Optogenetic control of phosphoinositide metabolism

Phosphoinositides (PIs) are lipid components of cell membranes that regulate a wide variety of cellular functions. Here we exploited the blue light-induced dimerization between two plant proteins, cryptochrome 2 (CRY2) and the transcription factor CIBN, to control plasma membrane PI levels rapidly,...

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Published in:Proceedings of the National Academy of Sciences - PNAS Vol. 109; no. 35; p. E2316
Main Authors: Idevall-Hagren, Olof, Dickson, Eamonn J, Hille, Bertil, Toomre, Derek K, De Camilli, Pietro
Format: Journal Article
Language:English
Published: United States 28.08.2012
Subjects:
Actins - metabolism
Amino Acid Motifs - physiology
Animals
Arabidopsis Proteins - genetics
Arabidopsis Proteins - metabolism
Binding Sites - physiology
Cell Membrane - metabolism
Cell Membrane - radiation effects
Cercopithecus aethiops
COS Cells
Cryptochromes - genetics
Cryptochromes - metabolism
Endocytosis - physiology
Endocytosis - radiation effects
Humans
KCNQ2 Potassium Channel - physiology
KCNQ3 Potassium Channel - physiology
Light
Membrane Potentials - physiology
Membrane Potentials - radiation effects
PC12 Cells
Phosphatidylinositol 3-Kinases - metabolism
Phosphatidylinositol 4,5-Diphosphate - metabolism
Phosphatidylinositols - metabolism
Phosphoric Monoester Hydrolases - genetics
Phosphoric Monoester Hydrolases - metabolism
Phosphorylation - physiology
Phosphorylation - radiation effects
Rats
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Signal Transduction - physiology
Signal Transduction - radiation effects
ISSN:1091-6490, 1091-6490
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Summary:Phosphoinositides (PIs) are lipid components of cell membranes that regulate a wide variety of cellular functions. Here we exploited the blue light-induced dimerization between two plant proteins, cryptochrome 2 (CRY2) and the transcription factor CIBN, to control plasma membrane PI levels rapidly, locally, and reversibly. The inositol 5-phosphatase domain of OCRL (5-ptase(OCRL)), which acts on PI(4,5)P(2) and PI(3,4,5)P(3), was fused to the photolyase homology region domain of CRY2, and the CRY2-binding domain, CIBN, was fused to plasma membrane-targeting motifs. Blue-light illumination (458-488 nm) of mammalian cells expressing these constructs resulted in nearly instantaneous recruitment of 5-ptase(OCRL) to the plasma membrane, where it caused rapid (within seconds) and reversible (within minutes) dephosphorylation of its targets as revealed by diverse cellular assays: dissociation of PI(4,5)P(2) and PI(3,4,5)P(3) biosensors, disappearance of endocytic clathrin-coated pits, nearly complete inhibition of KCNQ2/3 channel currents, and loss of membrane ruffling. Focal illumination resulted in local and transient 5-ptase(OCRL) recruitment and PI(4,5)P(2) dephosphorylation, causing not only local collapse and retraction of the cell edge or process but also compensatory accumulation of the PI(4,5)P(2) biosensor and membrane ruffling at the opposite side of the cells. Using the same approach for the recruitment of PI3K, local PI(3,4,5)P(3) synthesis and membrane ruffling could be induced, with corresponding loss of ruffling distally to the illuminated region. This technique provides a powerful tool for dissecting with high spatial-temporal kinetics the cellular functions of various PIs and reversibly controlling the functions of downstream effectors of these signaling lipids.
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ISSN:1091-6490
1091-6490
DOI:10.1073/pnas.1211305109