PMC

β-Galactosidase staining of aggregates formed by a mixture of 90% cotB::rd28 and 10% TL6 cells (ecmA::lacZ). Cells were developed on filters for 24 hr, and whole mounts were fixed, permeabilized, and stained with X-Gal (blue staining indicates enzymatic activity). The lack of solid staining of the stalk suggests that cotB::rd28 cells participate in stalk formation.

Effect of mixing control (TL6) and cotB::rd28 cells on fruiting body formation. TL6 cells express lacZ gene under the ecmA promoter. Morphology after 24 hr of development of 100% cotB::rd28 cells (A), 90% cotB::rd28 and 10% TL6 cells (B), 80% cotB::rd28 and 20% TL6 cells (C), 50% cotB::rd28 and 50% TL6 cells (D), and 100% TL6 cells (E).

Immunoblot of cellular extracts showing that the abundance of RD28 is differentially regulated by the two promoters (actin15 and cotB). Proteins (50 μg) were analyzed as in Fig. . (A) Crude extract of act15::rd28 cells developed on filter and collected at the times indicated. (B) Crude extracts of cotB::rd28 cells developed on filter and collected at the times indicated. With the actin15 promoter, the abundance of RD28 is greatest at the beginning of the developmental cycle. With the cotB promoter, the picture is reversed.

Whole mount in situ hybridization of rd28, cotB, and ecmA mRNA in cellular aggregates. AX4 and cotB::rd28 cells were allowed to develop for 16 hr (AX4 and cotB::rd28), fixed, permeabilized, and probed with antisense mRNA for rd28 (A and D), cotB (B and E), or ecmA (C and F). rd28 is not expressed in AX4 cells (D) and is only expressed in prespore cells in the transformed line (A); cotB is only expressed in prespore cells (B and E); and ecmA is only expressed in prestalk cells (C and F).

Immunoblot of membrane fractions showing that RD28 is present in a plasma membrane-enriched fraction. Proteins (50 μg) were analyzed as in Fig. . Lanes: 1, H = total cellular extract; 2, P1 = crude membrane pellet (20 min at 5,900 × g); 3, P2 = plasma membrane-enriched fraction from a sucrose gradient; 4, PM = plasma membrane-enriched fraction from a Renografin gradient. As in Fig. , cross-reacting polypeptides at 28 and 56 kDa are visible.

Developmental morphology of cotB::rd28 strain and the effect of 100 mM sorbitol on this process. Cells were allowed to aggregate and form fruiting bodies synchronously on filters (PDF medium). (H) A typical fruiting body of the control line (AX4) after 24 hr of development. (A–F) Aggregates and fruiting bodies of the cotB::rd28 cells after 20 hr (A), 24 hr (B and C), and 40 hr (D–F). (G) The results shown are similar to those shown in D–F, but 100 mM sorbitol has been added to the PDF medium. This addition ameliorates the development of fruiting bodies in the RD28-expressing cells.

Immunoblot of Dictyostelium extracts showing that RD28 is present in Dictyostelium transfected with the pact15::rd28 plasmid. Proteins (50 μg) were fractionated by SDS/PAGE, transferred to nitrocellulose membrane, and incubated with a polyclonal antibody raised against a carboxyl-terminal peptide of RD28. Lanes: 1, A. thaliana microsome fraction (positive control); 2 and 3, crude cellular extract of Dictyostelium transfected with the pact15::rd28 plasmid; 4, crude cellular extract of Dictyostelium parental strain (AX4; negative control). The band at 28 kDa represents monomeric RD28. This hydrophobic protein readily forms dimers under SDS/PAGE conditions (band at 56 kDa).

Responses of act15::rd28 cells to different osmotic conditions. (A) Cells were incubated at 1 × 10⁶ cells per ml with the indicated sorbitol concentration, and intact cells were counted after 1, 2.5, 5, and 10 min. Sorbitol is needed to stabilize the cells. (B) Cells were preincubated (open bars) or not (solid bars) with 50 mM sorbitol for 5 min, centrifuged, and exposed to the indicated sorbitol concentration. Intact cells were counted after 10 min. Incubation with sorbitol protects them against subsequent exposure to a low-osmotic-strength medium. (C) Effect of hyperosmotic shock on cell viability; act15::rd28 and AX4 cells were exposed at a concentration of 1 × 10⁶ cells per ml to a medium (PDF) supplemented or not (control) with 400 mM sorbitol (sorbitol). At the times indicated, the cells were removed, and their viability was tested after 2–3 days of growth on agar in association with K. aerogenes.