PILS-Nir1 is a sensitive phosphatidic acid biosensor that reveals mechanisms of lipid production

Claire C. Weckerly; Taylor A. Rahn; Max Ehrlich; Rachel C. Wills; Joshua G. Pemberton; Michael V. Airola; Gerald R.V. Hammond

doi:10.1083/jcb.202405174

PILS-Nir1 is a sensitive phosphatidic acid biosensor that reveals mechanisms of lipid production

Claire C. Weckerly
, Taylor A. Rahn
, Max Ehrlich
, Rachel C. Wills
, Joshua G. Pemberton
, Michael V. Airola
, Gerald R.V. Hammond

Biochemistry and Cell Biology

University of Pittsburgh
Stony Brook University
National Institutes of Health

Research output: Contribution to journal › Article › peer-review

7 Scopus citations

Abstract

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.

Original language	English
Journal	Journal of Cell Biology
Volume	224
Issue number	11
DOIs	https://doi.org/10.1083/jcb.202405174
State	Published - Nov 3 2025

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title = "PILS-Nir1 is a sensitive phosphatidic acid biosensor that reveals mechanisms of lipid production",

abstract = "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.",

author = "Weckerly, \{Claire C.\} and Rahn, \{Taylor A.\} and Max Ehrlich and Wills, \{Rachel C.\} and Pemberton, \{Joshua G.\} and Airola, \{Michael V.\} and Hammond, \{Gerald R.V.\}",

note = "Publisher Copyright: {\textcopyright} 2025 Weckerly et al.",

year = "2025",

month = nov,

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doi = "10.1083/jcb.202405174",

language = "English",

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T1 - PILS-Nir1 is a sensitive phosphatidic acid biosensor that reveals mechanisms of lipid production

AU - Weckerly, Claire C.

AU - Rahn, Taylor A.

AU - Ehrlich, Max

AU - Wills, Rachel C.

AU - Pemberton, Joshua G.

AU - Airola, Michael V.

AU - Hammond, Gerald R.V.

PY - 2025/11/3

Y1 - 2025/11/3

N2 - 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.

AB - 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.

UR - https://www.scopus.com/pages/publications/105015640420

U2 - 10.1083/jcb.202405174

DO - 10.1083/jcb.202405174

M3 - Article

C2 - 40923975

AN - SCOPUS:105015640420

SN - 0021-9525

VL - 224

JO - Journal of Cell Biology

JF - Journal of Cell Biology

IS - 11

ER -

PILS-Nir1 is a sensitive phosphatidic acid biosensor that reveals mechanisms of lipid production

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