A PI(3,5)P2 reporter reveals PIKfyve activity and dynamics on macropinosomes and phagosomes

James H. Vines, Hannes Maib, Catherine M. Buckley, Aurelie Gueho, Zhou Zhu, Thierry Soldati, David H.. Murray, Jason S. King (Lead / Corresponding author)

    Research output: Contribution to journalArticlepeer-review

    5 Citations (Scopus)
    60 Downloads (Pure)

    Abstract

    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.

    Original languageEnglish
    Article numbere202209077
    Number of pages22
    JournalJournal of Cell Biology
    Volume222
    Issue number9
    DOIs
    Publication statusPublished - 29 Jun 2023

    Keywords

    • Animals
    • Dictyostelium/genetics
    • Phagosomes
    • Endosomes
    • Phosphatidylinositols
    • Mammals

    ASJC Scopus subject areas

    • Cell Biology

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