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doi: 10.1242/10.1242/jcs.00039

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Evidence for a role for the Dictyostelium Rap1 in cell viability and the response to osmotic stress

Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Blvd., Vancouver, BC V6T 1Z3, Canada

View larger version (11K): [in a new window]   Fig. 1. Growth and viability of the pVEII-AS5' transformants. (A) The pVEII-AS5'-1 transformant () and the Ax-2 parent ([UNK]), which were grown in shake suspension HL-5 growth media in the presence of 1 mM folate, were washed and transferred to fresh HL5 media in the absence of folate at a cell density of 1x105 cells/ml. After three days of growth in shake suspension, when the population was in late exponential phase at a density of 5x106 cells/ml, it was diluted into fresh HL-5 media to a density of 1x105 cells/ml and growth continued. Growth was monitored by hemocytometer counts ([UNK],). (B) The viability of the pVEII-AS5' transformant () and Ax-2 ([UNK]) was determined by plating on rich nutrient agar plates in association with Klebsiella oxytoca. Cell viability is expressed relative to the viability at the start of the experiment and is the mean±s.e. for two experiments.

View larger version (32K): [in a new window]   Fig. 2. Rap1 levels during growth of the pVEII-AS5'-1 transformant in the absence of folate. Cells were harvested at the day indicated following the removal of folate, and the total cellular protein was fractionated by SDS-PAGE. Rap1 levels were determined by western blot analysis using a Dictyostelium Rap1-specific antibody. The RasG levels were determined with a RasG-specific antibody.

View larger version (14K): [in a new window]   Fig. 3. Response to osmotic shock. (A) Ax-2 ([UNK],) and pVEII-AS5'-1 (,) cells were shaken in KK2 buffer containing 0.4 M sorbitol ([UNK],) or 0.4 M sorbitol and 100 µM 8Br-cAMP (,) for the indicated time periods and then diluted and plated in association with K.oxytoca to determine cell viability. The number of plaques that were formed on the bacterial lawns were counted are expressed as a percentage of the zero time value and are the means±s.e. for two experiments. (B) Ax-2 ([UNK]), pVEII-AS5'-1 transformant () and pVEII-Rap1 (G12V) transformant () cells were shaken in KK2 in the presence of 0.4 M sorbitol for the indicated times. Aliquots were then treated with 3.5% perchloric acid and the amount of cGMP determined. The results are the mean±s.d. of five experiments for Ax-2, three experiments for pVEII-AS5' and the mean±s.e. for two experiments for pVEII-Rap1(G12V).

View larger version (37K): [in a new window]   Fig. 4. Tyrosine phosphorylation of actin. Ax-2 and pVEII-AS5'-1 cells were shaken in KK2 buffer in the presence or absence of 0.4 M sorbital for the indicated times and lysed in 1% SDS buffer containing 100 mM Na2VO4. The cell lysates were separated by SDS-PAGE, blotted onto nitrocellulose and probed with a phosphotyrosine-specific monoclonal antibody.

View larger version (48K): [in a new window]   Fig. 5. GST-RalGDS(RBD) binds to the activated form of Rap1. (A) Ax-2 and pVEII-Rap1 (G12V) transformant cells were lysed, and the indicated amounts of cell lysate were incubated with 20 µg GST-RalGDS(RBD) that had been precoupled to glutathione-Sepharose beads for 1 hour at 4°C. The beads were pelleted by centrifugation, and the bound Rap1 was released by adding SDS sample buffer. The samples were fractionated by SDS-PAGE, and Rap1 was detected by western blotting using the Dictyostelium Rap1-specific antibody. (B) Bacterially expressed Rap1 was pre-equilibrated with 1 mM GDP (lanes 1, 2, 5 and 6) or 1 mM GTP (lanes 3, 4, 7 and 8) and incubated with GST-RalGDS(RBD) bound to glutathione-Sepharose beads (lanes 1-4) or GST bound to glutathione-Sepharose beads (lanes 5-8). The Sepharose beads were pelleted by centrifugation, and both the bound Rap1 in the pellets (lanes 2, 4, 6 and 8) and the unbound Rap1 in the supernatants (lanes 1, 3, 5 and 7) were fractionated by SDS-PAGE and detected by western blotting.

View larger version (46K): [in a new window]   Fig. 6. Rap1 activation in response to hyperosmolarity. Ax-2 cells were withdrawn at the indicated times (lanes 0, 5, 10, 20, 30 and 60 minutes) after the addition of 0.4 M sorbitol and lysed. Cell lysate (100 µg) was incubated with GST-RalGDS(RBD)-glutathione-Sepharose beads, and the amounts of bound Rap1 were determined by western blotting. A sample was also taken immediately following the wash procedure, that is, immediately before the 1 hour adaptation in KK2 prior to sorbitol addition (lane OK).

View larger version (56K): [in a new window]   Fig. 7. Effect of EDTA on Rap1 activation. Ax-2 cells were incubated in the presence or absence of 2 mM EDTA immediately prior to the addition of sorbitol and then treated as described in the legend for Fig. 6.

View larger version (32K): [in a new window]   Fig. 8. Rap1 activation in response to different stress conditions. After a 1 hour adaptation at 22°C in KK2 buffer, vegetative Ax-2 cells were subjected to cold shock, heat shock or hypo-osmotic shock, and samples were taken at the time points indicated. Cell lysates were incubated with GST-RalGDS(RBD) as described in the Methods and Materials, and levels of Rap1 bound to GST-RalGDS(RBD) were determined by western blotting.