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The effect of overexpression of activated rasB, rasC and rasS on Dictyostelium discoideum

University of British Columbia

Dictyostelium discoideum contains at least 5 ras genes. It had been previously demonstrated that the expression of an activated rasG gene in D. discoideum resulted in cells which were unable to aggregate and a subset of which had a large, flattened morphology (Khosla et al, 1996. Mol. and Cell. Biol. 16: 4156-62). To investigate the role of the rasB, rasC, and rasS genes, an activating mutation (a glycine to threonine substitution at position 12) was made in each of the genes. The mutated genes were then expressed under the control of the inducible discoidin promoter. Overexpression of the rasC-G12T and rasS-G12T genes produced no evident abnormalities in cell morphology, growth, or development. Thus, rasC and rasS are not functionally equivalent to rasG, although no clues as to their roles in the cell were revealed. Overexpression of the rasB-G12T gene resulted in the appearance of morphologically aberrent amoebae: a large proportion of the cells were large and flat with an increase in cell surface projections known as crowns. These cells also became multinucleate and failed to divide by traction-mediated cytofission when grown on a surface. The response of these cells to nutrient stimulation and to azide were normal, as was their motility. Growth and development of the cells was unaffected by overexpression of rasB-G12T. These data suggest that the defects observed in the rasB-G12T cells are due to altered regulation of cell division processes since other actin and myosin-dependent processes are unimpaired. The growth and developmental defects caused by overexpression of rasG-G12T were not seen in the rasB-Gl2T cells. Comparison of the morphological phenotypes of rasB-G12T and rasG-G12T cells revealed that the rasB-G12T cells were, on average, larger and more highly multinucleate than the rasG-GI2T cells. The predominant actin-based cell suface structure seen on each of the two was also different: rasB-G12T cells frequently possessed crowns, while rasG-G12T cells more commonly displayed filopodia. Thus, it appears that rasB and rasG have at least some distinct roles in the cell, although they may overlap in some cellular functions.

Thesis/Dissertation

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2009-05-22

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http://hdl.handle.net/2429/8060

Microbiology and Immunology

University of British Columbia

UBCV

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