Application of hydrodynamic mild shear stress to adherent Dictyostelium discoideum vegetative cells triggers active actin cytoskeleton remodeling resulting in net cell movement along the flow. The average cell speed is strongly stimulated by external calcium (Ca2+, K50%=22 μM), but the directionality of the movement is almost unaffected. This calcium concentration is ten times higher than the one promoting cell adhesion to glass surfaces (K50%=2 μM). Addition of the calcium chelator EGTA or the Ca2+-channel blocker gadolinium (Gd3+) transiently stops cell movement. Monitoring the evolution of cell-surface contact area with time reveals that calcium stimulates cell speed by increasing the amplitude of both protrusion and retraction events at the cell edge, but not the frequency. As a consequence, with saturating external calcium concentrations, cells are sensitive to very low shear forces (20 pN; σ=0.1 Pa). Moreover, a null-mutant lacking the unique Gβ subunit does not respond to external Ca2+ changes (K50%>1000 μM), although the directionality of the movement is comparable with that of wild-type cells. Furthermore, cells lacking the inositol 1,4,5-trisphosphate receptor (IP3-receptor) exhibit a markedly reduced Ca2+ sensitivity. Thus, calcium release from internal stores and calcium entry through the plasma membrane modulate cell speed in response to shear stress.
Membrane-associated NADPH oxidase complexes catalyse the production of the superoxide anion radical from oxygen and NADPH. In mammalian systems, NADPH oxidases form a family of at least seven isoforms that participate in host defence and signalling pathways. We report here the cloning and the characterisation of slime mould Dictyostelium discoideum homologs of the mammalian heme-containing subunit of flavocytochrome b (gp91(phox)) (NoxA, NoxB and NoxC), of the small subunit of flavocytochrome b (p22(phox)) and of the cytosolic factor p67(phox). Null-mutants of either noxA, noxB, noxC or p22(phox) show aberrant starvation-induced development and are unable to produce spores. The overexpression of NoxA(myc2) in noxA null strain restores spore formation. Remarkably, the gene alg-2B, coding for one of the two penta EF-hand proteins in Dictyostelium, acts as a suppressor in noxA, noxB, and p22(phox) null-mutant strains. Knockout of alg-2B allows noxA, noxB or p22(phox) null-mutants to return to normal development. However, the knockout of gene encoding NoxC, which contains two penta EF-hands, is not rescued by the invalidation of alg-2B. These data are consistent with a hypothesis connecting superoxide and calcium signalling during Dictyostelium development.
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