Highlights d We identify a new regulator that shapes macropinocytic and phagocytic cups d Shaping protrusions into cups requires differential regulation of Ras and Rac d Cups are organized by integrating interactions with phospholipids and multiple GTPases d Defective cup formation causes a target shape-specific defect in phagocytosis

The formation and processing of vesicles from the cell surface serves many important cellular functions ranging from nutrient acquisition to regulating the turnover of membrane components and signalling. In this article, we summarise the endocytic pathways of the social amoeba Dictyostelium from the clathrin-dependent and independent internalisation of surface components to the engulfment of bacteria or fluid by phagocytosis and macropinocytosis respectively. Due to similarities with the professional phagocytes of the mammalian immune system Dictyostelium has been extensively used to investigate the complex remodelling and trafficking events that occur as phagosomes and macropinosomes transit through the cell. Here we discuss what is known about this maturation process in order to kill any potential pathogens and obtain nutrients for growth. Finally, we aim to put these studies in evolutionary context and highlight some of the many questions that remain in our understanding of these complex and important pathways.

Phosphoinositide signaling lipids (PIPs) are key regulators of membrane identity and trafficking. Of these, PI(3,5)P2 is one of the least well-understood, despite key roles in many endocytic pathways including phagocytosis and macropinocytosis. PI(3,5)P2 is generated by the phosphoinositide 5-kinase PIKfyve, which is critical for phagosomal digestion and antimicrobial activity. However PI(3,5)P2 dynamics and regulation remain unclear due to lack of reliable reporters. Using the amoeba Dictyostelium discoideum, we identify SnxA as a highly selective PI(3,5)P2-binding protein and characterize its use as a reporter for PI(3,5)P2 in both Dictyostelium and mammalian cells. Using GFP-SnxA, we demonstrate that Dictyostelium phagosomes and macropinosomes accumulate PI(3,5)P2 3 min after engulfment but are then retained differently, indicating pathway-specific regulation. We further find that PIKfyve recruitment and activity are separable and that PIKfyve activation stimulates its own dissociation. SnxA is therefore a new tool for reporting PI(3,5)P2 in live cells that reveals key mechanistic details of the role and regulation of PIKfyve/PI(3,5)P2.

Phosphoinositide signalling lipids (PIPs) are key regulators of membrane identity and vesicle trafficking. Of these, PI(3,5)P2 is one of the least understood, despite key roles in many endocytic pathways including phagocytosis and macropinocytosis. PI(3,5)P2 is predominantly generated by the phosphoinositide 5-kinase PIKfyve, and is critical for phagosomal digestion and the killing of engulfed microbes. However, the regulation of PIKfyve activity, PI(3,5)P2 dynamics, and how they control phagosome maturation remains unclear. Using the model professional phagocyte Dictyostelium discoideum we have identified a highly selective PI(3,5)P2-binding protein that allows faithful observation of PI(3,5)P2 dynamics in live cells. Using this probe we demonstrate that PIKfyve recruitment and activity are separable, and that PIKfyve activation stimulates its own dissociation from membranes. We show that PI(3,5)P2 accumulates on phagosomes 2-3 minutes after engulfment drives fusion of a specific population of PI(3,5)P2 and Rab7-positive macropinosomes. We find PIKfyve is required for the fusion between macropinosomes and phagosomes, which enables phagosomes to efficiently accumulate Rab7 and other components of the lysosomal machinery. These findings uncover key mechanistic details of the role and regulation of PIKfyve/PI(3,5)P2 likely to have general relevance across endocytic pathways.