Simultaneous In Situ Quantification of Two Cellular Lipid Pools Using Orthogonal Fluorescent Sensors

Abstract

Keywordslipids; sensors; quantitative imaging; phosphatidylserine; phosphatidylinositol-(3,4,5)-trisphosphate Membrane lipids are one of the most important and ubiquitous regulatory molecules that control the localization, activity, and mutual interactions of a wide variety of cellular proteins. [1] Because local lipid concentrations are highly variable and may serve as activation thresholds for myriad biological processes mediated by these proteins, spatiotemporally resolved lipid quantification is essential for elucidating the diverse and complex mechanisms of biological regulation. [2] We recently developed a chemical strategy for in situ quantification of a single lipid species in live mammalian cells using a hybrid sensor constructed with an engineered lipid binding protein and an environmentally sensitive (or solvatochromatic) fluorophore (ESF). [2] Quantification of cellular phosphatidylinositol-4,5-bisphosphate (PIP 2 ) by ratiometric imaging analysis demonstrated the spatiotemporal dynamics of this important signaling lipid in unprecedented details and provided new insight into how it regulates such diverse biological processes. [2] Under physiological conditions, multiple regulatory lipids are metabolically and functionally linked to one another. [3] Also, a single lipid species can exist disproportionally in opposite faces of lipid bilayers, performing distinct functions. [4] Most notably, tightly controlled transbilayer asymmetry of lipids in the plasma membrane (PM) of mammalian cells is crucial for cell survival, function, and regulation. [4] Thus, a new technology is needed for simultaneous in situ quantification of multiple lipids in the same membrane leaflet or a lipid species in two opposite leaflets of cell membranes. As a first step toward simultaneous quantification of multiple lipids, we developed a new strategy for dual in situ lipid quantification in live cells.Simultaneous dual lipid quantification would require orthogonal lipid sensors that allow robust dual ratiometric analysis. Unfortunately, limited availability of amphiphilic ESFs

Structural Basis for Cholesterol Transport-like Activity of the Hedgehog Receptor Patched

“…Detailed synthesis and spectral characterization of WCRX will be published elsewhere. All sensor calibration, microscopy measurements, and ratiometric imaging data analysis were performed as described (Liu et al, 2017;Liu et al, 2014;Yoon et al, 2011). In brief, YDA-WCRX and D4-DAN were calibrated using giant unilamellar vesicles whose lipid compositions recapitulate those of IPM (i.e., 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)/ 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS)/ liver phosphatidylinositol (PI)/cholesterol/ 1,2-dipalitoyl-phosphatidylinositol-4,5-bisphosphate (PI45P 2 ) (20/50-x/20/9/x/1: x = 0-20 mole%) and OPM (i.e., POPC/ porcine brain sphingomyelin (SM)/cholesterol (70-x/30/x: x = 0-60 mole%), respectively, with a custom-modified six-channel FV3000 (Olympus) confocal laser scanning microscope.…”

ORP2, a cholesterol transporter, regulates angiogenic signaling in endothelial cells

“…The PI(4,5)P 2 sensor was delivered into the cells by microinjection and PI(4,5)P 2 in the inner leaflets of the PM was quantified using a custom‐modified Olympus FV3000 confocal microscope as described previously 20 . All data acquisition and ratiometric imaging data analyses were performed as described in 20‐22 . The PI(4,5)P 2 concentration at the PM of a single HUVEC cell was determined in a spatiotemporally resolved manner and spatially averaged at a given time.…”

“…20 All data acquisition and ratiometric imaging data analyses were performed as described in. [20][21][22] The PI(4,5)P 2 concentration at the PM of a single HUVEC cell was determined in a spatiotemporally resolved manner and spatially averaged at a given time. Each data set was collected from a minimum of 15 different cells and averaged.…”

A fluorescent acrylamide-type monomer bearing an environment-sensitive methoxybenzocoumarin structure for the development of functional polymeric sensors

“…Intracellular thermometry can now be performed with these fluorescent polymeric thermometers. 6,9 Fluorescent polymeric sensors for potassium ions, 10 sulfate ions, 11 phosphatidylinositol-4,5-bisphosphate, 12 and phosphatidylserine 13 are also examples of macromolecular materials created from fluorescent monomers containing an environment-sensitive fluorophore. The use of some of these sensors 12,13 has been explored to monitor biologically important molecules in live cell imaging.…”

“…6,9 Fluorescent polymeric sensors for potassium ions, 10 sulfate ions, 11 phosphatidylinositol-4,5-bisphosphate, 12 and phosphatidylserine 13 are also examples of macromolecular materials created from fluorescent monomers containing an environment-sensitive fluorophore. The use of some of these sensors 12,13 has been explored to monitor biologically important molecules in live cell imaging. It should be noted that all of the fluorescent monomers in the functional polymers described above bear an environment-sensitive fluorophore, which shows stronger fluorescence in less protic, polar environments (e.g., in an aprotic organic solvent, such as ethyl acetate).…”