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. 2025 Jun 2;224(6):e202408158.
doi: 10.1083/jcb.202408158. Epub 2025 Mar 26.

An advanced toolset to manipulate and monitor subcellular phosphatidylinositol 3,5-bisphosphate

Joshua G Pemberton  1   2   3 , Isobel Barlow-Busch  4 , Meredith L Jenkins  4 , Matthew A H Parson  4 , Farkas Sarnyai  1 , Seyma Nur Bektas  1 , Yeun Ju Kim  1 , John E Heuser  1 , John E Burke  4   5 , Tamas Balla  1
Affiliations

An advanced toolset to manipulate and monitor subcellular phosphatidylinositol 3,5-bisphosphate

Joshua G Pemberton et al. J Cell Biol. .

Abstract

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.

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Conflict of interest statement

Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. J. Burke reported personal fees from Scorpion Therapeutics, other from Calico Life Sciences, personal fees from Reactive Therapeutics, and personal fees from Olema Oncology outside the submitted work. No other disclosures were reported.

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