RESUMO
RhoA and RhoC are highly related Rho GTPases, but differentially control cellular behaviour. We combined molecular, cellular, and biochemical experiments to characterise differences between these highly similar GTPases. Our findings demonstrate that enhanced expression of RhoC results in a striking increase in the migration and invasion of pancreatic carcinoma cells, whereas forced expression of RhoA decreases these actions. These isoform-specific functions correlate with differences in the cellular activity of RhoA and RhoC in human cells, with RhoC being more active than RhoA in activity assays and serum-response factor-dependent gene transcription. Subcellular localisation studies revealed that RhoC is predominantly localised in the membrane-containing fraction, whereas RhoA is mainly localised in the cytoplasmic fraction. These differences are not mediated by a different interaction with RhoGDIs. In vitro GTP/GDP binding analyses demonstrate different affinity of RhoC for GTP[S] and faster intrinsic and guanine nucleotide exchange factor (GEF)-stimulated GDP/GTP exchange rates compared to RhoA. Moreover, the catalytic domains of SopE and Dbs are efficacious GEFs for RhoC. mRNA expression of RhoC is markedly enhanced in advanced pancreatic cancer stages, and thus the differences discovered between RhoA and RhoC might provide explanations for their different influences on cell migration and tumour invasion.
Assuntos
Movimento Celular , Invasividade Neoplásica , Neoplasias Pancreáticas/patologia , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Estimulação Encefálica Profunda , Humanos , Neoplasias Pancreáticas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas rap de Ligação ao GTP/química , Proteínas rap de Ligação ao GTP/genética , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/genética , Proteína de Ligação a GTP rhoCRESUMO
Bacterial ghosts are nondenaturated empty cell envelopes of Gram-negative bacteria produced by E-mediated lysis. Such envelopes from the plant-adhering bacterium Pectobacterium cypripedii were tested for their ability to adhere to plant material and to be used as carriers for pesticide delivery. We show, using fluorescence-labeled P. cypripedii ghosts, that depending on the target plants 55 or 10% (rice or soya, respectively) of the applied bacterial ghosts was retained on the leaves after heavy simulated rain (84 mm). Furthermore, the bacterial ghosts could be loaded with the lipophilic triazole fungicide tebuconazole. In subsequent plant experiments in the glass house, the efficacy of the loaded bacterial ghost for resistance to rainfall and the protective and curative effects against the pathogens Erysiphe graminis, Leptosphaeria nodorum, and Pyrenophora teres on barley and wheat and against Sphaerotheca fuliginea on cucumber were tested. The bacterial ghosts were compared primarily with a commercial tebuconazole formulation, a wettable powder, as it has similar physical characteristics. The comparison revealed similar effects and showed consistently higher or comparable efficacy against the pathogens. The standard operational comparison with the most protective, cereal specific emulsion of oil in water displayed that the bacterial ghosts had equal to or lower efficacy than the emulsion. This study confirmed the potential of bacterial ghost platform technology as a new alternative carrier system for pesticides.