A PI(3,5)P2 probe reveals PIKfyve is required for Rab7 acquisition and the delivery and fusion of early macropinosomes to phagosomes

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. ### Competing Interest Statement The authors have declared no competing interest.