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bioRxiv · 10.1101/2024.02.28.582557

Nir1-LNS2 is a novel phosphatidic acid biosensor that reveals mechanisms of lipid production

Weckerly, C. C.·Rahn, T. A.·Ehrlich, M.·Wills, R. C.·Pemberton, J. G.·Airola, M. V.·Hammond, G. R.
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Abstract

Despite various roles of phosphatidic acid (PA) in cellular functions such as lipid homeostasis and vesicular trafficking, there is a lack of high-affinity tools to study PA in live cells. After analyzing the predicted structure of the LNS2 domain from the lipid transfer protein Nir1, we suspected that this phosphatidic acid-interacting Lipin-like sequence of Nir1 (PILS-Nir1) could serve as a novel PA biosensor. We then performed liposome binding assays as well as pharmacological and genetic manipulations of HEK293A cells expressing a fluorescent PILS-Nir1 to determine how specific lipids affect the interaction of PILS-Nir1 with membranes. We found that PILS-Nir1 bound to both PA and PIP2 in vitro. However, only PA was necessary and sufficient to localize PILS-Nir1 to membranes in cells. PILS-Nir1 also showed a heightened responsiveness to PA produced in various organelles when compared to biosensors using the Spo20 PA binding domain. PILS-Nir1s high sensitivity revealed a modest but discernible contribution of PLD to PA production downstream of muscarinic receptors, which has not been visualized with previous Spo20-based probes. In summary, PILS-Nir1 emerges as a versatile and sensitive biosensor, offering a new powerful tool for real-time investigation of PA dynamics in live cells.

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Weckerly, C. C., Rahn, T. A., Ehrlich, M., Wills, R. C., Pemberton, J. G., Airola, M. V., Hammond, G. R.. 2024-02-28. Nir1-LNS2 is a novel phosphatidic acid biosensor that reveals mechanisms of lipid production. https://doi.org/10.1101/2024.02.28.582557

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