|Abstract:Macropinocytosis involves the uptake of large volumes of fluid, which is regulated by various small GTPases. The Dictyostelium discoideum protein GflB is a guanine nucleotide exchange factor (GEF) of Rap1, involved in chemotaxis. Here we studied the role of GflB in macropinocytosis, phagocytosis, and cytokinesis. In plate culture of vegetative cells, compared with the parental strain AX2, gflB KO cells were flatter and more polarized, whereas GflB-overproducing cells were rounder. The gflB KO cells exhibited impaired crown formation and retraction, particularly retraction, resulting in more crowns (macropinocytic cups) per cell and longer crown lifetimes. Accordingly, gflB KO cells showed defects in macropinocytosis and also in phagocytosis and cytokinesis. F-actin was elevated in gflB KO cells. GflB localized to the actin cortex especially at crowns and phagocytic cups. The villin headpiece domain (VHP)-like N-terminal domain of GflB directly interacted with F-actin in vitro Furthermore, a domain enriched in basic amino acids interacted with specific membrane cortex structures such as the cleavage furrow. In conclusion, GflB acts as a key local regulator of actin-driven membrane protrusion possibly by modulating Rap1 signaling pathways.