Dataset Information

Inducible depletion of PI(4,5)P₂ by the synthetic iDePP system in Arabidopsis.

ABSTRACT: Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is a low-abundance membrane lipid essential for plasma membrane function^1,2. In plants, mutations in phosphatidylinositol 4-phosphate (PI4P) 5-kinases (PIP5K) suggest that PI(4,5)P₂ production is involved in development, immunity and reproduction^3-5. However, phospholipid synthesis is highly intricate⁶. It is thus likely that steady-state depletion of PI(4,5)P₂ triggers confounding indirect effects. Furthermore, inducible tools available in plants allow PI(4,5)P₂ to increase^7-9 but not decrease, and no PIP5K inhibitors are available. Here, we introduce iDePP (inducible depletion of PI(4,5)P₂ in plants), a system for the inducible and tunable depletion of PI(4,5)P₂ in plants in less than three hours. Using this strategy, we confirm that PI(4,5)P₂ is critical for various aspects of plant development, including root growth, root-hair elongation and organ initiation. We show that PI(4,5)P₂ is required to recruit various endocytic proteins, including AP2-µ, to the plasma membrane, and thus to regulate clathrin-mediated endocytosis. Finally, we find that inducible PI(4,5)P₂ perturbation impacts the dynamics of the actin cytoskeleton as well as microtubule anisotropy. Together, we propose that iDePP is a simple and efficient genetic tool to test the importance of PI(4,5)P₂ in given cellular or developmental responses, and also to evaluate the importance of this lipid in protein localization.

SUBMITTER: Doumane M

PROVIDER: S-EPMC7610831 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Inducible depletion of PI(4,5)P2 by the synthetic iDePP system in Arabidopsis.

Doumane Mehdi M Lebecq Alexis A Colin Léia L Fangain Aurélie A Stevens Floris D FD Bareille Joseph J Hamant Olivier O Belkhadir Youssef Y Munnik Teun T Jaillais Yvon Y Caillaud Marie-Cécile MC

Nature plants 20210517 5

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a low-abundance membrane lipid essential for plasma membrane function1,2. In plants, mutations in phosphatidylinositol 4-phosphate (PI4P) 5-kinases (PIP5K) suggest that PI(4,5)P2 production is involved in development, immunity and reproduction3-5. However, phospholipid synthesis is highly intricate6. It is thus likely that steady-state depletion of PI(4,5)P2 triggers confounding ...[more]

PMID: 34007035

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Project description:The cold- and menthol-sensitive transient receptor potential melastatin 8 (TRPM8) channel is important for both physiological temperature detection and cold allodynia. Activation of G-protein-coupled receptors (GPCRs) by proinflammatory mediators inhibits these channels. It was proposed that this inhibition proceeds via direct binding of G αq to the channel. TRPM8 requires the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2 or PIP2] for activity. However, it was claimed that a decrease in cellular levels of this lipid upon receptor activation does not contribute to channel inhibition. Here, we show that supplementing the whole-cell patch pipette with PI(4,5)P2 reduced inhibition of TRPM8 by activation of Gαq-coupled receptors in mouse dorsal root ganglion (DRG) neurons isolated from both sexes. Stimulating the same receptors activated phospholipase C (PLC) and decreased plasma membrane PI(4,5)P2 levels in these neurons. PI(4,5)P2 also reduced inhibition of TRPM8 by activation of heterologously expressed muscarinic M1 receptors. Coexpression of a constitutively active G αq protein that does not couple to PLC inhibited TRPM8 activity, and in cells expressing this protein, decreasing PI(4,5)P2 levels using a voltage-sensitive 5'-phosphatase induced a stronger inhibition of TRPM8 activity than in control cells. Our data indicate that, upon GPCR activation, G αq binding reduces the apparent affinity of TRPM8 for PI(4,5)P2 and thus sensitizes the channel to inhibition induced by decreasing PI(4,5)P2 levels.SIGNIFICANCE STATEMENT Increased sensitivity to heat in inflammation is partially mediated by inhibition of the cold- and menthol-sensitive transient receptor potential melastatin 8 (TRPM8) ion channels. Most inflammatory mediators act via G-protein-coupled receptors that activate the phospholipase C pathway, leading to the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. How receptor activation by inflammatory mediators leads to TRPM8 inhibition is not well understood. Here, we propose that direct binding of G αq both reduces TRPM8 activity and sensitizes the channel to inhibition by decreased levels of its cofactor, PI(4,5)P2 Our data demonstrate the convergence of two downstream effectors of receptor activation, G αq and PI(4,5)P2 hydrolysis, in the regulation of TRPM8.

Dataset Information

Inducible depletion of PI(4,5)P₂ by the synthetic iDePP system in Arabidopsis.

Publications

Inducible depletion of PI(4,5)P<sub>2</sub> by the synthetic iDePP system in Arabidopsis.

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Dataset Information

Inducible depletion of PI(4,5)P2 by the synthetic iDePP system in Arabidopsis.

Publications

Inducible depletion of PI(4,5)P&lt;sub&gt;2&lt;/sub&gt; by the synthetic iDePP system in Arabidopsis.

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OmicsDI is part of the ELIXIR infrastructure

Tweets

Inducible depletion of PI(4,5)P₂ by the synthetic iDePP system in Arabidopsis.

Inducible depletion of PI(4,5)P<sub>2</sub> by the synthetic iDePP system in Arabidopsis.