RESUMO
Germ cells are the only cell type capable of generating an entirely new organism. In order to execute germline-specific functions and to retain the capacity for totipotency, germ cells repress somatic differentiation, interact with a specialized microenvironment, and use germline-specific networks of RNA regulation.
Assuntos
Células Germinativas/citologia , Células Germinativas/metabolismo , Animais , Regulação da Expressão Gênica , Interferência de RNA , Transcrição GênicaRESUMO
Growth factor withdrawal results in the endocytosis and degradation of transporter proteins for glucose and amino acids. Here, we show that this process is under the active control of the small GTPase Rab7. In the presence of growth factor, Rab7 inhibition had no effect on nutrient transporter expression. In growth factor-deprived cells, however, blocking Rab7 function prevented the clearance of glucose and amino acid transporter proteins from the cell surface. When Rab7 was inhibited, growth factor deprived cells maintained their mitochondrial membrane potential and displayed prolonged, growth factor-independent, nutrient-dependent cell survival. Thus, Rab7 functions as a proapoptotic protein by limiting cell-autonomous nutrient uptake. Consistent with this, dominant-negative Rab7 cooperated with E1A to promote the transformation of p53(-/-) mouse embryonic fibroblasts (MEFs). These results suggest that proteins that limit nutrient transporter expression function to prevent cell-autonomous growth and survival.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Substâncias de Crescimento/fisiologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas rab de Ligação ao GTP/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Proteínas E1A de Adenovirus/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Aminoácidos/farmacologia , Animais , Western Blotting , Carbonil Cianeto m-Clorofenil Hidrazona/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Sobrevivência Celular/fisiologia , Cloroquina/metabolismo , Cães , Embrião de Mamíferos , Fibroblastos , Citometria de Fluxo , Privação de Alimentos , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Glucose/farmacologia , Transportador de Glucose Tipo 1 , Proteínas de Fluorescência Verde , Humanos , Imuno-Histoquímica , Interleucina-3/farmacologia , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia Confocal , Proteínas de Transporte de Monossacarídeos/genética , Mutação , Testes de Precipitina , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fatores de Tempo , Transformação Genética , Proteína Supressora de Tumor p53/metabolismo , Proteína bcl-X , proteínas de unión al GTP Rab7RESUMO
The serine/threonine kinase Akt is a component of many receptor signal transduction pathways and can prevent cell death following growth factor withdrawal. Here, we show that Akt inhibition of cell death is not dependent on new protein translation. Instead, Akt inhibition of cell death requires glucose hydrolysis through glycolysis. Akt was found to regulate multiple steps in glycolysis via posttranscriptional mechanisms that included localization of the glucose transporter, Glut1, to the cell surface and maintenance of hexokinase function in the absence of extrinsic factors. To test the role of glucose uptake and phosphorylation in growth factor-independent survival, cells were transfected with Glut1 and hexokinase 1 (Glut1/HK1) cells. Glut1/HK1 cells accumulated Glut1 on the cell surface and had high glucose uptake capacity similar to that of cells with constitutively active Akt (mAkt). Unlike mAkt-expressing cells, however, they did not consume more glucose, did not maintain prolonged phosphofructokinase-1 protein levels and activity, and did not maintain pentose phosphate shuttle activity in the absence of growth factor. Nevertheless, expression of Glut1 and HK1 promoted increased cytosolic NADH and NADPH levels relative to those of the control cells upon growth factor withdrawal, prevented activation of Bax, and promoted growth factor-independent survival. These data indicate that Bax conformation is sensitive to glucose metabolism and that maintaining glucose uptake and phosphorylation can promote cell survival in the absence of growth factor. Furthermore, Akt required glucose and the ability to perform glycolysis to prevent Bax activation. The prevention of Bax activation by posttranscriptional regulation of glucose metabolism may, therefore, be a required aspect of the ability of Akt to maintain long-term cell survival in the absence of growth factors.
Assuntos
Glucose/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Animais , Western Blotting , Morte Celular , Divisão Celular , Linhagem Celular , Sobrevivência Celular , DNA Complementar/metabolismo , Citometria de Fluxo , Glucose/farmacocinética , Transportador de Glucose Tipo 1 , Glicólise , Hexoquinase/metabolismo , Hidrólise , Interleucina-3/metabolismo , Camundongos , Microscopia de Fluorescência , Proteínas de Transporte de Monossacarídeos/metabolismo , NAD/metabolismo , NADP/metabolismo , Conformação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Frações Subcelulares , Fatores de Tempo , Transfecção , Proteína X Associada a bcl-2RESUMO
The separation of germline from somatic lineages is fundamental to reproduction and species preservation. Here, we show that Drosophila Germ cell-less (GCL) is a critical component in this process by acting as a switch that turns off a somatic lineage pathway. GCL, a conserved BTB (Broad-complex, Tramtrack, and Bric-a-brac) protein, is a substrate-specific adaptor for Cullin3-RING ubiquitin ligase complex (CRL3GCL). We show that CRL3GCL promotes PGC fate by mediating degradation of Torso, a receptor tyrosine kinase (RTK) and major determinant of somatic cell fate. This mode of RTK degradation does not depend upon receptor activation but is prompted by release of GCL from the nuclear envelope during mitosis. The cell-cycle-dependent change in GCL localization provides spatiotemporal specificity for RTK degradation and sequesters CRL3GCL to prevent it from participating in excessive activities. This precisely orchestrated mechanism of CRL3GCL function and regulation defines cell fate at the single-cell level.
Assuntos
Linhagem da Célula , Proteínas Culina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Proteínas Nucleares/metabolismo , Proteólise , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Membrana Celular/metabolismo , Sequência Conservada , Proteínas de Drosophila/química , Células Germinativas/citologia , Células Germinativas/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Mitose , Membrana Nuclear/metabolismo , Sinais de Localização Nuclear/metabolismo , Proteínas Nucleares/química , Oogênese , Domínios Proteicos , Transdução de Sinais , Especificidade por Substrato , UbiquitinaçãoRESUMO
Activation of the FGF signaling pathway during preimplantation development of the mouse embryo is known to be essential for differentiation of the inner cell mass and the formation of the primitive endoderm (PrE). We now show using fluorescent reporter knockin lines that Fgfr1 is expressed in all cell populations of the blastocyst, while Fgfr2 expression becomes restricted to extraembryonic lineages, including the PrE. We further show that loss of both receptors prevents the development of the PrE and demonstrate that FGFR1 plays a more prominent role in this process than FGFR2. Finally, we document an essential role for FGFRs in embryonic stem cell (ESC) differentiation, with FGFR1 again having a greater influence than FGFR2 in ESC exit from the pluripotent state. Collectively, these results identify mechanisms through which FGF signaling regulates inner cell mass lineage restriction and cell commitment during preimplantation development.
Assuntos
Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Endoderma/citologia , Fator 4 de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/fisiologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/fisiologia , Animais , Massa Celular Interna do Blastocisto/citologia , Massa Celular Interna do Blastocisto/metabolismo , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de SinaisRESUMO
Pim-2 is a transcriptionally regulated oncogenic kinase that promotes cell survival in response to a wide variety of proliferative signals. Deregulation of Pim-2 expression has been documented in several human malignancies, including leukemia, lymphoma, and multiple myeloma. Here, we show that the ability of Pim-2 to promote survival of cells is dependent on nuclear factor (NF)-kappaB activation. Pim-2 activates NF-kappaB-dependent gene expression by inducing phosphorylation of the oncogenic serine/threonine kinase Cot, leading to both augmentation of IkappaB kinase activity and a shift in nuclear NF-kappaB from predominantly p50 homodimers to p50/p65 heterodimers. Blockade of NF-kappaB function eliminates Pim-2-mediated survival in both cell lines and primary cells, and both Cot phosphorylation and expression are required for the prosurvival effects of Pim-2. Although Pim-2 cooperates with Myc to promote growth factor-independent cell proliferation, this feature is abrogated by NF-kappaB blockade. The ability of Pim-2 to serve as an oncogene in vivo depends on sustained NF-kappaB activity. Thus, the transcriptional induction of Pim-2 initiates a novel NF-kappaB activation pathway that regulates cell survival.
Assuntos
Ativação Linfocitária/fisiologia , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Sequência de Bases , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Hidrólise , Proteínas I-kappa B/metabolismo , Células Jurkat , Camundongos , Camundongos Transgênicos , FosforilaçãoRESUMO
Cancer cells frequently display high rates of aerobic glycolysis in comparison to their nontransformed counterparts, although the molecular basis of this phenomenon remains poorly understood. Constitutive activity of the serine/threonine kinase Akt is a common perturbation observed in malignant cells. Surprisingly, although Akt activity is sufficient to promote leukemogenesis in nontransformed hematopoietic precursors and maintenance of Akt activity was required for rapid disease progression, the expression of activated Akt did not increase the proliferation of the premalignant or malignant cells in culture. However, Akt stimulated glucose consumption in transformed cells without affecting the rate of oxidative phosphorylation. High rates of aerobic glycolysis were also identified in human glioblastoma cells possessing but not those lacking constitutive Akt activity. Akt-expressing cells were more susceptible than control cells to death after glucose withdrawal. These data suggest that activation of the Akt oncogene is sufficient to stimulate the switch to aerobic glycolysis characteristic of cancer cells and that Akt activity renders cancer cells dependent on aerobic glycolysis for continued growth and survival.
Assuntos
Glioblastoma/metabolismo , Leucemia/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Aerobiose , Animais , Linhagem Celular Tumoral , Ativação Enzimática , Glioblastoma/enzimologia , Glioblastoma/genética , Glucose/metabolismo , Glucose/farmacologia , Glicólise , Humanos , Leucemia/enzimologia , Leucemia/genética , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , TransfecçãoRESUMO
The primordial germ cells (PGCs) are the first cells to form during Drosophila melanogaster embryogenesis. Whereas the process of somatic cell formation has been studied in detail, the mechanics of PGC formation are poorly understood. Here, using four-dimensional multi-photon imaging combined with genetic and pharmacological manipulations, we find that PGC formation requires an anaphase spindle-independent cleavage pathway. In addition to using core regulators of cleavage, including the small GTPase RhoA (Drosophila rho1) and the Rho-associated kinase, ROCK (Drosophila drok), we show that this pathway requires Germ cell-less (GCL), a conserved BTB-domain protein not previously implicated in cleavage mechanics. This alternative form of cell formation suggests that organisms have evolved multiple molecular strategies for regulating the cytoskeleton during cleavage.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Embrião não Mamífero/citologia , Desenvolvimento Embrionário/fisiologia , Células Germinativas/citologia , Fuso Acromático/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Drosophila melanogaster/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Microscopia de Fluorescência por Excitação Multifotônica , Imagem com Lapso de TempoRESUMO
Early embryos of some metazoans polarize radially to facilitate critical patterning events such as gastrulation and asymmetric cell division; however, little is known about how radial polarity is established. Early embryos of Caenorhabditis elegans polarize radially when cell contacts restrict the polarity protein PAR-6 to contact-free cell surfaces, where PAR-6 regulates gastrulation movements. We have identified a Rho guanosine triphosphatase activating protein (RhoGAP), PAC-1, which mediates C. elegans radial polarity and gastrulation by excluding PAR-6 from contacted cell surfaces. We show that PAC-1 is recruited to cell contacts, and we suggest that PAC-1 controls radial polarity by restricting active CDC-42 to contact-free surfaces, where CDC-42 binds and recruits PAR-6. Thus, PAC-1 provides a dynamic molecular link between cell contacts and PAR proteins that polarizes embryos radially.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Comunicação Celular , Membrana Celular/metabolismo , Polaridade Celular , Embrião não Mamífero/citologia , Proteínas Ativadoras de GTPase/metabolismo , Animais , Padronização Corporal , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Citoplasma/metabolismo , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Gastrulação , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Signals that regulate T cell homeostasis are not fully understood. G protein-coupled receptors (GPCR), such as the chemokine receptors, may affect homeostasis by direct signaling or by guiding T cell migration to distinct location-restricted signals. Here, we show that blockade of Galphai-associated GPCR signaling by treatment with pertussis toxin led to T cell atrophy and shortened life-span in T cell-replete hosts and prevented T cell homeostatic growth and proliferation in T cell-deficient hosts. In vitro, however, neither GPCR inhibition nor chemokine stimulation affected T cell atrophy, survival, or proliferation. These findings suggest that GPCR signals are not trophic stimuli, but instead may be required for migration to distinct trophic signals, such as IL-7 or self-peptide/MHC. Surprisingly, while chemokines did not affect atrophy, atrophic T cells displayed increased chemokine-induced chemotaxis that was prevented by IL-7 and submitogenic anti-CD3 antibody treatment. This increase in migration was associated with increased levels of GTP-bound Rac and the ability to remodel actin. These data suggest a novel mechanism of T cell homeostasis wherein GPCR may promote T cell migration to distinct location-restricted homeostatic trophic cues for T cell survival and growth. Homeostatic trophic signals, in turn, may suppress chemokine sensitivity and cytoskeletal remodeling, to inhibit further migration.
Assuntos
Quimiotaxia/imunologia , Substâncias de Crescimento/imunologia , Homeostase/imunologia , Receptores Acoplados a Proteínas G/imunologia , Linfócitos T/imunologia , Actinas/imunologia , Actinas/metabolismo , Transferência Adotiva , Animais , Atrofia/imunologia , Western Blotting , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/imunologia , Citoesqueleto/metabolismo , Citometria de Fluxo , Interleucina-7/imunologia , Interleucina-7/metabolismo , Tecido Linfoide/patologia , Camundongos , Toxina Pertussis/farmacologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Citocinas/imunologia , Linfócitos T/efeitos dos fármacosRESUMO
MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally. To block all miRNA formation in zebrafish, we generated maternal-zygotic dicer (MZdicer) mutants that disrupt the Dicer ribonuclease III and double-stranded RNA-binding domains. Mutant embryos do not process precursor miRNAs into mature miRNAs, but injection of preprocessed miRNAs restores gene silencing, indicating that the disrupted domains are dispensable for later steps in silencing. MZdicer mutants undergo axis formation and differentiate multiple cell types but display abnormal morphogenesis during gastrulation, brain formation, somitogenesis, and heart development. Injection of miR-430 miRNAs rescues the brain defects in MZdicer mutants, revealing essential roles for miRNAs during morphogenesis.
Assuntos
Encéfalo/embriologia , MicroRNAs/fisiologia , Morfogênese , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Padronização Corporal , Diferenciação Celular , Sistema Nervoso Central/embriologia , Gástrula/fisiologia , Inativação Gênica , Coração/embriologia , MicroRNAs/genética , MicroRNAs/metabolismo , Mutação , Neurônios/citologia , Fenótipo , Processamento Pós-Transcricional do RNA , RNA de Cadeia Dupla/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Transdução de Sinais , Somitos/citologia , Somitos/fisiologia , Medula Espinal/embriologiaRESUMO
The mechanisms that regulate basal T cell size and metabolic activity are uncertain. Since the phosphatidylinositol-3 phosphate kinase (PI3 K) and Akt (PKB) pathway has been shown in model organisms to regulate both cell size and metabolism, we generated transgenic mice expressing a constitutively active form of Akt (myristoylated Akt, mAkt) in T cells. Naive transgenic T cells were enlarged and had increased rates of glycolysis compared to control T cells. In addition, mAkt transgenic T cells resisted death-by-neglect upon in vitro culture. Upon activation, mAkt-transgenic T cells were less dependent than control cells on costimulation through CD28 and could both grow rapidly and secrete cytokines in the absence of CD28 ligation. In addition, transgenic expression of mAkt led to the accumulation of CD4 T cells and B cells with age. Many aged mAkt-transgenic mice also developed autoimmunity with immunoglobulin deposits on kidney glomeruli and displayed increased incidence of lymphoma. Together, these data show that Akt activation is sufficient to increase basal T cell size and metabolism. Enhancement of T cell metabolism by Akt and more rapid CD28-independent T cell growth may contribute to the accumulation of excess immune cells and the development of lymphoma and autoimmunity.
Assuntos
Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Autoimunidade , Antígenos CD28/metabolismo , Divisão Celular , Tamanho Celular , Glicólise , Humanos , Interferon gama/biossíntese , Interleucina-2/biossíntese , Interfase , Linfoma de Células T/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , Linfócitos T/citologiaRESUMO
Bak and Bax are required and redundant regulators of an intrinsic mitochondrial cell death pathway. To analyze this pathway in T cell development and homeostasis, we reconstituted mice with Bak(-/-)Bax<(-/-) hematopoietic cells. We found that the development and selection of Bak(-/-)Bax(-/-) thymocytes was disrupted, with altered representation of thymic subsets and resistance to both death-by-neglect and antigen receptor-induced apoptosis. Elimination of Bak(-/-)Bax(-/-) T cells that responded to endogenous superantigen was also reduced. Despite more efficient early reconstitution and apoptotic resistance of Bak(-/-)Bax(-/-) thymocytes, thymic cellularity declined over time. Reduced thymic cellularity resulted from a progressive cessation of thymopoiesis. However, animals developed splenomegaly as a result of accumulated memory T cells that were not deleted after antigen-driven expansion. These data indicate that Bak and Bax are required for thymic selection and peripheral lymphoid homeostasis and suggest that thymopoiesis can be negatively regulated by the accumulation of cells that would normally be eliminated by pro-apoptotic Bcl-2-related genes.
Assuntos
Apoptose/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Proteínas de Membrana/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Proteínas Proto-Oncogênicas/imunologia , Animais , Diferenciação Celular , Células Cultivadas , Feminino , Hematopoese , Imunofenotipagem , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Timo/citologia , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína X Associada a bcl-2RESUMO
Growth factor withdrawal results in the termination of factor-dependent transcription. One transcript that declines rapidly following growth factor deprivation of hematopoietic cells is the serine/threonine kinase pim-2. When constitutively expressed, Pim-2 conferred long-term resistance to a variety of apoptotic stimuli including growth factor withdrawal and endogenous levels of Pim-2 contributed to growth factor-mediated apoptotic resistance. Pim-2 expression maintained cell size and mitochondrial potential independently of the PI3K/Akt/TOR pathway. Pim-2-dependent maintenance of cell size and survival correlated with its ability to maintain rapamycin-resistant phosphorylation of the translational repressor 4E-BP1 and phosphorylation of the BH3 protein BAD. These results establish Pim-2 as a direct link between growth factor-induced transcription and a novel, kinase-dependent pathway that promotes cell-autonomous survival.
Assuntos
Apoptose , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/fisiologia , Transcrição Gênica , Animais , Antibióticos Antineoplásicos/farmacologia , Northern Blotting , Western Blotting , Divisão Celular , Linhagem Celular , Sobrevivência Celular , Relação Dose-Resposta a Droga , Citometria de Fluxo , Regulação da Expressão Gênica , Vetores Genéticos , Glucose/metabolismo , Glucose/farmacologia , Interleucina-3/metabolismo , Lactatos/metabolismo , Potenciais da Membrana , Camundongos , Mitocôndrias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Fosforilação , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Estaurosporina/farmacologia , Tapsigargina/farmacologia , Fatores de Tempo , TransgenesRESUMO
Despite evidence that protein kinases are regulators of apoptosis, a specific role for phosphatases in regulating cell survival has not been established. Here we show that alpha4, a noncatalytic subunit of protein phosphatase 2A (PP2A), is required to repress apoptosis in murine cells. alpha4 is a nonredundant regulator of the dephosphorylation of the transcription factors c-Jun and p53. As a result of alpha4 deletion, multiple proapoptotic genes were transcribed. Either inhibition of new protein synthesis or Bcl-xL overexpression suppressed apoptosis initiated by alpha4 deletion. Thus, mammalian cell viability depends on repression of transcription-initiated apoptosis mediated by a component of PP2A.