Deciphering many roles played by inositol lipids in signal transduction and membrane function demands experimental approaches that can detect their dynamic accumulation with subcellular accuracy and exquisite sensitivity. The former criterion is met by imaging of fluorescence biosensors in living cells, whereas the latter is facilitated by biochemical measurements from populations. Here, we introduce BRET-based biosensors able to detect rapid changes in inositol lipids in cell populations with both high sensitivity and subcellular resolution in a single, convenient assay. We demonstrate robust and sensitive measurements of PtdIns4P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics, as well as changes in cytoplasmic Ins(1,4,5)P3 levels. Measurements were made during either experimental activation of lipid degradation, or PI 3-kinase and phospholipase C mediated signal transduction. Our results reveal a previously unappreciated synthesis of PtdIns4P that accompanies moderate activation of phospholipase C signaling downstream of both EGF and muscarinic M3 receptor activation. This signaling-induced PtdIns4P synthesis relies on protein kinase C, and implicates a feedback mechanism in the control of inositol lipid metabolism during signal transduction.

SUMMARY One of the largest challenges in cell biology is to map the lipid composition of the membranes of various organelles and define the exact location of processes that control the synthesis and distribution of lipids between cellular compartments. The critical role of phosphoinositides, low-abundant lipids with rapid metabolism and exceptional regulatory importance in the control of almost all aspects of cellular functions created the need for tools to visualize their localizations and dynamics at the single cell level. However, there is also an increasing need for methods to determine the cellular distribution of other lipids regulatory or structural, such as diacylglycerol, phosphatidic acid, or other phospholipids and cholesterol. This review will summarize recent advances in this research field focusing on the means by which changes can be described in more quantitative terms.

Receptor endocytosis plays an important role in regulating the responsiveness of cells to specific ligands. Phosphatidylinositol 4,5-bisphosphate (PtdInsP2) has been shown to be critical for endocytosis of some cell surface receptors, such as EGF and transferrin receptor, but its role in G protein-coupled receptor internalization has not been investigated. By employing luciferase-labeled type 1 angiotensin II (AT1), type 2C serotonin (5HT2C) or β2 adrenergic (β2A) receptors and fluorescently tagged proteins (β-arrestin 2, plasma membrane targeted Venus, Rab5) we were able to follow the sequence of molecular interactions along the endocytic route of the receptors in HEK 293 cells using the highly sensitive method of bioluminescence resonance energy transfer and confocal microscopy. To study the role of plasma membrane PtdInsP2 in receptor endocytosis, we used our previously developed rapamycin-inducible heterodimerization system, in which the recruitment of a 5-phosphatase domain to the plasma membrane degrades PtdInsP2. Here we show that ligand-induced interaction of AT1, 5HT2C and β2A receptors with β-arrestin 2 was unaffected by PtdInsP2 depletion. However, arrival of the receptors to Rab5-positive early endosomes was completely abolished in the absence of PtdInsP2. Remarkably, removal of the receptors from the plasma membrane was reduced but not eliminated after PtdInsP2 depletion. Under these conditions, stimulated AT1 receptors clustered along the plasma membrane but did not enter the cells. Our data suggest that in the absence of PtdInsP2, these receptors move into clathrin-coated membrane structures, but these are not cleaved efficiently and hence cannot reach the early endosomal compartment.

Oxysterol-binding protein (OSBP)-related proteins (ORPs) mediate non-vesicular lipid transfer between intracellular membranes. Phosphoinositide (PI) gradients play important roles in the ability of OSBP and some ORPs to transfer cholesterol and phosphatidylserine between the endoplasmic reticulum (ER) and other organelle membranes. Here, we show that plasma membrane (PM) association of ORP3 (also known as OSBPL3), a poorly characterized ORP family member, is triggered by protein kinase C (PKC) activation, especially when combined with Ca 2+ increases, and is determined by both PI(4,5)P 2 and PI4P. After activation, ORP3 efficiently extracts PI4P and to a lesser extent phosphatidic acid from the PM, and slightly increases PM cholesterol levels. Full activation of ORP3 resulted in decreased PM PI4P levels and inhibited Ca 2+ entry via the store-operated Ca 2+ entry pathway. The C-terminal region of ORP3 that follows the strictly defined lipid transfer domain was found to be critical for the proper localization and function of the protein.