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...

The study of free-living amoebae has proven valuable to explain the molecular mechanisms controlling phagocytosis, cell adhesion and motility. In this study, we identified a new adhesion molecule in Dictyostelium amoebae. The SibA (Similar to Integrin Beta) protein is a type I transmembrane protein, and its cytosolic, transmembrane and extracellular domains contain features also found in integrin b chains. In addition, the conserved cytosolic domain of SibA interacts with talin, a well-characterized partner of mammalian integrins. Finally, genetic inactivation of SIBA affects adhesion to phagocytic particles, as well as cell adhesion and spreading on its substrate. It does not visibly alter the organization of the actin cytoskeleton, cellular migration or multicellular development. Our results indicate that the SibA protein is a Dictyostelium cell adhesion molecule presenting structural and functional similarities to metazoan integrin b chains. This study sheds light on the molecular mechanisms controlling cell adhesion and their establishment during evolution.

Molecular mechanisms of endocytosis in the genetically and biochemically tractable professional phagocyte Dictyostelium discoideum reveal a striking degree of similarity to higher eukaryotic cells. Pulse-chase feeding with latex beads allowed purification of phagosomes at different stages of maturation. Gentle ATP stripping of an actin meshwork entrapping contaminating organelles resulted in a 10-fold increase in yield and purity, as confirmed by electron microscopy. Temporal profiling of signaling, cytoskeletal, and trafficking proteins resulted in a complex molecular fingerprint of phagosome biogenesis and maturation. First, nascent phagosomes were associated with coronin and rapidly received a lysosomal glycoprotein, LmpB. Second, at least two phases of delivery of lysosomal hydrolases (cathepsin D [CatD] and cysteine protease [CPp34]) were accompanied by removal of plasma membrane components (PM4C4 and biotinylated surface proteins). Third, a phase of late maturation, preparing for final exocytosis of undigested material, included quantitative recycling of hydrolases and association with vacuolin. Also, lysosomal glycoproteins of the Lmp family showed distinct trafficking kinetics. The delivery and recycling of CatD was directly visualized by confocal microscopy. This heavy membrane traffic of cargos was precisely accompanied by regulatory proteins such as the Rab7 GTPases and the endosomal SNAREs Vti1 and VAMP7. This initial molecular description of phagocytosis demonstrates the feasibility of a comprehensive analysis of phagosomal lipids and proteins in genetically modified strains.