Optogenetic Control of Phosphatidylinositol (3,4,5)-Triphosphate Production by Light-Sensitive Cryptochrome Proteins on the Plasma Membrane

Phosphatidylinositol(3,4,5)-triphosphate(PIP3),acting as a fundamental second messenger,is emerging as a promising biomarker for disease diagnosis and prognosis.However,the real time analysis of phosphoinositide in living cells remains a key challenge owing to the low basal abundance and its fast metabolic rate.Herein,we design an optogenetic system that uses light sensitive protein-protein interaction between Arabidopsis cryptochrome 2(CRY2)and CIB1 to spatiotemporally visualize the PIP3 production with sub-second timescale.In this system,a CIBN is anchored on the plasma membrane,whereas a CRY2 fused with a constitutively active PI3-kinase(acPI3K)would be driven from the cytosol to the membrane by the blue-light-activated CRY2-CIB1 interaction upon light irradiation.The PIP3 production is visualized via a fused fluorescent protein by the translocation of a Pleckstrin Homology(PH)domain(GRP1)from the cytosol to the plasma membrane with high specificity.We demonstrated the fast dynamics and reversibility of the optogenetic system initiated PIP3 synthesis on the plasma membrane.Notably,the real-time cell movements were also observed upon localized light stimulation.The established optogenetic method provides a novel spatiotemporal strategy for specific PIP3 visualization,which is beneficial to improve the understanding of PIP3 functions.