Co-loss of profilin I, II and cofilin with actin from maturing phagosomes in Dictyostelium discoideum

Although it is known that actin polymerizes rapidly at the plasma membrane during the ingestion phase of phagocytosis, not yet fully understood are th e mechanisms by which actin is recruited to form a phagocytic cup and subse quently is dissociated from the phagosome. The aim of this study was to ide ntify actin-binding proteins that mediated actin filament dynamics during p hagosome formation and processing. We report that profilins I and II, which promote filament assembly, and cofilin, which stimulates filament disassem bly, were constituents of phagosomes isolated from Dictyostelium discoideum fed latex beads, and associated with actin. Biochemical analyses detected one isoform only of cofilin, which bound actin in unstimulated cells as wel l as in cells engaged in phagocytosis, subjected to various stress treatmen ts, and through development. At membranes of young phagosomes, profilins I and II colocalized with monomeric actin labeled with fluorescent DNase I, a nd cofilin colocalized with filamentous actin labeled with rhodamine phallo idin. Both immunocytochemical and quantitative immunoblotting data indicate d that the kinetic loss of profilins I, II, and cofilin of maturing phagoso mes closely followed the falling levels of actin associated with the vesicl es. As evidence of vesicle processing, D. discoideum crystal protein tan es terase) was recruited rapidly to phagosomes and its levels increased while those of actin, profilins I,II, and cofilin jointly decreased. The localiza tion data and concurrent losses of profilins and cofilin with actin from ph agosomes are consistent with the roles of these actin-binding proteins in f ilament dynamics and indicated that they were involved in regulating the as sembly and disassembly of the actin coat of phagosomes.