RESUMO
Paraspeckles are mammalian-specific nuclear bodies built on the long noncoding RNA NEAT1_2 The molecular mechanisms of paraspeckle formation have been mainly studied using human or mouse cells, and it is not known if the same molecular components are involved in the formation of paraspeckles in other mammalian species. We thus investigated the expression pattern of NEAT1_2 in naked mole-rats (nNEAT1_2), which exhibit extreme longevity and lower susceptibility to cancer. In the intestine, nNEAT1_2 is widely expressed along the entire intestinal epithelium, which is different from the expression of mNeat1_2 that is restricted to the cells of the distal tip in mice. Notably, the expression of FUS, a FET family RNA binding protein, essential for the formation of paraspeckles both in humans and mice, was absent in the distal part of the intestinal epithelium in naked mole-rats. Instead, mRNAs of other FET family proteins EWSR1 and TAF15 were expressed in the distal region. Exogenous expression of these proteins in Fus-deficient murine embryonic fibroblast cells rescued the formation of paraspeckles. These observations suggest that nNEAT1_2 recruits a different set of RNA binding proteins in a cell type-specific manner during the formation of paraspeckles in different organisms.
Assuntos
Paraspeckles , RNA Longo não Codificante , Animais , Humanos , Mucosa Intestinal/metabolismo , Camundongos , Ratos-Toupeira/genética , Ratos-Toupeira/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
NEAT1_2 long noncoding RNA (lncRNA) is the molecular scaffold of paraspeckle nuclear bodies. Here, we report an improved RNA extraction method: extensive needle shearing or heating of cell lysate in RNA extraction reagent improved NEAT1_2 extraction by 20-fold (a property we term "semi-extractability"), whereas using a conventional method NEAT1_2 was trapped in the protein phase. The improved extraction method enabled us to estimate that approximately 50 NEAT1_2 molecules are present in a single paraspeckle. Another architectural lncRNA, IGS16, also exhibited similar semi-extractability. A comparison of RNA-seq data from needle-sheared and control samples revealed the existence of multiple semi-extractable RNAs, many of which were localized in subnuclear granule-like structures. The semi-extractability of NEAT1_2 correlated with its association with paraspeckle proteins and required the prion-like domain of the RNA-binding protein FUS This observation suggests that tenacious RNA-protein and protein-protein interactions, which drive nuclear body formation, are responsible for semi-extractability. Our findings provide a foundation for the discovery of the architectural RNAs that constitute nuclear bodies.
Assuntos
Núcleo Celular/química , RNA Longo não Codificante/análise , RNA Longo não Codificante/isolamento & purificação , Animais , Humanos , Biologia Molecular/métodos , Nucleoproteínas/análise , Nucleoproteínas/isolamento & purificação , Ligação Proteica , Análise de Sequência de RNARESUMO
Methylglyoxal (MG) is a precursor for the generation of endogenous advanced glycation end-products involved in various diseases, including infertility. The present study evaluated the motility and developmental competence after in vitro fertilization of mouse sperm which were exposed to MG in the capacitation medium for 1.5 h. Sperm motility was analyzed using an SQA-V automated sperm quality analyzer. Intracellular reactive oxygen species (ROS), membrane integrity, mitochondrial membrane potential, and DNA damage were assessed using flow cytometry. The matured oocytes were inseminated with MG-exposed sperm, and subsequently, the fertilization and embryonic development in vitro were evaluated in vitro. The exposure of sperm to MG did not considerably affect the swim-up of sperm but resulted in a deteriorated sperm motility in a concentration-dependent manner, which was associated with a decreased mitochondrial activity. However, these effects was not accompanied by obvious ROS accumulation or DNA damage. Furthermore, MG diminished the fertilization rate and developmental competence, even after normal fertilization. Collectively, a short-term exposure to MG during sperm capacitation had a critical impact on sperm motility and subsequent embryonic development after fertilization. Considering that sperm would remain in vivo for up to 3 days until fertilization, our findings suggest that sperm can be affected by MG in the female reproductive organs, which may be associated with infertility.
Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Fertilização/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Aldeído Pirúvico/metabolismo , Capacitação Espermática , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Animais , Cromatina/química , Dano ao DNA , Feminino , Fertilização in vitro , Masculino , Camundongos , Camundongos Endogâmicos ICR , Oócitos , Espécies Reativas de Oxigênio/metabolismo , Análise do Sêmen , Espermatozoides/fisiologiaRESUMO
ß-actin plays key roles in cell migration. Our previous work demonstrated that ß-actin in migratory non-muscle cells is N-terminally arginylated and that this arginylation is required for normal lamellipodia extension. Here, we examined the function of ß-actin arginylation in cell migration. We found that arginylated ß-actin is concentrated at the leading edge of lamellipodia and that this enrichment is abolished after serum starvation as well as in contact-inhibited cells in confluent cultures, suggesting that arginylated ß-actin at the cell leading edge is coupled to active migration. Arginylated actin levels exhibit dynamic changes in response to cell stimuli, lowered after serum starvation and dramatically elevating within minutes after cell stimulation by readdition of serum or lysophosphatidic acid. These dynamic changes require active translation and are not seen in confluent contact-inhibited cell cultures. Microinjection of arginylated actin antibodies into cells severely and specifically inhibits their migration rates. Together, these data strongly suggest that arginylation of ß-actin is a tightly regulated dynamic process that occurs at the leading edge of locomoting cells in response to stimuli and is integral to the signaling network that regulates cell migration.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Arginina/metabolismo , Movimento Celular/fisiologia , Animais , Células Cultivadas , Camundongos , Processamento de Proteína Pós-Traducional/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Coordinated cell migration during development is crucial for morphogenesis and largely relies on cells of the neural crest lineage that migrate over long distances to give rise to organs and tissues throughout the body. Recent studies of protein arginylation implicated this poorly understood posttranslational modification in the functioning of actin cytoskeleton and in cell migration in culture. Knockout of arginyltransferase (Ate1) in mice leads to embryonic lethality and severe heart defects that are reminiscent of cell migration-dependent phenotypes seen in other mouse models. To test the hypothesis that arginylation regulates cell migration during morphogenesis, we produced Wnt1-Cre Ate1 conditional knockout mice (Wnt1-Ate1), with Ate1 deletion in the neural crest cells driven by Wnt1 promoter. Wnt1-Ate1 mice die at birth and in the first 2-3 weeks after birth with severe breathing problems and with growth and behavioral retardation. Wnt1-Ate1 pups have prominent defects, including short palate and altered opening to the nasopharynx, and cranial defects that likely contribute to the abnormal breathing and early death. Analysis of neural crest cell movement patterns in situ and cell motility in culture shows an overall delay in the migration of Ate1 knockout cells that is likely regulated by intracellular mechanisms rather than extracellular signaling events. Taken together, our data suggest that arginylation plays a general role in the migration of the neural crest cells in development by regulating the molecular machinery that underlies cell migration through tissues and organs during morphogenesis.
Assuntos
Arginina/metabolismo , Movimento Celular , Crescimento e Desenvolvimento , Crista Neural/patologia , Aminoaciltransferases/metabolismo , Animais , Animais Recém-Nascidos , Osso e Ossos/anormalidades , Osso e Ossos/enzimologia , Osso e Ossos/patologia , Adesão Celular , Células Cultivadas , Técnicas de Cocultura , Anormalidades Craniofaciais/enzimologia , Anormalidades Craniofaciais/patologia , Técnicas de Inativação de Genes , Mesoderma/enzimologia , Mesoderma/patologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Crista Neural/crescimento & desenvolvimento , Palato/anormalidades , Palato/enzimologia , Palato/patologia , Análise de Sobrevida , Proteína Wnt1/metabolismoRESUMO
Protein arginylation mediated by arginyltransferase (ATE1) is essential for heart formation during embryogenesis, however its cell-autonomous role in cardiomyocytes and the differentiated heart muscle has never been investigated. To address this question, we generated cardiac muscle-specific Ate1 knockout mice, in which Ate1 deletion was driven by α-myosin heavy chain promoter (αMHC-Ate1 mouse). These mice were initially viable, but developed severe cardiac contractility defects, dilated cardiomyopathy, and thrombosis over time, resulting in high rates of lethality after 6months of age. These symptoms were accompanied by severe ultrastructural defects in cardiac myofibrils, seen in the newborns and far preceding the onset of cardiomyopathy, suggesting that these defects were primary and likely underlay the development of the future heart defects. Several major sarcomeric proteins were arginylated in vivo. Moreover, Ate1 deletion in the hearts resulted in a significant reduction of active and passive myofibril forces, suggesting that arginylation is critical for both myofibril structural integrity and contractility. Thus, arginylation is essential for maintaining the heart function by regulation of the major myofibril proteins and myofibril forces, and its absence in the heart muscle leads to progressive heart failure through cardiomyocyte-specific defects.
Assuntos
Cardiomiopatia Dilatada/metabolismo , Coração/fisiologia , Miofibrilas/metabolismo , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Animais , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/prevenção & controle , Genes Letais , Camundongos , Camundongos Knockout , Contração Miocárdica/genética , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Miofibrilas/fisiologia , Sarcômeros/metabolismoRESUMO
ß- and γ-cytoplasmic actins are ubiquitously expressed in every cell type and are nearly identical at the amino acid level but play vastly different roles in vivo. Their essential roles in embryogenesis and mesenchymal cell migration critically depend on the nucleotide sequences of their genes, rather than their amino acid sequences; however, it is unclear which gene elements underlie this effect. Here we address the specific role of the coding sequence in ß- and γ-cytoplasmic actins' intracellular functions, using stable polyclonal populations of immortalized mouse embryonic fibroblasts with exogenously expressed actin isoforms and their 'codon-switched' variants. When targeted to the cell periphery using ß-actin 3'UTR; ß-actin and γ-actin have differential effects on cell migration. These effects directly depend on the coding sequence. Single-molecule measurements of actin isoform translation, combined with fluorescence recovery after photobleaching, demonstrate a pronounced difference in ß- and γ-actins' translation elongation rates in cells, leading to changes in their dynamics at focal adhesions, impairments in actin bundle formation, and reduced cell anchoring to the substrate during migration. Our results demonstrate that coding sequence-mediated differences in actin translation play a key role in cell migration.
Most mammalian cells make both ß- and γ-actin, two proteins which shape the cell's internal skeleton and its ability to migrate. The molecules share over 99% of their sequence, yet they play distinct roles. In fact, deleting the ß-actin gene in mice causes death in the womb, while the animals can survive with comparatively milder issues without their γ-actin gene. How two similar proteins can have such different biological roles is a long-standing mystery. A closer look could hold some clues: ß- and γ-actin may contain the same blocks (or amino acids), but the genetic sequences that encode these proteins differ by about 13%. This is because different units of genetic information known as synonymous codons can encode the same amino acid. These 'silent substitutions' have no effect on the sequence of the proteins, yet a cell reads synonymous codons (and therefore produces proteins) at different speeds. To find out the impact of silent substitutions, Vedula et al. swapped the codons for the two proteins, forcing mouse cells to produce ß-actin using γ-actin codons, and vice versa. Cells with non-manipulated γ-actin and those with ß-actin made using γ-actin codons could move much faster than cells with ß-actin. This suggested that silent substitutions were indeed affecting the role of the protein. Vedula et al. found that cells read γ-codons and therefore made γ-actin much more slowly than ß-codons: this also affected how quickly the protein could be dispatched where it was needed in the cell. Slower production meant that bundles of γ-actin were shorter, which allowed cells to move faster by providing a weaker anchoring system. Overall, this work provides new links between silent substitutions and protein behavior, a relatively new research area which is likely to shed light on other protein families.
Assuntos
Actinas/metabolismo , Movimento Celular/fisiologia , Biossíntese de Proteínas/fisiologia , Actinas/genética , Substituição de Aminoácidos , Animais , Sequência de Bases , Adesões Focais , Regulação da Expressão Gênica , Camundongos , Isoformas de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Uniparental zygotes with two paternal (androgenetic [AG]) or two maternal (gynogenetic [GG]; parthenogenetic [PG]) genomes are not able to develop into viable offspring but can form blastocysts from which embryonic stem cells (ESCs) can be derived. Although some aspects of the in vitro and in vivo differentiation potential of PG and GG ESCs of several species have been studied, the developmental capacity of AG ESCs is much less clear. Here, we investigate the potential of murine AG ESCs to undergo neural differentiation. We observed that AG ESCs differentiate in vitro into pan-neural progenitor cells (pnPCs) that further give rise to cells that express neuronal- and astroglial-specific markers. Neural progeny of in vitro-differentiated AG ESCs exhibited fidelity of expression of six imprinted genes analyzed, with the exception of Ube3a. Bisulfite sequencing for two imprinting control regions suggested that pnPCs predominantly maintained their methylation pattern. Following blastocyst injection of AG and biparental (normal fertilized [N]) ESCs, we found widespread and evenly distributed contribution of ESC-derived cells in both AG and N chimeric early fetal brains. AG and N ESC-derived cells isolated from chimeric fetal brains by fluorescence-activated cell sorting exhibited similar neurosphere-initiating cell frequencies and neural multilineage differentiation potential. Our results indicate that AG ESC-derived neural progenitor/stem cells do not differ from N neural progenitor/stem cells in their self-renewal and neural multilineage differentiation potential. Disclosure of potential conflicts of interest is found at the end of this article.
Assuntos
Androgênios/fisiologia , Blastocisto/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Animais , Blastocisto/citologia , Encéfalo/citologia , Encéfalo/fisiologia , Técnicas de Cultura de Células , Diferenciação Celular , Divisão Celular , Feminino , Genes Reporter , Genoma , Impressão Genômica , Masculino , Camundongos , Camundongos Endogâmicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitina-Proteína Ligases/genética , ZigotoRESUMO
The 28,000-year-old remains of a woolly mammoth, named 'Yuka', were found in Siberian permafrost. Here we recovered the less-damaged nucleus-like structures from the remains and visualised their dynamics in living mouse oocytes after nuclear transfer. Proteomic analyses demonstrated the presence of nuclear components in the remains. Nucleus-like structures found in the tissue homogenate were histone- and lamin-positive by immunostaining. In the reconstructed oocytes, the mammoth nuclei showed the spindle assembly, histone incorporation and partial nuclear formation; however, the full activation of nuclei for cleavage was not confirmed. DNA damage levels, which varied among the nuclei, were comparable to those of frozen-thawed mouse sperm and were reduced in some reconstructed oocytes. Our work provides a platform to evaluate the biological activities of nuclei in extinct animal species.
Assuntos
Núcleo Celular/metabolismo , Fósseis/diagnóstico por imagem , Mamutes/metabolismo , Proteômica , Animais , Núcleo Celular/química , Feminino , Masculino , Mamutes/genética , Camundongos , Técnicas de Transferência Nuclear , Oócitos/metabolismoRESUMO
Cell migration is an evolutionarily conserved mechanism that underlies the development and functioning of uni- and multicellular organisms and takes place in normal and pathogenic processes, including various events of embryogenesis, wound healing, immune response, cancer metastases, and angiogenesis. Despite the differences in the cell types that take part in different migratory events, it is believed that all of these migrations occur by similar molecular mechanisms, whose major components have been functionally conserved in evolution and whose perturbation leads to severe developmental defects. These mechanisms involve intricate cytoskeleton-based molecular machines that can sense the environment, respond to signals, and modulate the entire cell behavior. A big question that has concerned the researchers for decades relates to the coordination of cell migration in situ and its relation to the intracellular aspects of the cell migratory mechanisms. Traditionally, this question has been addressed by researchers that considered the intra- and extracellular mechanisms driving migration in separate sets of studies. As more data accumulate researchers are now able to integrate all of the available information and consider the intracellular mechanisms of cell migration in the context of the developing organisms that contain additional levels of complexity provided by extracellular regulation. This review provides a broad summary of the existing and emerging data in the cell and developmental biology fields regarding cell migration during development.
Assuntos
Movimento Celular/fisiologia , Embrião de Mamíferos/fisiologia , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário/fisiologia , Animais , Encéfalo/embriologia , Adesão Celular/fisiologia , Citoesqueleto/fisiologia , Células Germinativas/fisiologia , Coração/embriologia , Humanos , Mesoderma/citologia , Mesoderma/fisiologia , Modelos Biológicos , Movimento , Crista Neural/fisiologia , Processamento de Proteína Pós-TraducionalRESUMO
The inefficiency of mammalian somatic cell cloning is associated with abnormal gene expression presumably caused by errors in reprogramming of the transplanted genome. In the mouse, aggregation of four-cell stage clones leads to an improvement of both gene expression and development. To determine whether clone-clone aggregation at postgenomic activation stages influences gene expression in bovine clones, we profiled, in single and aggregated embryos at the blastocyst stage, expression of developmentally relevant genes namely Oct4, Dnmt1, Dnmt3, Glut1, Glut3, and a housekeeping gene, Poly(A) polymerase (PolyA) by real-time RT-PCR. Compared to embryos generated by in vitro fertilization (IVF), individual clones more frequently exhibited transcript levels that were more than twofold higher or lower than the average value of IVF embryos. This was observed less often in clone aggregates for Oct4, Dnmt1, Dnmt3, and PolyA, but not for Glut1 and Glut3. The analysis of interferon tau bioactivity as a marker of trophectoderm function in blastocyst outgrowths showed that both single clones and clone aggregates have less extraembryonic potential in vitro compared to IVF embryos, with no apparent consequence of aggregation. These findings indicate that aggregation of bovine clones with each other at later cleavage stages can change gene expression patterns at preimplantation stages, but does not rescue trophectoderm function in vitro.
Assuntos
Blastocisto/citologia , Clonagem de Organismos/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas tau/metabolismo , Animais , Bovinos , Linhagem Celular , Núcleo Celular/metabolismo , Primers do DNA/química , Ectoderma/metabolismo , Implantação do Embrião , Fertilização in vitro , Oócitos/metabolismo , RNA Mensageiro/metabolismoRESUMO
ß- and γ-cytoplasmic actin are nearly indistinguishable in their amino acid sequence, but are encoded by different genes that play non-redundant biological roles. The key determinants that drive their functional distinction are unknown. Here, we tested the hypothesis that ß- and γ-actin functions are defined by their nucleotide, rather than their amino acid sequence, using targeted editing of the mouse genome. Although previous studies have shown that disruption of ß-actin gene critically impacts cell migration and mouse embryogenesis, we demonstrate here that generation of a mouse lacking ß-actin protein by editing ß-actin gene to encode γ-actin protein, and vice versa, does not affect cell migration and/or organism survival. Our data suggest that the essential in vivo function of ß-actin is provided by the gene sequence independent of the encoded protein isoform. We propose that this regulation constitutes a global 'silent code' mechanism that controls the functional diversity of protein isoforms.
Assuntos
Actinas/genética , Actinas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Edição de Genes , CamundongosRESUMO
Alpha synuclein (α-syn) is a central player in neurodegeneration, but the mechanisms triggering its pathology are not fully understood. Here we found that α-syn is a highly efficient substrate for arginyltransferase ATE1 and is arginylated in vivo by a novel mid-chain mechanism that targets the acidic side chains of E46 and E83. Lack of arginylation leads to increased α-syn aggregation and causes the formation of larger pathological aggregates in neurons, accompanied by impairments in its ability to be cleared via normal degradation pathways. In the mouse brain, lack of arginylation leads to an increase in α-syn's insoluble fraction, accompanied by behavioral changes characteristic for neurodegenerative pathology. Our data show that lack of arginylation in the brain leads to neurodegeneration, and suggests that α-syn arginylation can be a previously unknown factor that facilitates normal α-syn folding and function in vivo.
Assuntos
Arginina/metabolismo , Encéfalo/fisiologia , Doenças Neurodegenerativas/metabolismo , alfa-Sinucleína/metabolismo , Sequência de Aminoácidos , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Humanos , Espectrometria de Massas , Camundongos , Camundongos Knockout , Modelos Biológicos , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/prevenção & controle , Neurônios/metabolismo , Neurônios/patologia , Peptídeos/química , Peptídeos/metabolismo , Agregados Proteicos , Agregação Patológica de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Proteínas Recombinantes , Especificidade por Substrato , alfa-Sinucleína/químicaRESUMO
Translocated in liposarcoma/fused in sarcoma (TLS/FUS) is an RNA-binding protein that regulates the splicing pattern of mRNA transcripts and is known to cause a type of familial amyotrophic lateral sclerosis (ALS). In the absence of TLS, Mammalian enabled (Mena), an actin-regulatory protein and a target of TLS, undergoes preferential alternative splicing. In the present study, we show that the ablation of TLS dysregulates the subcellular location and functions of Mena. When TLS knockout (KO) mouse embryonic fibroblasts (MEFs) were transfected with wild-type Mena, it no longer accumulated at focal adhesions and peripheral structures, whereas the localization of the alternatively spliced form was maintained. Additionally, the ability of Mena to suppress the motility of cells was lost in TLS KO MEFs. Moreover, Mena failed to promote neurite outgrowth in TLS KO primary neurons. Taken together, TLS is intimately involved in the local cytoskeletal dynamics surrounding Mena in both fibroblasts and neurons. The robust change in cytoskeletal dynamics, as indicated by the dysregulation of Mena in TLS KO cells, provides a new insight into the pathogenesis of certain types of ALS.
Assuntos
Citoesqueleto de Actina/fisiologia , Proteínas do Citoesqueleto/metabolismo , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Neurônios/metabolismo , Proteína FUS de Ligação a RNA/fisiologia , Sequência de Aminoácidos , Animais , Western Blotting , Diferenciação Celular , Células Cultivadas , Proteínas do Citoesqueleto/genética , Embrião de Mamíferos/citologia , Feminino , Fibroblastos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Neurônios/citologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Paraspeckles are nuclear bodies built on the long noncoding RNA Neat1, which regulates a variety of physiological processes including cancer progression and corpus luteum formation. To obtain further insight into the molecular basis of the function of paraspeckles, we performed fine structural analyses of these nuclear bodies using structural illumination microscopy. Notably, paraspeckle proteins are found within different layers along the radially arranged bundles of Neat1 transcripts, forming a characteristic core-shell spheroidal structure. In cells lacking the RNA binding protein Fus, paraspeckle spheroids are disassembled into smaller particles containing Neat1, which are diffusely distributed in the nucleoplasm. Sequencing analysis of RNAs purified from paraspeckles revealed that AG-rich transcripts associate with Neat1, which are distributed along the shell of the paraspeckle spheroids. We propose that paraspeckles sequester core components inside the spheroids, whereas the outer surface associates with other components in the nucleoplasm to fulfill their function.
Assuntos
Corpos de Inclusão Intranuclear/metabolismo , Microscopia/métodos , Animais , Sequência de Bases , Feminino , Fibroblastos/metabolismo , Hibridização in Situ Fluorescente , Camundongos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Análise de Sequência de RNARESUMO
BACKGROUND: Contractile properties of myofibrils from the myocardium and diaphragm in chronic heart failure are not well understood. We investigated myofibrils in a knockout (KO) mouse model with cardiac-specific deletion of arginyl-tRNA-protein transferase (α-MHCAte1), which presents dilated cardiomyopathy and heart failure. OBJECTIVE: The aim of this study was to test the hypothesis that chronic heart failure in α-MHCAte1 mice is associated with abnormal contractile properties of the heart and diaphragm. METHODS: We used a newly developed system of atomic force cantilevers (AFC) to compare myofibrils from α-MHCAte1 and age-matched wild type mice (WT). Myofibrils from the myocardium and the diaphragm were attached to the AFC used for force measurements during activation/deactivation cycles at different sarcomere lengths. RESULTS: In the heart, α-MHCAte1 myofibrils presented a reduced force during full activation (89±9 nN/µm(2)) when compared to WT (132±11 nN/µm(2)), and the decrease was not influenced by sarcomere length. These myofibrils presented similar kinetics of force development (K(act)), redevelopment (K(tr)), and relaxation (K(rel)). In the diaphragm, α-MHCAte1 myofibrils presented an increased force during full activation (209±31 nN/µm(2)) when compared to WT (123±20 nN/µm(2)). Diaphragm myofibrils of α-MHCAte1 and WT presented similar K(act), but α-MHCAte1 myofibrils presented a faster K(rel) (6.11±0.41s(-1) vs 4.63±0.41 s(-1)). CONCLUSION: Contrary to our working hypothesis, diaphragm myofibrils from α-MHCAte1 mice produced an increased force compared to myofibrils from WT. These results suggest a potential compensatory mechanism by which the diaphragm works under loading conditions in the α-MHCAte1 chronic heart failure model.
Assuntos
Aminoaciltransferases/genética , Diafragma/fisiologia , Deleção de Genes , Contração Muscular/genética , Miocárdio , Miofibrilas/genética , Aminoaciltransferases/deficiência , Animais , Fenômenos Biomecânicos/genética , Modelos Animais de Doenças , Coração/fisiologia , Camundongos , Camundongos Knockout , Contração Miocárdica/genética , Miocárdio/enzimologiaAssuntos
Ciclo Celular/genética , Núcleo Celular/genética , Clonagem de Organismos , Animais , Cromatina/genética , Clonagem de Organismos/efeitos adversos , Clonagem de Organismos/métodos , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Transferência Nuclear , Óvulo/citologia , GravidezRESUMO
Posttranslational protein arginylation mediated by Ate1 is essential for cardiovascular development, actin cytoskeleton functioning, and cell migration. Ate1 plays a role in the regulation of cytoskeleton and is essential for cardiovascular development and angiogenesis--capillary remodeling driven by in-tissue migration of endothelial cells. To address the role of Ate1 in cytoskeleton-dependent processes and endothelial cell function during development, we produced a conditional mouse knockout with Ate1 deletion driven by Tek endothelial receptor tyrosine kinase promoter expressed in the endothelium and in the germ line. Contrary to expectations, Tek-Ate1 mice were viable and had no visible angiogenesis-related phenotypes; however, these mice showed reproductive defects, with high rates of embryonic lethality in the second generation, at stages much earlier than the complete Ate1 knockout strain. While some of the early lethality originated from the subpopulation of embryos with homozygous Tek-Cre transgene--a problem that has not previously been reported for this commercial mouse strain--a distinct subpopulation of embryos had lethality at early post-implantation stages that could be explained only by a previously unknown defect in gametogenesis originating from Tek-driven Ate1 deletion in premeiotic germs cells. These results demonstrate a novel role of Ate1 in germ cell development.
Assuntos
Aminoaciltransferases/genética , Gametogênese/genética , Deleção de Genes , Regiões Promotoras Genéticas , Receptores Proteína Tirosina Quinases/genética , Actinas/metabolismo , Animais , Movimento Celular , Citoesqueleto/metabolismo , Implantação do Embrião , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas , Masculino , Camundongos , Camundongos Knockout , Receptor TIE-2RESUMO
The biological role of genomic imprinting in adult tissue is central to the consideration of transplanting uniparental embryonic stem (ES) cell-derived tissues. We have recently shown that both maternal (parthenogenetic/gynogenetic) and paternal (androgenetic) uniparental ES cells can differentiate, both in vivo in chimeras and in vitro, into adult-repopulating hematopoietic stem and progenitor cells. This suggests that, at least in some tissues, the presence of two maternal or two paternal genomes does not interfere with stem cell function and tissue homeostasis in the adult. Here, we consider implications of the contribution of uniparental cells to hematopoiesis and to development of other organ systems, notably neural tissue for which consequences of genomic imprinting are associated with a known bias in development and behavioral disorders. Our findings so far indicate that there is little or no limit to the differentiation potential of uniparental ES cells outside the normal developmental paradigm. As a potentially donor MHC-matching source of tissue, uniparental transplants may provide not only a clinical resource but also a unique tool to investigate aspects of genomic imprinting in adults.
RESUMO
The development of somatic cell nuclear transfer (SCNT) embryos critically depends on appropriate reprogramming and expression of pluripotency genes, such as Pou5f1/POU5F1 (previously known as Oct4/OCT4). To study POU5F1 transcription activation in living bovine SCNT embryos without interference by maternal POU5F1 mRNA, we generated chromosomally normal fetal fibroblast donor cells stably carrying a mouse Pou5f1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene at a single integration site without detectable EGFP expression. Morphologic and quantitative analyses of whole-mount SCNT embryos by confocal microscopy revealed robust initial activation of the Pou5f1 reporter gene during the fourth cell cycle. In Day 6 SCNT embryos EGFP expression levels were markedly higher than in Day 4 embryos but varied substantially between individual embryos, even at comparable cell numbers. Embryos with low EGFP levels had far more morphologically abnormal cell nuclei than those with high EGFP levels. Our data strongly suggest that bovine SCNT embryos consistently start activation of the POU5F1 promoter during the fourth cell cycle, whereas later in development the expression level substantially differs between individual embryos, which may be associated with developmental potential. In fibroblasts from phenotypically normal SCNT fetuses recovered on Day 34, the Pou5f1 reporter promoter was silent but was activated by a second round of SCNT. The restoration of pluripotency can be directly observed in living cells or SCNT embryos from such Pou5f1-EGFP transgenic fetuses, providing an attractive model for systematic investigation of epigenetic reprogramming in large mammals.