Identification of actin nucleation activity and polymerization inhibitor in ameboid cells: their regulation by chemotactic stimulation

Abstract

Actin polymerization occurs in amebae of Dictyostelium discoideum after chemotactic stimulation (Hall, A. L., A. Schlein, and J. Condeelis. 1988. J. Cell. Biochem. 37:285-299). When cells are lysed with Triton X-100 during stimulation, an actin nucleation activity is detected in lysates by measuring the rate of pyrene-labeled actin polymerization. This stimulated nucleation activity is closely correlated with actin polymerization observed in vivo in its kinetics, developmental regulation, and cytochalasin D sensitivity. Actin polymerization is coordinate with pseudopod extension in synchronized populations of cells and is correlated with the accumulation of F actin in pseudopods. The stimulated actin nucleation activity is present in low-speed pellets from Triton lysates (cytoskeletons) within 3 s of stimulation and is stable compared with the nucleation activity of whole cell lysates. Low-speed supernatants contain a reversible inhibitor of the actin nucleation activity that is itself regulated by chemotactic stimulation. Neither activity requires Ca2+ and both are fully expressed in 10 mM EGTA. Fractions containing the inhibitor do not sever actin filaments but do inhibit actin polymerization that is seeded by fragments of purified F actin. These results indicate that chemotactic stimulation of Dictyostelium discoideum generates both an actin-nucleating activity and an actin-polymerization inhibitor, and suggest that the parallel regulation of these two activities leads to the transient phases of actin polymerization observed in vivo. The different compartmentation of these two activities may account for polarized pseudopod extension in gradients of chemoattractant.