PIKfyve is required for phagosomal Rab7 acquisition and the delivery and fusion of early macropinosomes to phagosomes

Abstract

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.

PI(3,5)P2Controls the Signaling Activity of Class I PI3K

“…In general, PtdInsPs operate by recruiting effector proteins to defined membrane sites where they regulate the cellular activities of these proteins (Balla, 2013; Di Paolo and De Camilli, 2006; Posor et al, 2022). To date, only a limited number of proteins have been reported to bind PI(3,5)P 2 (Rivero-Ríos and Weisman, 2022) and even for those reported (Li et al, 2013; Vines et al, 2022), their specificity for PI(3,5)P 2 remains controversial (Hammond et al, 2015; Hasegawa et al, 2017), which has hampered development of a PI(3,5)P 2 -specific biosensors. The lack of a reliable PI(3,5)P 2 biosensor has made it difficult to determine the exact cellular functions of PI(3,5)P 2 in health and disease.…”