ABSTRACT Macropinocytosis is an actin-driven process of large-scale and non-specific fluid uptake used for feeding by some cancer cells and the macropinocytosis model organism Dictyostelium discoideum . In Dictyostelium , macropinocytic cups are organized by ‘macropinocytic patches’ in the plasma membrane. These contain activated Ras, Rac and phospholipid PIP3, and direct actin polymerization to their periphery. We show that a Dictyostelium Akt (PkbA) and an SGK (PkbR1) protein kinase act downstream of PIP3 and, together, are nearly essential for fluid uptake. This pathway enables the formation of larger macropinocytic patches and macropinosomes, thereby dramatically increasing fluid uptake. Through phosphoproteomics, we identify a RhoGAP, GacG, as a PkbA and PkbR1 target, and show that it is required for efficient macropinocytosis and expansion of macropinocytic patches. The function of Akt and SGK in cell feeding through control of macropinosome size has implications for cancer cell biology.

ABSTRACT Macropinocytosis is a conserved endocytic process used by Dictyostelium amoebae for feeding on liquid medium. To further Dictyostelium as a model for macropinocytosis, we developed a high-throughput flow cytometry assay to measure macropinocytosis, and used it to identify inhibitors and investigate the physiological regulation of macropinocytosis. Dictyostelium has two feeding states: phagocytic and macropinocytic. When cells are switched from phagocytic growth on bacteria to liquid media, the rate of macropinocytosis slowly increases, due to increased size and frequency of macropinosomes. Upregulation is triggered by a minimal medium containing three amino acids plus glucose and likely depends on macropinocytosis itself. The presence of bacteria suppresses macropinocytosis while their product, folate, partially suppresses upregulation of macropinocytosis. Starvation, which initiates development, does not of itself suppress macropinocytosis: this can continue in isolated cells, but is shut down by a conditioned-medium factor or activation of PKA signalling. Thus macropinocytosis is a facultative ability of Dictyostelium cells, regulated by environmental conditions that are identified here. This article has an associated First Person interview with the first author of the paper.