Figure 1.

Architecture and expression of RasGEF Q and localization of RasGEF Q domains. (A) Schematic diagram of RasGEF Q depicting its domain organization and constructs used in the study. (B) Accumulation of RasGEF Q protein during development. Total cell lysates prepared at the indicated time points were immunoblotted and probed using mAb K-70-187-1 raised against RasGEF Q. *, smaller form of the protein. Expression of the developmentally regulated cell adhesion protein csA is shown for control at the bottom. (C) Localization of GFP-RasGEF Q¹⁷³ (green) in wild-type cells. In both vegetative (0 h; bottom) and aggregation-competent (6 h; top) cells, GFP-RasGEF Q¹⁷³ was present throughout the cytosol and showed specific enrichment with cortical actin (arrowheads). Actin (red) was recognized by mAb Act 1–7 followed by cy3-labeled anti–mouse secondary antibody. Cells were fixed with cold methanol. (D) Localization of GFP-tagged RasGEF Q domains. The top row shows that gfp-gef localizes to the cell cortex (carat) and nucleus (arrowhead). The second row shows that GFP-Δ-GEF-GEFQ localizes throughout the cytosol but is enriched in the cell cortex. The third row shows that the DEP domain is present throughout the cytoplasm. The fourth row shows that GFP-Δ-DEP-GEFQ localizes throughout the cytosol with slight enrichment in the cell cortex. F-actin was stained by TRITC-labeled phalloidin and DNA was stained with DAPI. Cells were fixed with picric acid/formaldehyde. Bars, 10 μm.