Protein kinase B gene homologue pkbR1 performs one of its roles at first finger stage of Dictyostelium

Abstract

Dictyostelium discoideum has protein kinases AKT/PKBA and PKBR1 that belong to the AGC family of kinases. The protein kinase B-related kinase (PKBR1) has been studied with emphasis on its role in chemotaxis, but its roles in late development remained obscure. The pkbR1 null mutant stays in the first finger stage for about 16 h or longer. Only a few aggregates continue to the migrating slug stage; however, the slugs immediately go back probably to the previous first finger stage and stay there for approximately 37 h. Finally, the mutant fingers diversify into various multicellular bodies. The expression of the pkbR1 finger protein probably is required for development to the slug stage and to express ecmB, which is first observed in migrating slugs. The mutant also showed no ST-lacZ expression, which is of the earliest step in differentiation to one of the stalk cell subtypes. The pkbR1 null mutant forms a small number of aberrant fruiting bodies, but in the presence of 10% of wild-type amoebae the mutant preferentially forms viable spores, driving the wild type to form nonviable stalk cells. These results suggest that the mutant has defects in a system that changes the physiological dynamics in the prestalk cell region of a finger. We suggest that the arrest of its development is due to the loss of the second wave of expression of a protein kinase A catalytic subunit gene (pkaC) only in the prestalk region of the pkbR1 null mutant.

Fig. 1.

Ax2 (A) and pkbR1 null (B) cells developed in a phototactic chamber in a highly synchronized fashion. The Ax2 and pkbR1 cells were grown in modified HL5 medium. Five drops of 50 μl of suspended cells (∼3.5 × 10⁶ cells/drop) were placed on a nonnutrient agar plate, and the plate was placed in a phototactic chamber. The pkbR1 null cells showed delayed development of around 36 h. At 26 h, some slugs migrated coordinately toward a light source, but the slugs immediately came back to the fingers within 20 min. At 35 to ∼37 h, the mutant cells begin to exhibit diverse morphology, including various multicellular bodies and a few fruiting bodies.

Fig. 2.

pkbR1 null mutant expresses ecmA-lacZ normally but not ecmB-lacZ. Ax2 and pkbR1 null cells were grown in HL5 medium. The pkbR1 null strain expresses ecmA-lacZ and ecmO-lacZ (data not shown) constructs with normal timing but not ecmB at the core of the prestalk A region of fingers. The pkbR1 null mutant forms rarely migrating slugs on 13 mM phosphate-buffered agar plates. (A to F) ecmA-lacZ expression; (G to L) ecmB-lacZ expression. (A to C and G to I) Ax2 strain; (D to F and J to L) pkbR1 null strain; (A, D, G, and J) mound; (B, E, and K) tipped mound; (H) Mexican hat; (C, F, and L) first finger; (I) migrating slug on H₂O agar; (L) first finger showing the slight expression of ecmB at the basal disc but not at the core. Black bar, 100 μm.

Fig. 3.

SP60-lacZ and ST-lacZ expression patterns in Ax2 and pkbR1 null cells. (A to F) SP60-lacZ expression; (G to L) ST-lacZ expression. (A to C and G to I) Ax2; (D to F and J to L) pkbR1 null strain; (A, D) mound; (B, E) tipped mound; (C, F) fingers (note the expanded prespore region in the mutant pkbR1 finger [F] compared to that of the parental strain [C]); (G, H) slug at 17 h; (I) fruiting body at 17 h; (J, K) finger at 20 h (there is very weak dispersed expression, probably in ALC cells, but no ST-expressing cells at the core of the anterior tip of pkbR1 null fingers [compare G and H to J and K]); (L) fruiting body with a short stalk, 40 h (some of the multicellular bodies become fruiting bodies). Black bar, 100 μm.

Fig. 4.

In chimera, pkbR1 null cells preferentially sort to the spore region. The results show that pkbR1 mutant cells localize preferably to the posterior spore region rather than to the stalk region during culmination. pkbR1 null cells constitutively expressing lacZ from an act15 promoter were mixed with the parental Ax2 cells in ratios of 1:9 (A to D) and 9:1 (E to H). (A, E) Mound; (B, F) tipped aggregate; (C, G) first finger; (D, H) fruiting body. Black bars, 100 μm.

Fig. 5.

pkbR1 null cells are able to differentiate into stalk cells by DIF-1. Ax2 and pkbR1 null cells were incubated in tissue culture dishes (24-well plate) at a density of 10⁵ cells/cm² in stalk medium supplemented with 5 mM cAMP. After 20 h, the medium was removed and cells attached on wells were washed with stalk buffer twice and incubated in fresh stalk medium containing 100 nM DIF-1. After 22 h, stalk cell differentiation was scored by phase-contrast microscopy. Results are the averages from three assays.

Fig. 6.

pkaC transcription analysis by in situ hybridization. For in situ hybridization, pkbR1 null and Ax2 cells were grown in modified HL5 medium, harvested, and developed on agar plates for 12 h (G, H), 21 h (A to C), or 34 h (D to F), and samples were prepared as described in Materials and Methods. Hybridization was done by a modification of the method described previously (9). S, sense RNA probe; AS, antisense mRNA probe. (A) pkbR1 null, S, 21 h; (B, C) pkbR1 null, AS, 21 h; (D) pkbR1 null, S, 34 h; (E, F) pkbR1 null, AS, 34 h; (G) Ax2, S, 12 h; (H) Ax2, AS, 12 h.