Visualization and manipulation of phosphoinositide dynamics in live cells using engineered protein domains.
ABSTRACT There is hardly a membrane-associated molecular event that is not regulated by phosphoinositides, a minor but critically important class of phospholipids of cellular membranes. The rapid formation, elimination, and conversion of these lipids in specific membrane compartments are ensured by a wealthy number of inositol lipid kinases and phosphatases with unique localization and regulatory properties. The existence of multiple inositol lipid pools have been indicated by metabolic labeling studies, but the level of functional compartmentalization revealed by the identification of numerous protein effectors acted upon by phosphoinositides could not have been foreseen. The changing perception of inositides from just serving as lipid precursors of second messengers to becoming highly dynamic local membrane-bound regulators poses new challenges concerning the detection of their rapid localized changes. Moreover, it is increasingly evident that manipulation of lipids in highly defined compartments would be a highly superior approach to soaking the cells with a particular phosphoinositide when studying the local regulation of the lipid on any effectors. In this review, we will summarize our efforts to improve our tools in studying phosphoinositide dynamics and discuss our views on the values of these methods compared to other options currently used or being explored.
SourceAvailable from: Olof Idevall-Hagren[Show abstract] [Hide abstract]
ABSTRACT: Phosphoinositides, the phosphorylated products of inositol phospholipids, play critical regulatory roles in cell physiology. The elucidation of their functions will greatly benefit from the methodology to manipulate their local concentrations within membranes with high spatial and temporal precision. Recently developed genetically encoded and light-regulated dimerization modules, in combination with the use of fluorescence-tagged lipid-binding domains and live-cell imaging, provide an attractive means to achieve this goal. Here we describe a protocol for blue light-dependent conversion of one phosphoinositide species into another based on the light-regulated dimerization between cryptochrome 2 (CRY2) and its ligand, CIB1. We describe the development of these tools using the dephosphorylation of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) as an example and show how they can be used to rapidly and reversibly deplete the plasma membrane of this lipid. We also provide instructions for image analysis. The CRY2-CIB1 dimerization method has also already been adapted for the acute and spatially restricted generation of PI(3,4,5)P3 in the plasma membrane. More generally, this methodology should be broadly applicable to studies of the spatiotemporal regulation of membrane lipid metabolism in many types of cells.Methods in molecular biology (Clifton, N.J.) 01/2014; 1148:109-28. DOI:10.1007/978-1-4939-0470-9_8 · 1.29 Impact Factor
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ABSTRACT: Chemically inducible rapid manipulation of small GTPase activity has proven a powerful approach to dissect complex spatiotemporal signaling of these molecular switches. However, overexpression of these synthetic molecular probes freely in the cytosol often results in elevated background activity before chemical induction, which perturbs the cellular basal state and thereby limits their wide application. As a fundamental solution, we have rationally designed and newly developed a strategy to remove unwanted background activity without compromising the extent of induced activation. By exploiting interaction between a membrane lipid and its binding protein, target proteins were translocated from one organelle to another on a time scale of seconds. This improved strategy now allows for rapid manipulation of small GTPases under a physiological state, thus enabling fine dissection of sophisticated signaling processes shaped by these molecules.ACS Chemical Biology 09/2012; 7(12):1950–1955. DOI:10.1021/cb300280k · 5.36 Impact Factor
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ABSTRACT: Local accumulation of phosphoinositides (PIPs) is an important factor for a broad range of cellular events including membrane trafficking and cell signaling. The negatively charged phosphoinositide headgroups can interact with cations or cationic proteins and this electrostatic interaction has been identified as the main phosphoinositide clustering mechanism. However, an increasing number of reports show that phosphoinositide-mediated signaling events are at least in some cases cholesterol dependent, suggesting other possible contributors to the segregation of phosphoinositides. Using fluorescence microscopy on giant unilamellar vesicles and monolayers at the air/water interface, we present data showing that cholesterol stabilizes fluid phosphoinositide-enriched phases. The interaction with cholesterol is observed for all investigated phosphoinositides (PI(4)P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2 and PI(3,4,5)P3) as well as phosphatidylinositol. We find that cholesterol is present in the phosphoinositide-enriched phase and that the resulting phase is fluid. Cholesterol derivatives modified at the hydroxyl group (cholestenone, cholesteryl ethyl ether) do not promote formation of phosphoinositide domains, suggesting an instrumental role of the cholesterol hydroxyl group in the observed cholesterol/phosphoinositide interaction. This leads to the hypothesis that cholesterol participates in an intermolecular hydrogen bond network formed among the phosphoinositide lipids. We had previously reported that the intra- and intermolecular hydrogen bond network between the phosphoinositide lipids leads to a reduction of the charge density at the phosphoinositide phosphomonoester groups (Kooijman et al. Biochemistry 48, (2009) 9360). We believe that cholesterol acts as a spacer between the phosphoinositide lipids, thereby reducing the electrostatic repulsion, while participating in the hydrogen bond network, leading to its further stabilization. To illustrate the effect of phosphoinositide segregation on protein binding, we show that binding of the tumor suppressor protein PTEN to PI(5)P and PI(4,5)P2 is enhanced in the presence of cholesterol. These results provide new insights into how phosphoinositides mediate important cellular events.Chemistry and Physics of Lipids 09/2014; DOI:10.1016/j.chemphyslip.2014.02.003 · 2.59 Impact Factor