To identify the molecular mechanisms involved in phagocytosis, we generated random insertion mutants of Dictyostelium discoideum and selected two mutants defective for phagocytosis. Both represented insertions in the same gene, named PHG1. This gene encodes a polytopic membrane protein with an N-terminal lumenal domain and nine potential transmembrane segments. Homologous genes can be identified in many species; however, their function is yet to be elucidated. Disruption of PHG1 caused a selective defect in phagocytosis of latex beads and Escherichia coli, but not Klebsiella aerogenes bacteria. This defect in phagocytosis was caused by a decrease in the adhesion of mutant cells to phagocytosed particles. These results indicate that the Phg1 protein is involved in the adhesion of Dictyostelium to various substrates, a crucial event of phagocytosis and demonstrate the usefulness of a genetic approach to dissect the molecular events involved in the phagocytic process.Phagocytosis is the process by which cells internalize large particles (typically Ͼ1 m diameter), such as bacteria or cell debris. In higher eucaryotes, phagocytic cells are essential players of the host defense against invading pathogens and tissue remodeling (for review see Ref. 1). Phagocytosis involves adhesion of the phagocytic cell to the particle, and reorganization of the actin cytoskeleton to allow engulfment. A number of receptors required for the recognition of particles to be phagocytosed have been identified in mammalian cells, e.g. Fc receptors involved in the phagocytosis of opsonized particles (2). These receptors presumably transduce a local activation signal upon recognition of their ligand, leading to reorganization of the actin cytoskeleton. Protein kinases such as Syk (3) as well as GTP-binding proteins of the Rho family (4) have been implicated in the transduction of the activation signal.The cellular slime mold Dictyostelium discoideum has been used previously as a model organism to study phagocytosis (5,6). Vegetative Dictyostelium amoebae multiply as single cells feeding phagocytically on bacteria. The mechanisms involved in phagocytosis by Dictyostelium cells are very similar to those used by mammalian phagocytes, and involve notably the actin cytoskeleton and RacF1, a member of the Rho family of GTPbinding proteins (7). The receptors responsible for phagocytosis have not yet been identified. However, previous studies suggest that there are at least two receptors, one a lectin and the other one a "nonspecific" receptor, which accounts for the phagocytosis of most hydrophilic particles in HL5 medium (5).Here we describe the identification of a new gene implicated in phagocytosis in Dictyostelium. The phenotype of the phg1 mutants suggests a role for Phg1p in adhesion to phagocytic substrates. EXPERIMENTAL PROCEDURESCell Culture and Internalization Assays-Wild-type cells used in this study are DH1-10 cells, a subclone of DH1 cells. They were grown at 21°C in HL5 medium (8) and subcultured twice a week. Cells were typically not a...

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Spalten (Spn), a member of the PP2C family of Ser/Thr protein phosphatases, is required for Dictyostelium cell-type differentiation and morphogenesis. We have identified a new protein kinase, ARCK-1, through a second site suppressor screen for mutants that allow spn null cells to proceed further through development. ARCK-1 has a C-terminal kinase domain most closely related to Ser/Thr protein kinases and an N-terminal putative regulatory domain with ankyrin repeats, a 14-3-3 binding domain, and a C1 domain, which is required for binding to RasBGTP in a two-hybrid assay. Disruption of the gene encoding ARCK-1 results in weak, late developmental defects. However, overexpression of ARCK-1 phenocopies the spn null phenotype, consistent with Spn and ARCK-1 being on the same developmental pathway. Our previous analyses of Spn and the present analysis of ARCK-1 suggest a model in which Spn and ARCK-1 differentially control the phosphorylation state of a protein that regulates cell-type differentiation. Dephosphorylation of the substrate by Spn is required for cell-type differentiation. Control of ARCK-1 and Spn activities by upstream signals is proposed to be part of the developmental regulatory program mediating cell-fate decisions in Dictyostelium.