Figure 7.
Complementation of a Dictyostelium RhoGDI1 null mutant with T. borchii and mammalian RhoGDIs. GDI1 null cells were transfected with plasmids that allow expression (indicated by an R, for rescue) of GFP fusions of Dictyostelium RhoGDI1(GDI1), TbRhoGDI, bovine RhoGDI1 and human RhoGDI2 (LyGDI). The wild-type Dictyostelium strain AX2 was used as reference. A. Total cell homogenates of 4 × 105 cells were resolved in 12% polyacrylamide gels and blotted onto nitrocellulose. The blot of the upper panel was incubated with a GFP-specific mAb K3-184-2. The blot of the lower panel was incubated with Dictyostelium GDI1-specific mAb K8-322-2. All GFP fusions are expressed at levels comparable to those of the endogenous Dictyostelium GDI1. B. Growth of GDI1- and complementation mutants in shaking suspension. GDI1- mutant cells have a reduced growth rate and reach lower cell densities than the wild type. This defect was restored after expression of bovine RhoGDI1, but not by TbRhoGDI or human LyGDI. Curves are representative of at least three independent determinations, each done in duplicate. C. Distribution of the number of nuclei in GDI1- and complementation mutants. Cells were allowed to grow on coverslips, then fixed with cold methanol and stained with DAPI. 300 cells of each population were scored. Photographs show DAPI staining (left panels) and phase contrast (right panels). Arrows point at examples of giant multinucleate cells. Scale bar, 50 μm. In the GDI1- mutant, cells with four or more nuclei account for about 10% of the population. This defect was restored completely after expression of bovine RhoGDI1 and partially after expression of TbRhoGDI, whereas human LyGDI had no effect.