonistas & inibidores , Proteínas Aviárias/genética , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Células Cultivadas , Embrião de Galinha , Citoplasma/metabolismo , Citoplasma/ultraestrutura , França , Proteínas de Fluorescência Verde/antagonistas & inibidores , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Recém-Nascido , Proteínas com Domínio LIM/antagonistas & inibidores , Proteínas com Domínio LIM/genética , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/ultraestrutura , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/ultraestrutura , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sarcolema/metabolismo , Sarcolema/ultraestrutura , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Via de Sinalização WntRESUMO
BACKGROUND: Insect embryonic dorso-ventral patterning depends greatly on two pathways: the Toll pathway and the Bone Morphogenetic Protein pathway. While the relative contribution of each pathway has been investigated in holometabolous insects, their role has not been explored in insects with a hemimetabolous type of development. The hemimetabolous insect Rhodnius prolixus, an important vector of Chagas disease in the Americas, develops from an intermediate germ band and displays complex movements during katatrepsis that are not observed in other orders. However, little is known about the molecular events that regulate its embryogenesis. Here we investigate the expression and function of genes potentially involved in the initial patterning events that establish the embryonic dorso-ventral axis in this hemipteran. RESULTS: We establish a staging system for early embryogenesis that allows us to correlate embryo morphology with gene expression profiles. Using this system, we investigate the role of Toll pathway genes during embryogenesis. Detailed analyses of gene expression throughout development, coupled with functional analyses using parental RNA interference, revealed that maternal Toll is required to establish germ layers along the dorso-ventral axis and for embryo placement along the anterior-posterior axis. Interestingly, knockdown of the Toll pathway effector Rp-dorsal appears to regulate the expression of the Bone Morphogenetic Protein antagonist Rp-short-gastrulation. CONCLUSIONS: Our results indicate that Toll signals are the initiating event in dorso-ventral patterning during Rhodnius embryogenesis, and this is the first report of a conserved role for Toll in a hemipteran. Furthermore, as Rp-dorsal RNA interference generates anteriorly misplaced embryos, our results indicate a novel role for Toll signals in establishment of the anterior-posterior axis in Rhodnius.
RESUMO
Calcium-dependent cysteine proteases of the calpain family are modulatory proteases that cleave their substrates in a limited manner. Among their substrates, calpains target vertebrate and invertebrate IκB proteins. Because proteolysis by calpains potentially generates novel protein functions, it is important to understand how this affects NFκB activity. We investigate the action of Calpain A (CalpA) on the Drosophila melanogaster IκB homologue Cactus in vivo. CalpA alters the absolute amounts of Cactus protein. Our data indicate, however, that CalpA uses additional mechanisms to regulate NFκB function. We provide evidence that CalpA interacts physically with Cactus, recognizing a Cactus pool that is not bound to Dorsal, a fly NFκB/Rel homologue. We show that proteolytic cleavage by CalpA generates Cactus fragments lacking an N-terminal region required for Toll responsiveness. These fragments are generated in vivo and display properties distinct from those of full-length Cactus. We propose that CalpA targets free Cactus, which is incorporated into and modulates Toll-responsive complexes in the embryo and immune system.