PIKY Easters: How PIKfyve and PI(3,5) P2 Regulate Macropinosome and Phagosome Maturation in Dictyostelium

Endocytosis is the uptake of extracellular material into the cell. Once internalized, endosomes undergo a series of complex regulatory steps to facilitate the breakdown and/or delivery of different cargoes to different cellular locations - a process called maturation. Successful maturation relies upon phosphoinositide signalling lipids (PIPs) and regulatory proteins to aid in membrane identity and coordinate complex trafficking events. PIKfyve is phosphoinositide 5-kinase essential for efficient maturation of phagosomes and is responsible for synthesis of PI(3,5) P2, a key membrane marker in endocytic pathways including phagocytosis and macropinocytosis. Despite its importance, PI(3,5) P2 is one of the least well understood regulators of trafficking. Here, we have used the model professional phagocyte Dictyostelium discoideum to identify a novel PI(3,5) P2 biosensor, SnxA, which can faithfully report PI(3,5) P2 dynamics in live cells. We find PI(3,5) P2 binding is conferred through a PX-domain within the reporter, and validate the probe in mammalian cells and D. discoideum, showing it is recruited to macropinosomes in a PIKfyve dependent manner. In D. discoideum we employ an automated image analysis pipeline to examine SnxA enrichment to phagosomal membranes over time. We quantify PI(3,5) P2 arrival ∼3-minutes after phagosome engulfment and show this is completely lost in ∆PIKfyve cells. To further characterise the role of PIKfyve and PI(3,5) P2, we quantify the enrichment of other phagosomal maturation regulators. We find that, while Rab5 and PI(3)P dynamics are unchanged in ∆PIKfyve cells, Rab7 delivery to phagosomal membranes is severally perturbed. By coexpressing fusion proteins, we go on to delineate a subpopulation of Rab7/PI(3,5) P2-positive vesicles which are delivered to phagosomes. We find these vesicles contain macropinocytic material which is delivered to phagosomes shortly after their engulfment in a PIKfyve-dependant manner. These findings uncover key mechanistic details of the role and regulation of PIKfyve and PI(3,5) P2.