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First published online September 3, 2008
doi: 10.1242/10.1242/jcs.030056

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The GPI-anchored superoxide dismutase SodC is essential for regulating basal Ras activity and for chemotaxis of Dictyostelium discoideum

Department of Biological Sciences, Florida International University, Miami, FL 33199, USA

View larger version (66K): [in this window] [in a new window]   Fig. 1. Generation of REMI mutants exhibiting higher basal levels of GFP-PHcrac at the plasma membrane. (A) remi56 cells displayed more plasma membrane localization of GFP-PHcrac, which suggests higher PtdIns(3,4,5)P3 levels in the membrane of remi56. (B) Wild type and remi56 lines were tested to see whether they are able to form developmental territorial streams. remi56 lines failed to aggregate in submerged culture at the cell density of 2.5x10 cells/cm, whereas wild type cells aggregated normally.

View larger version (65K): [in this window] [in a new window]   Fig. 2. Identification of the insertional mutation in the REMI mutants and generation of sodC– cells. (A) remi56 displayed an insertion at a locus encoding a superoxide dismutase (SOD) domain-containing protein, SodC. SodC encodes an N-terminal signal peptide with the C-terminal GPI-anchoring sequence (omega domain), and a partial and a full Cu/Zn-superoxide dismutase (SOD) domain. (B) sodC– cells were generated by homologous recombination from wild-type cells, and confirmed by genomic PCR. (C) sodC– cells showed increased basal membrane localization of GFP-PHcrac compared with wild-type cells. (D) Western blot analysis of the cytosolic and membrane fractions of the wild-type and sodC– cells showed more GFP-PHcrac localization at the membrane in sodC than in wild-type cells. Ras proteins are shown as a loading control for membrane fractions.

View larger version (18K): [in this window] [in a new window]   Fig. 3. SodC has SOD activity. (A) The SodC SOD domain was expressed in Dictyostelium as a GFP fusion protein under the Actin 15 promoter. GFP and GFP-SOD proteins were purified by immunoprecipitation with anti-GFP antibody, and normalized by western blot analysis using anti-GFP antibody. (B) Relative superoxide levels were compared after incubation with equal amounts of purified GFP or GFP-SOD protein. The relative superoxide level from GFP was set as 1.0. GFP-SOD samples exhibited an average level of 0.3 (standard deviation of 0.06) from three independent experiments.

View larger version (30K): [in this window] [in a new window]   Fig. 4. SodC is a GPI-anchored membrane protein. (A) A Myc epitope tag was inserted in-frame after the SodC signal peptide. Myc-SodC was expressed in the wild-type background, and detected by western blotting (bottom left). Membrane and cytosolic fractions were prepared, and Myc-SodC was detected only from the membrane fraction (bottom right). (B) Membrane localization of Myc-SodC was also confirmed by indirect immunofluorescence. (C) Log phase wild-type and sodC– cells were plated at a density of 1x10 cells/cm, and treated with or without 1 unit of GPI-specific PI-PLC for 5 minutes at 25°C. Extracellular SOD activities were compared. (D) The relative levels of extracellular superoxide in wild-type and sodC– cells were measured using XTT reduction, as described in the Materials and Methods. Virtually identical levels of the radical were detected from three independent experiments. (E) The relative intracellular superoxide levels were determined using NBT. Cell-trapped NBT levels were 18±5% (s.d.) higher in sodC than in wild-type cells (data are from three independent experiments).

View larger version (62K): [in this window] [in a new window]   Fig. 5. sodC– cells were defective in aggregation, but not in development. (A) sodC– cells failed to aggregate in submerged culture at low cell density (2.5x10 cells/cm), where wild-type cells aggregate normally. (B) sodC– cells displayed normal development when plated at high cell densities (1.25x10 cells/cm) at which chemotaxis can be bypassed.

View larger version (13K): [in this window] [in a new window]   Fig. 6. sodC– cells were defective in chemotaxis. (A-C) Cells were challenged with a point source of either 0.1 µM or 10 µM cAMP. Tracing images of chemotaxing cells were arranged to demonstrate relative directional movement, cell shape and distances traveled towards the cAMP point source (circle). Superimposed tracing images were grouped as early (0 to 20 minutes) and late (21-40 minutes) duration as marked. Each tracing image is a 1-minute interval. (D) The roundness of chemotaxing cells is summarized as defined in the Materials and Methods.

View larger version (21K): [in this window] [in a new window]   Fig. 7. Defects in sodC– cells were partially rescued by SodC but not by a SodC(H245R,H247Q) double-point mutation. (A) RT-PCR experiment with a primer set specific for SodC was used to detect the level of SodC transcript. A specific RT-PCR product was obtained from wild type, but not from sodC– cells as expected. Ig7 transcripts were used as a control. Similar levels of the transcripts were observed in sodC– cells expressing wild-type SodC and the SodC(H245R,H247Q) mutant under Actin-15 promoter. (B) sodC– cells expressing wild-type SodC and SodC(H245R,H247Q) mutant were challenged with a micropipette filled with 0.1 µM cAMP for 20 minutes. Twenty stacks of tracing images are shown with a 100 µm scale bar. Summary of the roundness (C) is shown.

View larger version (25K): [in this window] [in a new window]   Fig. 8. LY294002 treatment attenuated chemotaxis defects of sodC– cells. (A) Cells expressing the PtdIns(3,4,5)P3 marker GFP-PHcrac were pulsed for 4 hours with 50 nM cAMP, and either left in DB buffer or treated with 15 µM or 50 µM LY294002 (LY) for 20 minutes. GFP-PHcrac aberrantly localized at the plasma membrane of sodC– cells but not of wild type. Membrane localization of GFP-PHcrac in sodC– cells largely disappeared after LY treatment. GFP signals at the membrane of sodC– cells were reminiscent of fine filopodial extensions, which also disappeared after LY treatment. (B) Wild-type and sodC– cells were pulsed for 4 hours with 50 nM cAMP, and treated with and without 15 µM LY294002 (LY) for 20 minutes. Cells were then challenged with micropipettes filled with 10 µM cAMP for 20 minutes. Twenty stacks of cell tracing images 1 minute apart, are shown with a 100 µm scale bar. (C) Roundness values of the analyzed cells are shown.

View larger version (55K): [in this window] [in a new window]   Fig. 9. Aberrant localization of PI3K in sodC– cells. (A) The membrane localization domain of PI3K1 (N-PI3K1) fused with GFP was expressed in wild-type and sodC– cells. Aggregation-competent polarized wild-type cells clearly demonstrated localized PI3K membrane translocation at the leading edge, whereas sodC– cells showed no PI3K polarization around the membrane. By contrast, GFP-PTEN localization was indistinguishable between wild-type and sodC– cells. (B) 0.01% Triton X100 fraction showed that more N-PI3K1-GFP proteins were aberrantly enriched in the membrane fraction of sodC– cells than of wild-type cells. (C) Cells expressing PI3K1-LD-GFP proteins were pulsed with 50 nM cAMP for 4 hours, and stimulated with 10 µM cAMP. Membrane translocation of PI3K1-LD protein was recorded at 10-second intervals. Clear membrane localization of PI3K1-LD was observed in wild-type cells, but no such changes were seen in sodC– cells. (D) Cells were pulsed for 4 hours, fixed and stained with TRITC-phalloidin, as described in the Materials and Methods. Two representative images are shown for each wild-type and sodC– cell. Wild-type cells displayed more polarized cell bodies with a leading edge enriched with F-Actin. By contrast, sodC– cells were much more round than the wild type and showed numerous filopodia-like structures instead of a dominant pseudopodium. (E) Pulsed cells were stimulated with 10 µM cAMP as indicated, and lysed with a F-Actin buffer containing 0.2 % of Triton X-100 and TRITC-phalloidin, and the F-Actin levels were measured as described in the Materials and Methods. sodC– cells displayed a higher basal level of F-Actin compared with wild type, but no wild-type-like response was observed after cAMP stimulation.

View larger version (52K): [in this window] [in a new window]   Fig. 10. Ras proteins were not properly regulated in sodC– cells. (A) Active Ras proteins are visualized by GFP-RBD signals on the plasma membrane. Wild-type cells often displayed well-organized RBD signal on one side of a cell, whereas sodC exhibited broad GFP-RBD signal around cellular peripheries. Images were captured after 4 hours of cAMP pulsing. (B) A significantly higher basal level of active Ras was detected in extracts from sodC– cells compared with wild-type cells after 4 hours of pulsing. (C) Cells were pulsed with 50nM cAMP for 4 hours, and then stimulated with 10 µM cAMP as indicated. Total Ras protein levels were first normalized by western blot using anti-Pan-Ras antibody. Active Ras proteins were determined by GST-RBD assay (Sasaki et al., 2004). (D) A higher basal level of active Ras in sodC– cells was significantly decreased upon depletion of superoxide by incubation with radical scavenger XTT (4 mM) for 10 minutes at room temperature. As in C, wild-type cells showed a lower level of active Ras. (E) sodC– cells displayed higher basal level of active GFP-RasG than did wild-type cells. (F) GFP-RasG was modestly activated by conditioned medium (CM) and was susceptible to XTT in wild-type cells. In sodC– cells, GFP-RasG showed higher basal activity and was susceptible to XTT, but no further stimulation of GFP-RasG was observed with CM.

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