Coordinated Ras and Rac Activity Shapes Macropinocytic Cups and Enables Phagocytosis of Geometrically Diverse Bacteria

Engulfment of extracellular material by phagocytosis or macropinocytosis depends on the ability of cells to generate specialized cup-shaped protrusions. To effectively capture and internalize their targets, these cups are organized into a ring or ruffle of actin-driven protrusion encircling a non-protrusive interior domain. These functional domains depend on the combined activities of multiple Ras and Rho family small GTPases, but how their activities are integrated and differentially regulated over space and time is unknown. Here, we show that the amoeba Dictyostelium discoideum coordinates Ras and Rac activity using the multidomain protein RGBARG (RCC1, RhoGEF, BAR, and RasGAP-containing protein). We find RGBARG uses a tripartite mechanism of Ras, Rac, and phospholipid interactions to localize at the protruding edge and interface with the interior of both macropinocytic and phagocytic cups. There, we propose RGBARG shapes the protrusion by expanding Rac activation at the rim while suppressing expansion of the active Ras interior domain. Consequently, cells lacking RGBARG form enlarged, flat interior domains unable to generate large macropinosomes. During phagocytosis, we find that disruption of RGBARG causes a geometry-specific defect in engulfing rod-shaped bacteria and ellipsoidal beads. This demonstrates the importance of coordinating small GTPase activities during engulfment of more complex shapes and thus the full physiological range of microbes, and how this is achieved in a model professional phagocyte. [Display omitted] •We identify a new regulator that shapes macropinocytic and phagocytic cups•Shaping protrusions into cups requires differential regulation of Ras and Rac•Cups are organized by integrating interactions with phospholipids and multiple GTPases•Defective cup formation causes a target shape-specific defect in phagocytosis Forming cup-shaped protrusions allows cells to engulf extracellular fluid and particles by macropinocytosis and phagocytosis, respectively. Buckley et al. identify a new regulator that differentially regulates small GTPases to generate the cup shape. They propose a model whereby this coordinates the shape and allows cells to engulf different shapes.