Wilkins, Andrew; (2000) The function of Ras proteins in Dictyostelium discoideum. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The Ras family of GTP-binding proteins has been implicated in the control of cellular growth, differentiation and motility. They are molecular switches which are active when bound to GTP and inactive when GDP-bound. Dictyostelium cells express five different Ras family members. Ras activation is mediated by guanine nucleotide exchange factors (RasGEFs) which facilitate exchange the of GDP for GTP by Ras proteins. The identification and molecular genetic characterisation of novel Dictyostelium RasGEFs is the main subject of this thesis. The discovery of a physiological function for the Dictyostelium RasD protein is also described. I have identified five partial cDNAs from the Tsukuba Dictyostelium cDNA project which have strong sequence homology to known RasGEFs. To investigate the function of these proteins, Dictyostelium cells with single gef gene disruptions were generated by homologous recombination. Four of the five mutants appeared to grow and develop normally but one mutant, gefB-, showed growth and developmental abnormalities. gefB- cells are unable to proliferate in axenic culture and are impaired in phagocytosis and fluid-phase endocytosis. Conversely, gefB- cells show an enhanced rate of migration, moving twice as fast as wild-type cells. The mutant cells have abnormal morphology; they are highly polarised, have many elongated microspikes and have an absence of pinocytic crowns on the cell surface. In addition, gefB- cells show a cell autonomous impairment in multicellular development. Although the phenotype of vegetative gefB- cells is similar to that of rasS- cells, it was not possible to show a direct interaction between RasS and GefB. However, GefB does exhibit the catalytic properties of a RasGEF in vivo, since it is able to complement the temperature sensitive cdc25-5 S.cerevisiae mutant. The Dictyostelium RasD protein is a small GTP-binding protein closely related to the mammalian Ras proteins Ha-, Ki- and N-Ras, and is maximally expressed during the multicellular stage of Dictyostelium development. Previous work had predicted that RasD was essential for correct differentiation and pattern formation in Dictyostelium aggregates. To further investigate the function of RasD, Dictyostelium cells containing a disrupted rasD- gene were generated by homologous recombination. Surprisingly, rasD- cells proliferate, aggregate and develop indistinguishably from wild-type cells. However rasD slugs exhibit a clear defect in phototaxis and thermotaxis exhibiting an approximately thirty-fold decrease in the efficiency of orientation towards a light or heat source relative to wild-type slugs. RasD is the first signalling protein shown to be necessary for phototaxis in Dictyostelium slugs.

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