Expression of Gα subunit transcripts in wild-type and Gα subunit mutants. Total RNA was isolated from cells in late log phase of growth and subjected to RNA blot analysis using Gα2- or Gα5-specific probes as describe in Materials and methods. Each lane was loaded with 4 μg of total RNA. All lanes from each panel were run on same gel and then blotted and hybridized together but the scanned image of the autoradiograph was spliced to bring relevant lanes together. (A) Endogenous Gα2 (e) and vector-expressed Gα2/4 or Gα2 transcripts (v) in wild-type, gα2⁻, and gα4⁻ cells with no vector (−), the Gα2/4 vector (2/4), or the Gα2 vector (2). (B) Endogenous Gα5 (e) and vector-expressed Gα5/4 or Gα5 transcripts (v) in wild-type and gα4⁻ cells with the Gα5/4 vector (5/4), Gα5 vector (5), or no vector (−). Variability in the level of vector-expressed transcripts is likely due to gel loading inconsistencies but in each case the level of vector-expressed transcripts is higher than the level of endogenous gene transcripts. Endogenous Gα2 and Gα5 transcripts were more visible upon longer exposures.

Development of gα2⁻ cells with or without Gα subunit expression vectors. Cells were grown in axenic medium and washed in phosphate buffer and then cell suspensions (2 × 10⁸ cells/ml) were plated for development as described in the Materials and methods section. Panel (A) gα2⁻ cells with no Gα subunit expression vector. Panel (B) gα2⁻ cells with Gα2/4 subunit expression vector. Panel (C) aggregates of gα2⁻ cells with Gα2 subunit expression vector. Panel (D) aggregates of wild-type cells with no Gα subunit expression vector. Developing cells were photographed 12 hrs after initial plating using a dissecting microscope (20X magnification).

Chemotaxis of gα4⁻ cells with or without Gα subunit expression vectors. Cells were grown in axenic medium, washed in phosphate buffer, and plated for chemotaxis as described in the Materials and methods section. Droplets of cell suspensions (2 × 10⁷ cells/ml) were spotted onto nonnutrient plates followed by the spotting of one microliter droplets of 1 mM folate solution approximately 2–3 mm away from the cell droplet. (A) Micrographs of chemotaxis assays. In all panels the source of folate is above (upper side) cell droplet. Deformations in agar were created by a needle and used as a reference points to align images captured at different times during the assay. Cell distribution was photographed 3 hrs later using a dissecting microscope (20X magnification) and indirect light. Original cell droplet perimeter is indicated by drawn circle. Developing cell aggregates were photographed 23 hrs after initial plating using a dissecting microscope (20X magnification). Data is representative of one of several assays with similar results. (B) Chemotaxis index of gα4⁻ cells with or without Gα subunit expression vectors. Cell suspensions (1 × 10⁷ cells/ml) were plated for chemotaxis assays and cell distribution was photographed after 3 hrs. Chemotaxis index was determined as the number cells outside the original drop perimeter on the side facing the source of folate divided by the total number of cells outside the original droplet perimeter. Each index was determined as the mean of six separate droplets. Data is representative of one of three different experiments with similar results and the vertical error bars represent the standard deviation. (C) Migration maps of gα4⁻ cells with or without Gα subunit expression vectors. Cell suspension (5 × 10⁶ cells/ml) were plated for chemotaxis assays as described above and then 2.5 hours after the initial plating time-lapse photography was used to follow the movement of cells over a 30 minute period. Images corresponding to 5 min intervals were used to trace the movement of individual cells. Only cells with minimal or no cell-cell contact were chosen for creating maps. Multiple paths representing different cells of the each strain are shown in each panel and the initial point of each path is designated with an open circle. All initial points were positioned at a central location in each panel with the orientation of the folate source at the top of each panel. Data is representative of one of two experiments with similar results. In all panels and graph, gα4⁻ cells without Gα subunit expression vector (none), gα4⁻ cells with the Gα2/4 subunit (Gα2/4), Gα2 subunit (Gα2), Gα5/4 subunit (Gα5/4), or Gα5 subunit (Gα5) expression vectors, and wild-type cells with no Gα subunit expression vector

Growth and development phenotypes on bacterial lawns. (A) Plaque edge phenotype of wild-type and gα4⁻ cells with chimeric Gα subunit vectors on bacterial lawns. Wild-type cells without Gα subunit (none) expression vector or wild-type and gα4⁻ cells with the Gα2/4 subunit (Gα2/4) or Gα5/4 subunit (Gα5/4) expression vector were placed on a bacterial lawns and allowed to form plaques. Plaque edges were photographed after 4 days using the same magnification (20X) on a dissecting microscope. (B) Wild-type cells with the Gα5/4 subunit expression vector developing within a plaque on a bacterial lawn. Developing aggregates were photographed using a dissecting microscope (40X). Arrows indicate mature fruiting body formation.

Comparison of Dictyostelium Gα subunits. (A) Alignment of Dictyostelium Gα4, Gα5 and Gα2 subunit amino acid sequences using ClustalW program and formatted using BOXSHADE program to highlight identical (black background) and similar residues (gray background). The number (2) above the aligned sequences indicates residues identical between the Gα4 and Gα2 but not Gα5 subunit. The number (5) above the aligned sequences indicates identity between the Gα4 and Gα5 but not Gα2 subunit. (B) Unrooted phylogenic tree of Dictyostelium Gα subunits generated at http://clustalw.genome.ad.jp/ by the neighbor-joining method from a ClustalW alignment. Sequence information was obtained using dictyBase at http://www.dictybase.org/. (C) Alignment of wild-type and chimeric Gα subunit carboxyl terminal amino acid sequences formatted using the BOXSHADE program as described above.

Development of gα4⁻ cells with or without Gα subunit expression vectors. Cells were grown in axenic medium and washed in phosphate buffer and then cell suspensions (2 × 10⁸ cells/ml) were plated for development as described in the Materials and methods section. Panel (A) aggregates of gα4⁻ cells with no Gα subunit expression vector. Insert displays a side view of a typical aggregate with the round mound and extended tip morphology. Panel (B) aggregates of gα4⁻ cells with Gα4 subunit expression vector in the early stages of culmination. Insert displays a side view of a typical aggregate displaying wild-type slug morphology. Panel (C) gα4⁻ cells with Gα2 subunit expression vector. Panel (D) gα4⁻cells with Gα2/4 subunit expression vector. Panel E: gα4⁻ cells with Gα5 subunit expression vector. Panel F: gα4⁻ cells with Gα5/4 subunit expression vector. Developing cell aggregates were photographed 23 hrs after initial plating using a dissecting microscope (20X magnification).

Development of gα5⁻ cells with or without Gα subunit expression vectors. Cells were grown in axenic medium and washed in phosphate buffer and then cell suspensions (2 × 10⁸ cells/ml) were plated for development as described in the Materials and methods section. Panel (A) aggregates of gα5⁻ cells with no Gα subunit expression vector. Panel (B) aggregates of gα5⁻ cells with Gα5/4 subunit expression vector. Panel (C) aggregates of gα5⁻ cells with Gα5 subunit expression vector. Panel (D) aggregates of wild-type cells with no Gα subunit expression vector. Developing cell aggregates were photographed 12 hrs after initial plating using a dissecting microscope (20X magnification).

Chemotaxis distance of gα4⁻ or wild-type cells with or without Gα subunit expression vectors. Chemotaxis assays were conducted as described in Figure 6 and the maximum distance from the original cell droplet perimeter to the leading edge of migrating cells in the direction of the folate source was recorded. (A) Chemotaxis distance traveled by gα4⁻ cells without a Gα subunit expression vector (none) and cells with the Gα2/4 subunit (Gα2/4), Gα2 subunit (Gα2), Gα5/4 subunit (Gα5/4), Gα5 subunit (Gα5), or Gα4 subunit (Gα4) expression vectors after 3.5 hrs. (B) Chemotaxis distance traveled by wild-type cells without a Gα subunit expression vector (none) and cells with the Gα2/4 subunit (Gα2/4), Gα2 subunit (Gα2), Gα5/4 subunit (Gα5/4), or Gα5 subunit (Gα5) expression vectors after 3 hrs. Each value is the mean of six independent cell droplets. Error represents the standard deviation. Data is representative of one of at least two assays with similar results.

Folate-stimulated cGMP accumulation in gα4⁻ cells with or without Gα expression vectors. Cells were grown, harvested, and stimulated with folate as described in the Materials and methods section. (A) cGMP accumulation in gα4⁻ cells with the Gα4 subunit (open squares) or without a Gα subunit (open circles) expression vector. (B) cGMP accumulation in gα4⁻ cells with the Gα2/4 subunit (open squares) or Gα2 subunit (open circles) expression vector. (C) cGMP accumulation in gα4⁻ cells with the Gα5/4 subunit (open squares) or Gα5 subunit (open circles) expression vector. Concentration of cGMP was normalized with respect to cell extract protein concentration and each cell extract was assayed in duplicate. The data represent the average of three individual experiments and the vertical error bars represent deviation from the mean value.