The role of aginactin in actin polymerization during Dictyostelium chemotaxis

Abstract

During the phenomenon of amoeboid chemotaxis, the binding of ligands to external cell surface receptors signals a complex series of intracellular processes that control cell shape and movement toward the source of the chemoattractant. Stimulation of starved Dictyostelium amoebae with the chemoattractant cAMP produces a rapid increase in actin nucleation activity which is cotemporal with an increase in actin assembly and a decrease in Ca{dollar}\sp{lcub}2+{rcub}{dollar}-insensitive barbed end capping activity at 5 seconds post-stimulation (Hall et al., 1989). The uncapping of actin filaments at their barbed ends has been proposed as a mechanism for increases in actin polymerization following chemotactic stimulation. In order to investigate this hypothesis, we employed a purification strategy that selects for capping activities that show a decrease following stimulation with cAMP in Dictyostelium as determined by an in vitro pyrene-labeled actin polymerization assay. A 70 kDa protein was found to be associated with this agonist-regulated capping activity or aginactin and was confirmed to be an Hsc70 (70 kDa heat shock cognate protein) based on sequence analysis. Further characterization has demonstrated that aginactin is a mixture of Hsc70 and capping protein, a heterodimeric barbed end capping protein, in a 2:1 molar ratio. Immunoprecipitation of capping protein from purified aginactin removes all capping activity while immunoprecitation of Hsc70 does not, indicating that the capping activity of aginactin is intrinsic property of capping protein. Gel filtration chromatography and immunoprecipitation assays fail to demonstrate the existence of a stable complex of Hsc70 and capping protein in either lysate supernatants or purified aginactin. We measure an apparent K{dollar}\sb{lcub}\rm d{rcub}{dollar} of approximately 1 nM for capping protein, indicating high affinity binding to F-actin. Furthermore, reconstitution experiments with purified native Hsc70 and capping protein demonstrate that Hsc70 neither stimulates nor inhibits the capping activity of capping protein. We conclude therefore that Hsc70 is not a regulatory cofactor for capping protein. Capping protein was found to leave the cytosolic fraction and enter the cytoskeleton at 5 seconds following cAMP stimulation. This finding does not support a model where the release of capping protein from barbed filament ends leads to an increase in nucleation activity and actin assembly in the cytoskeleton. Rather, capping protein acts to terminate the actin polymerization response following cAMP stimulation.