Phosphatidylinositol (PI) 3,5-bisphosphate (PI(3,5)P2) is a minor inositol-containing phospholipid that serves as an important regulator of endolysosomal functions. However, the precise sites of subcellular enrichment and molecular targets of this regulatory lipid are poorly understood. Here, we describe the generation and detailed characterization of a short engineered catalytic fragment of the human PIKfyve enzyme, which potently converts PI 3-phosphate to PI(3,5)P2. This novel tool allowed for the evaluation of reported PI(3,5)P2-sensitive biosensors and showed that the recently identified phox homology (PX) domain of the Dictyostelium discoideum (Dd) protein, SNXA, can be used to monitor the production of PI(3,5)P2 in live cells. Modification and adaptation of the DdSNXAPX-based probes into compartment-specific bioluminescence resonance energy transfer–based biosensors allows for the real-time monitoring of PI(3,5)P2 generation within the endocytic compartments of entire cell populations. Collectively, these molecular tools should allow for exciting new studies to better understand the cellular processes controlled by localized PI(3,5)P2 metabolism.

Subjects:
Biochemistry, Cell signaling, Membrane and lipid biology
© 2025 Crown copyright. The government of Australia, Canada, or the UK ("the Crown") owns the copyright interests of authors who are government employees. The Crown Copyright is not transferable.
2025
Crown copyright. The government of Australia, Canada, or the UK (“the Crown”) owns the copyright interests of authors who are government employees. The Crown Copyright is not transferable.
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