An in vivo analysis of the function of ABP-120 in Dictyostelium amoebae using molecular genetics

Abstract

The actin binding protein ABP-120 cross-links actin filaments into orthogonal networks in vitro, similar to networks seen in pseudopods in situ and has been localized to newly formed pseudopods following chemotactic stimulation in vivo. Therefore, ABP-120 has been proposed to play a role in cross-linking actin filaments during pseudopod formation in motile Dictyostelium amoebae. To test this hypothesis several independent cell lines, that lack ABP-120 were created by gene disruption using homologous recombination and then subsequently a cloned version of the gene was re-introduction into one of the mutant cell lines with the concomitant restoration of a wild type phenotype. The phenotypes of two different cell lines lacking ABP-120, generating in two different Dictyostelium strains, have been characterized and show identical results. The amount of F-actin cross-linked into the cytoskeleton following cAMP stimulation of ABP-120{dollar}\sp-{dollar} cells is reduced at times when ABP-120 has been shown to be incorporated into the cytoskeleton even though there is no change in the total amount of F-actin produced compared to ABP-120{dollar}\sp+{dollar} control cell lines. ABP-120{dollar}\sp-{dollar} cells translocating in buffer exhibit defects in both the rate and extent of pseudopod formation. In the confocal and electron microscope, pseudopods extended by ABP-120{dollar}\sp-{dollar} cells are small and flat. Protrusions of ABP-120{dollar}\sp-{dollar} cells contain actin filament aggregates with reduced pore sizes that are less three-dimensional than protrusions of ABP-120{dollar}\sp+{dollar} cells.;ABP-120 is also important in the formation of pseudopods produced in response to a phagocytic signal. ABP-120{dollar}\sp-{dollar} cells were analyzed for their ability to uptake either bacteria or latex beads and were found to have a 50% defect in comparison to control ABP-120{dollar}\sp+{dollar} cells. The defect in particle uptake has an effect on the ability of ABP-120{dollar}\sp-{dollar} cells to grow when bacteria is present as the sole supply of nutrition either in bacterial suspensions or when cells are grown on a bacterial lawn. It can be concluded that the cross-linking of actin filaments by ABP-120 is important not only in the formation of pseudopods during chemotactic stimulation but ABP-120 is also important for the formation of pseudopods produced during phagocytosis.