This research demonstrates PILS-Nir1's enhanced detection of phosphatidic acid in HEK293A cells, revealing insights into lipid production mechanisms.
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.
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Weckerly et al. (2025) studied this question.
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