Convergence of Ras- and Rac-regulated formin pathways is pivotal for phagosome formation and particle uptake in Dictyostelium

Abstract

AbstractMacroendocytosis comprising phagocytosis and macropinocytosis are actin-driven processes regulated by small GTPases that depend on the dynamic reorganization of the membrane that protrudes and internalizes extracellular material by cup-shaped structures. To effectively capture, enwrap, and internalize their targets, these cups are arranged into a peripheral ring or ruffle of protruding actin sheets emerging from an actin-rich, non-protrusive zone at its base. Despite extensive knowledge of the mechanism driving actin assembly of the branched network at the protrusive cup edge, which is initiated by the actin-related protein (Arp) 2/3 complex downstream of Rac signaling, our understanding of actin assembly in the base is still incomplete. In the Dictyostelium model system, the Ras-regulated formin ForG was previously shown to specifically contribute to actin assembly at the cup base. Loss of ForG is associated with a strongly impaired macroendocytosis and a 50% reduction of F-actin content at the base of phagocytic cups, in turn indicating the presence of additional factors that specifically contribute to actin formation at the base. Here, we show that ForG synergizes with the Rac-regulated formin ForB to form the bulk of linear filaments at the cup base. Consistently, combined loss of both formins virtually abolishes cup formation and leads to severe defects of macroendocytosis, emphasizing the relevance of converging Ras- and Rac-regulated formin pathways in assembly of linear filaments in the cup base, which apparently provide mechanical support to the entire structure. Remarkably, we finally show that active ForB, unlike ForG, additionally drives phagosome rocketing to aid particle internalization.Significance StatementCup formation in macroendocytosis is a decisive, actin-dependent process that relies on distinct actin assembly factors generating the necessary mechanical forces to drive rearrangements of the plasma membrane and engulfment of extracellular material. Hitherto, in Dictyostelium the Arp2/3 complex and VASP were shown to promote actin assembly at the protrusive rim of phagocytic cups, while the Ras-regulated formin ForG generates about half of the actin filament mass at the base. Here, we show that ForG synergizes with the Rac-regulated formin ForB to form the bulk of filaments at the cup base. Loss of both formins virtually abolishes cup formation and leads to dramatic defects in macroendocytosis, illustrating the relevance of converging Ras- and Rac-regulated signaling pathways in this process.