PILS-Nir1 is a sensitive phosphatidic acid biosensor that reveals mechanisms of lipid production
Phosphatidic acid (PA) regulates lipid homeostasis and vesicular trafficking, yet high-affinity tools to study PA in live cells are lacking. We identified the lipin-like sequence of Nir1 (PILS-Nir1) as a candidate PA biosensor based on structural analysis of Nir1's LNS2 domain. Using liposome-binding assays and pharmacological and genetic manipulations in HEK293A cells expressing fluorescent PILS-Nir1, we found that while PILS-Nir1 binds PA and PIP2in vitro, only PA is necessary and sufficient for membrane localization in cells. PILS-Nir1 displayed greater sensitivity to organelle-generated PA than Spo20-based probes, enabling visualization of modest PA production by PLD downstream of muscarinic receptors-previously undetectable with existing biosensors. Thus, PILS-Nir1 provides a versatile, sensitive tool for real-time PA dynamics in live cells.
Authors
- T. Rähn (ORCID: https://orcid.org/0000-0002-5031-6914)
- Gerald Hammond (ORCID: https://orcid.org/0000-0002-6660-3272)
- Joshua G. Pemberton (ORCID: https://orcid.org/0000-0002-1492-1471)
- Claire C. Weckerly (ORCID: https://orcid.org/0000-0003-2368-8429)
- Rachel C. Wills (ORCID: https://orcid.org/0000-0003-2161-9235)
- M. Ehrlich
- Michael V. Airola (ORCID: https://orcid.org/0000-0003-4981-1253)
Institutions
- National Institutes of Health (US)
- University of Pittsburgh (US)
- Eunice Kennedy Shriver National Institute of Child Health and Human Development (US)
- Stony Brook University (US)
Publication Details
- Journal
- The Journal of Cell Biology
- Published
- 2025-09-09
- DOI
- https://doi.org/10.1083/jcb.202405174
- Citations
- 8
- Primary Topic
- Lipid Membrane Structure and Behavior
- Type
- article
- Field-Weighted Citation Impact
- 6.45
Funders
- University of Pittsburgh
- Stony Brook University
- National Institutes of Health