RESUMO
The disruption of the gene encoding the Dictyostelium Ras subfamily protein, RasC, results in a strain that does not aggregate and has defects in both cAMP signal relay and cAMP chemotaxis. Disruption of a second gene in the rasC(-) strain by Restriction Enzyme Mediated Integration produced cells that were capable of forming multicellular structures in plaques on bacterial lawns. The disrupted gene (dmpA) encoded a novel membrane protein that was designated Dmp1. Although the rasC(-)/dmpA(-) cells progressed through early development, they did not form aggregation streams on a plastic surface under submerged starvation conditions. Phosphorylation of PKB in response to cAMP, which is significantly reduced in rasC(-) cells, remained low in the rasC(-)/dmpA(-) cells. However, in spite of this low PKB phosphorylation, the rasC(-)/dmpA(-) cells underwent efficient chemotaxis to cAMP in a spatial gradient. Cyclic AMP accumulation, which was greatly reduced in the rasC(-) cells, was restored in the rasC(-)/dmpA(-) strain, but cAMP relay in these cells was not apparent. These data indicate that although the rasC(-)/dmpA(-) cells were capable of associating to form multicellular structures, normal aggregative cell signaling was clearly not restored. Disruption of the dmpA gene in a wild-type background resulted in cells that exhibited a slight defect in aggregation and a more substantial defect in late development. These results indicate that, in addition to the role played by Dmp1 in aggregation, it is also involved in late development.
Assuntos
Quimiotaxia/genética , Dictyostelium/citologia , Dictyostelium/genética , Deleção de Genes , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Protozoários/genética , Proteínas ras/deficiência , Sequência de Aminoácidos , Animais , Northern Blotting , Southern Blotting , Agregação Celular/genética , AMP Cíclico/metabolismo , Dictyostelium/fisiologia , Genes de Protozoários , Genes Supressores , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Fenótipo , Fosforilação , Estrutura Terciária de Proteína/genética , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/fisiologia , Proteínas ras/biossíntese , Proteínas ras/genéticaRESUMO
The disruption of the gene encoding the Dictyostelium Ras subfamily protein, RasC results in a strain that fails to aggregate with defects in both cAMP signal relay and chemotaxis. Restriction enzyme mediated integration disruption of a second gene in the rasC(-) strain resulted in cells that were capable of forming multicellular structures in plaques on bacterial lawns. The disrupted gene, designated pikD(1), encodes a member of the phosphatidyl-inositol-4-kinase beta subfamily. Although the rasC(-)/pikD(1) cells were capable of progressing through early development, when starved on a plastic surface under submerged conditions, they did not form aggregation streams or exhibit pulsatile motion. The rasC(-)/pikD(1) cells were extremely efficient in their ability to chemotax to cAMP in a spatial gradient, although the reduced phosphorylation of PKB in response to cAMP observed in rasC(-) cells, was unchanged. In addition, the activation of adenylyl cyclase, which was greatly reduced in the rasC(-) cells, was only minimally increased in the rasC(-)/pikD(1) strain. Thus, although the rasC(-)/pikD(-) cells were capable of associating to form multicellular structures, normal cell signaling was clearly not restored. The disruption of the pikD gene in a wild type background resulted in a strain that was delayed in aggregation and formed large aggregation streams, when starved on a plastic surface under submerged conditions. This strain also exhibited a slight defect in terminal development. In conclusion, disruption of the pikD gene in a rasC(-) strain resulted in cells that were capable of forming multicellular structures, but which did so in the absence of normal signaling and aggregation stream formation.
Assuntos
1-Fosfatidilinositol 4-Quinase/genética , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/genética , Genes de Protozoários , Genes ras , Adenilil Ciclases/metabolismo , Animais , Adesão Celular/genética , Agregação Celular/genética , Quimiotaxia , AMP Cíclico/análogos & derivados , AMP Cíclico/análise , AMP Cíclico/biossíntese , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , DNA de Protozoário , Dictyostelium/citologia , Dictyostelium/enzimologia , Dictyostelium/fisiologia , Ativação Enzimática , Regulação da Expressão Gênica no Desenvolvimento/genética , Cinética , Mutagênese Insercional , Transdução de Sinais , InaniçãoRESUMO
In addition to its previously established roles in cAMP relay and cAMP chemotaxis, loss of signal transduction through the RasC protein was found to impact a number of vegetative cell functions. Vegetative rasC- cells exhibited reduced random motility, were less polarized and had altered F-actin distribution. Cells lacking RasC also contained more protein and were larger in size than wild type cells. These increases were associated with increased liquid phase endocytosis. Despite the increase in cell size, cytokinesis was relatively normal and there was no change in the rate of cell division. rasC- cells also chemotaxed poorly to folate and exhibited reduced F-actin accumulation, reduced ERK2 phosphorylation and reduced Akt/PKB phosphorylation in response to folate, indicating that RasC was also involved in transducing chemotactic signals in vegetative cells.
Assuntos
Actinas , Dictyostelium/fisiologia , Endocitose/fisiologia , Proteínas ras/fisiologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Actinas/metabolismo , Animais , Divisão Celular/genética , Divisão Celular/fisiologia , Quimiotaxia/genética , Quimiotaxia/fisiologia , Dextranos/metabolismo , Dictyostelium/citologia , Endocitose/genética , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Ácido Fólico/metabolismo , MAP Quinase Quinase 2/metabolismo , Fosforilação , Pinocitose/genética , Pinocitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/fisiologia , Transdução de Sinais/fisiologia , Proteínas ras/deficiência , Proteínas ras/genéticaRESUMO
The Cdk8 proteins are kinases which phosphorylate the carboxy terminal domain (CTD) of RNA polymerase II (Pol II) as well as some transcription factors and, therefore, are involved in the regulation of transcription. Here, we report that a Cdk8 homologue from Dictyostelium discoideum is localized in the nucleus where it forms part of a high molecular weight complex that has CTD kinase activity. Insertional mutagenesis was used to abrogate gene function, and analysis of the null strain revealed that the DdCdk8 protein plays an important role in spore formation during late development. As previously reported [Dev. Growth Differ. 44 (2002) 213] Ddcdk8- cells also exhibit impaired aggregation, although we report that the severity of the defect depends upon experimental conditions. When aggregation occurs, Ddcdk8- cells form abnormal terminally differentiated structures within which the Ddcdk8- cells differentiate into stalk cells but fail to form spores, indicating a role for DdCdk8 in cell differentiation. When Ddcdk8 is expressed from its own promoter, the protein is able to rescue both the late developmental defect and the impaired aggregation. However, when expressed from an heterologous promoter, only the impaired aggregation is rescued. This result demonstrates that the defect during late development is not a consequence of impaired aggregation and indicates a direct role for DdCdk8 in spore formation.
Assuntos
Dictyostelium/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Fenótipo , Esporos/crescimento & desenvolvimento , Animais , Northern Blotting , Quimiotaxia/fisiologia , Cromatografia em Gel , Dictyostelium/enzimologia , Imunofluorescência , Biblioteca Gênica , Mutagênese Insercional , Testes de Precipitina , Análise de Sequência de DNA , Transfecção , beta-GalactosidaseRESUMO
Dictyostelium Crp is a member of the cyclin-dependent kinase (Cdk) family of proteins. It is most related in sequence to mammalian Cdk5, which unlike other members of the family, has functions that are unrelated to the cell cycle. In order to better understand the function of Crp in Dictyostelium, we overexpressed a dominant negative form, Crp-D144N, under the control of the actin 15 promoter. Cells overexpressing Crp-D144N exhibit a reduced growth rate in suspension culture and reduced rates of fluid-phase endocytosis and phagocytosis. There is no reduction in Cdc2 kinase activity in extracts from cells overexpressing Crp-D144N, suggesting that the growth defect is not due to inhibition of Cdc2. In addition to the growth defect, the act15::crp-D144N transformants aggregate at a slower rate than wild-type cells and form large aggregation streams. These eventually break up to form small aggregates and most of these do not produce mature fruiting bodies. The aggregation defect is fully reversed in the presence of wild-type cells but terminal differentiation is only partially rescued. In act15::crp-D144N transformants, the countin component of the counting factor, a secreted protein complex that regulates the breakup of streams, mostly appears outside the cell as degradation products and the reduced level of the intact protein may at least partially account for the initial formation of the large aggregation streams. Our observations indicate that Crp is important for both endocytosis and efflux and that defects in these functions lead to reduced growth and aberrant development.