Absence of catalytic domain in a putative protein kinase C (PkcA) suppresses tip dominance in Dictyostelium discoideum

Abstract

A number of organisms possess several isoforms of protein kinase C but little is known about the significance of any specific isoform during embryogenesis and development. To address this we characterized a PKC ortholog (PkcA; DDB_G0288147) in Dictyostelium discoideum. pkcA expression switches from prestalk in mound to prespore in slug, indicating a dynamic expression pattern. Mutants lacking the catalytic domain of PkcA (pkcA(-)) did not exhibit tip dominance. A striking phenotype of pkcA- was the formation of an aggregate with a central hollow, and aggregates later fragmented to form small mounds, each becoming a fruiting body. Optical density wave patterns of cAMP in the late aggregates showed several cAMP wave generation centers. We attribute these defects in pkcA(-) to impaired cAMP signaling, altered cell motility and decreased expression of the cell adhesion molecules - CadA and CsaA. pkcA(-) slugs showed ectopic expression of ecmA in the prespore region. Further, the use of a PKC-specific inhibitor, GF109203X that inhibits the activity of catalytic domain phenocopied pkcA(-).

Keywords: Dictyostelium; Differentiation; Morphogenesis; PKC; Tip dominance.

Fig. 1

Conserved C1 and kinase domains of PkcA. Multiple sequence alignment showing conserved residues of PkcA in D. discoideum aligned with PKC isoforms of different organisms. Conserved regions are highlighted in yellow and conserved residues in red. C1 domain with aligned cysteine and histidine residues. Kinase domain with aligned ATP binding pocket, invariant lysine, gatekeeper residue, Mg²⁺ binding site and the activation loop.

Fig. 2

Dynamic expression pattern of pkcA. (A) pkcA-LacZ expression at various stages of development. Scale bar—0.2 mm. Semi-quantitative PCR of pkcA using RNA extracted from (B) prespore and prestalk enriched cell type, (C) pkcA expression throughout development.

Fig. 3

Fragmenting of late aggregate in pkcA⁻. (A) Absence of pkcA expression in pkcA⁻. (B) Developmental profiling of Ax2, pkcA⁻, Ax2+10 μM GF109203X and pkcA⁻/[act15:pkcA]. Scale bar—0.2 mm. (C) Number of tipped mounds formed per unit area by 14 h of development of Ax2, pkcA⁻, Ax2+10 μM GF109203X and pkcA⁻/[act15:pkcA], represented as a bar graph; error bars show the standard deviation. The number of tipped mounds was quantified from ten different frames. Three independent experiments in triplicate were carried out. Level of significance is indicated as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Fig. 4

pkcA⁻ is defective in cAMP relay. (A) cAMP wave generating centers are represented by optical density waves. Wave generating centers are indicated by asterixes. Scale bar—0.2 mm. (B) Bar graph representing the relative change in the expression levels of acaA, pde4, 5′nt and adk of pkcA⁻ in comparison to Ax2 at 9 h and 12 h of development. (C) cAMP levels of Ax2 and pkcA⁻ at 9 h and 12 h of development. The experiments were carried out thrice; mean and standard deviation are represented as error bars. Level of significance is indicated as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Fig. 5

cAMP chemotaxis. Average chemotactic velocity of Ax2, pkcA⁻ and pkcA⁻/[act15:pkcA] in response to cAMP; error bars show the standard deviation. Velocity is calculated by dividing the total displacement of the cells by time. A total of 32 cells from three independent experiments was used to calculate the average velocity. Level of significance is indicated as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Fig. 6

Actin polymerization. In vivo actin polymerization assay measuring F-actin levels in response to cAMP stimulation. After 10 s exposure to cAMP, actin polymerization in pkcA⁻ increased two-fold compared to Ax2 cells. Level of significance is indicated as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. The experiment was carried out thrice with two biological replicates.

Fig. 7

Cell adhesion properties of pkcA⁻.Cell–cell adhesion profiles of starved Ax2 and pkcA⁻ cells shaken in suspension. The assay was carried out thrice with three biological replicates both in the presence and absence of 10 mM EDTA. Mean values of percent aggregation; error bars show the standard deviation.

Fig. 8

Defective ecmA expression in pkcA⁻. (A) ecmA-GFP expression in Ax2 and pkcA⁻ cells at 16 h of development. Scale bar—0.5 mm. (B) Semi-quantitative PCR for ecmA and pspA of RNA isolated from Ax2 and pkcA⁻ at 10 h, 12 h, 14 h and 16 h of development.

Fig. 9

Cell autonomous and non-autonomous defects in pkcA⁻. (A) Reconstitution of pkcA⁻ with Ax2 cells in different ratios reduced the fragmentation defects of pkcA⁻ aggregates. (B) 50% unlabeled pkcA⁻, when reconstituted with 50% labeled Ax2 expressing act15-GFP and vice-versa show a prestalk cell fate bias. (C) Reconstitution of 20% labeled pkcA⁻ expressing ecmA-GFP with 80% unlabeled Ax2 cells show cell autonomous prestalk differentiation defect. Scale bar—1.0 mm. (D) Stalk cell induction assay performed with Ax2 and pkcA⁻ cells expressing ecmA-GFP in the presence of 0 nM, 1 nM, 10 nM, and 100 nM of DIF. Mean values of three independent experiments in triplicate; error bars show the standard deviation. Level of significance is indicated as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.