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.

Polyphosphoinositides (PPIs) are essential phospholipids located in the cytoplasmic leaflet of eukaryotic cell membranes. Despite contributing only a small fraction to the bulk of cellular phospholipids, they make remarkable contributions to practically all aspects of a cell's life and death. They do so by recruiting cytoplasmic proteins/effectors or by interacting with cytoplasmic domains of membrane proteins at the membrane–cytoplasm interface to organize and mold organelle identity. The present study summarizes aspects of our current understanding concerning the metabolism, manipulation, measurement, and intimate roles these lipids play in regulating membrane homeostasis and vital cell signaling reactions in health and disease.

Endoplasmic reticulum–plasma membrane (ER–PM) contact sites play an integral role in cellular processes such as excitation–contraction coupling and store-operated calcium entry (SOCE). Another ER–PM assembly is one tethered by the extended synaptotagmins (E-Syt). We have discovered that at steady state, E-Syt2 positions the ER and Sac1, an integral ER membrane lipid phosphatase, in discrete ER–PM junctions. Here, Sac1 participates in phosphoinositide homeostasis by limiting PM phosphatidylinositol 4-phosphate (PI(4)P), the precursor of PI(4,5)P2. Activation of G protein–coupled receptors that deplete PM PI(4,5)P2 disrupts E-Syt2–mediated ER–PM junctions, reducing Sac1’s access to the PM and permitting PM PI(4)P and PI(4,5)P2 to recover. Conversely, depletion of ER luminal calcium and subsequent activation of SOCE increases the amount of Sac1 in contact with the PM, depleting PM PI(4)P. Thus, the dynamic presence of Sac1 at ER–PM contact sites allows it to act as a cellular sensor and controller of PM phosphoinositides, thereby influencing many PM processes.