Calreticulin and calnexin in the endoplasmic reticulum are important for phagocytosis.

Calreticulin and calnexin are Ca2+-binding proteins with chaperone activity in the endoplasmic reticulum. These proteins have been eliminated by gene replacement in Dictyostelium, the only microorganism known to harbor both proteins; family members in Dictyostelium are located at the base of phylogenetic trees. A dramatic decline in the rate of phagocytosis was observed in double mutants lacking calreticulin and calnexin, whereas only mild changes occurred in single mutants. Dictyostelium cells are professional phagocytes, capable of internalizing particles by a sequence of activities: adhesion of the particle to the cell surface, actin-dependent outgrowth of a phagocytic cup, and separation of the phagosome from the plasma membrane. In the double-null mutants, particles still adhered to the cell surface, but the outgrowth of phagocytic cups was compromised. Green fluorescent protein-tagged calreticulin and calnexin, expressed in wild-type cells, revealed a direct link of the endoplasmic reticulum to the phagocytic cup enclosing a particle, such that the Ca2+ storage capacity of calreticulin and calnexin might directly modulate activities of the actin system during particle uptake.

Dynamics of the Dictyostelium Arp2/3 complex in endocytosis, cytokinesis, and chemotaxis.

The Arp2/3 complex is a ubiquitous and important regulator of the actin cytoskeleton. Here we identify this complex from Dictyostelium and investigate its dynamics in live cells. The predicted sequences of the subunits show a strong homology to the members of the mammalian complex, with the larger subunits generally better conserved than the smaller ones. In the highly motile cells of Dictyostelium, the Arp2/3 complex is rapidly re-distributed to the cytoskeleton in response to external stimuli. Fusions of Arp3 and p41-Arc with GFP reveal that in phagocytosis, macropinocytosis, and chemotaxis the complex is recruited within seconds to sites where actin polymerization is induced. In contrast, there is little or no localization to the cleavage furrow during cytokinesis. Rather the Arp2/3 complex is enriched in ruffles at the polar regions of mitotic cells, which suggests a role in actin polymerization in these ruffles.

DAip1, a Dictyostelium homologue of the yeast actin-interacting protein 1, is involved in endocytosis, cytokinesis, and motility.

The 64-kD protein DAip1 from Dictyostelium contains nine WD40-repeats and is homologous to the actin-interacting protein 1, Aip1p, from Saccharomyces cerevisiae, and to related proteins from Caenorhabditis, Physarum, and higher eukaryotes. We show that DAip1 is localized to dynamic regions of the cell cortex that are enriched in filamentous actin: phagocytic cups, macropinosomes, lamellipodia, and other pseudopodia. In cells expressing green fluorescent protein (GFP)-tagged DAip1, the protein rapidly redistributes into newly formed cortical protrusions. Functions of DAip1 in vivo were assessed using null mutants generated by gene replacement, and by overexpressing DAip1. DAip1-null cells are impaired in growth and their rates of fluid-phase uptake, phagocytosis, and movement are reduced in comparison to wild-type rates. Cytokinesis is prolonged in DAip1-null cells and they tend to become multinucleate. On the basis of similar results obtained by DAip1 overexpression and effects of latrunculin-A treatment, we propose a function for DAip1 in the control of actin depolymerization in vivo, probably through interaction with cofilin. Our data suggest that DAip1 plays an important regulatory role in the rapid remodeling of the cortical actin meshwork.