Ras proteins have multiple functions in vegetative cells of Dictyostelium

Abstract

During the aggregation of Dictyostelium cells, signaling through RasG is more important in regulating cyclic AMP (cAMP) chemotaxis, whereas signaling through RasC is more important in regulating the cAMP relay. However, RasC is capable of substituting for RasG for chemotaxis, since rasG⁻ cells are only partially deficient in chemotaxis, whereas rasC⁻/rasG⁻ cells are totally incapable of chemotaxis. In this study we have examined the possible functional overlap between RasG and RasC in vegetative cells by comparing the vegetative cell properties of rasG⁻, rasC⁻, and rasC⁻/rasG⁻ cells. In addition, since RasD, a protein not normally found in vegetative cells, is expressed in vegetative rasG⁻ and rasC⁻/rasG⁻ cells and appears to partially compensate for the absence of RasG, we have also examined the possible functional overlap between RasG and RasD by comparing the properties of rasG⁻ and rasC⁻/rasG⁻ cells with those of the mutant cells expressing higher levels of RasD. The results of these two lines of investigation show that RasD is capable of totally substituting for RasG for cytokinesis and growth in suspension, whereas RasC is without effect. In contrast, for chemotaxis to folate, RasC is capable of partially substituting for RasG, but RasD is totally without effect. Finally, neither RasC nor RasD is able to substitute for the role that RasG plays in regulating actin distribution and random motility. These specificity studies therefore delineate three distinct and none-overlapping functions for RasG in vegetative cells.

Fig. 1.

Cell growth in suspension culture. JH10 (⧫), rasC⁻ (▴), rasG^- (▪), rasC^-/rasG^- (•), rasG^-/[rasG]:rasD (□), and rasC^-/rasG^-/[rasG]:rasD (▵) strains were transferred from petri plates into axenic medium at time zero and counted at intervals thereafter. The plotted values are the means of duplicate cell counts. The data plotted are for a single experiment, but similar data were obtained for three independent experiments.

Fig. 2.

Western blot analysis of RasD protein level. Cell lysates from the JH10, rasG^-, rasG^-/[rasG]:rasD, rasC^-/rasG^-, rasC^-/rasG^-/[rasG]:rasD, rasC^-, and rasD^- strains were probed with RasD specific antibody. For the rasG^-/[rasG]:rasD and the rasC^-/rasG^-/[rasG]:rasD cells, lanes “a,” “b,” and “c” refer to independently clonal isolates.

Fig. 3.

Nuclear staining. Cells were grown in shaken suspension for 5 days and allowed to adhere to glass coverslips for 30 min, washed, and then fixed with formaldehyde. Fixed cells were stained with DAPI as described in Materials and Methods. Epifluorescence images of random fields of view were captured by using an Olympus IX-70 inverted microscope. The cells shown are representative of all of the cells in the population.

Fig. 4.

Chemotaxis in a folate spatial gradient. The indicated cells were grown to a density of ∼4 × 10⁵ cells/cm² in Nunc dishes. At t = 0, a micropipette filled with 25 mM folate was positioned in the field of view, and cell movements were monitored by time-lapse microscopy. The results of single experiments are shown, but the results for each strain were highly reproducible.

Fig. 5.

RasC and RasD activation in response to cAMP. Extracts from rasD^-, rasG^-/[rasG]:rasD, and rasC^-/rasG^-/[rasG]:rasD cells that had been pulsed with cAMP and then subjected to a single cAMP stimulus for the indicated times (in seconds) were bound to GST-Byr2-RBD as described in Materials and Methods. The bound material was analyzed by Western blotting, using antibodies specific for RasC (A) or RasD (B). The “Total” lanes represent the level of Ras protein in 8.3 μg of lysate protein used for the pull-down assay.