title: Simultaneous in situ quantification of two cellular lipid pools using orthogonal fluorescent sensors.
abstract: Lipids regulate a wide range of biological activities. Since their local concentrations are tightly controlled in a spatiotemporally specific manner, the simultaneous quantification of multiple lipids is essential for elucidation of the complex mechanisms of biological regulation. Here, we report a new method for the simultaneous in situ quantification of two lipid pools in mammalian cells using orthogonal fluorescent sensors. The sensors were prepared by incorporating two environmentally sensitive fluorophores with minimal spectral overlap separately into engineered lipid-binding proteins. Dual ratiometric analysis of imaging data allowed accurate, spatiotemporally resolved quantification of two different lipids on the same leaflet of the plasma membrane or a single lipid on two opposite leaflets of the plasma membrane of live mammalian cells. This new imaging technology should serve as a powerful tool for systems-level investigation of lipid-mediated cell signaling and regulation.
authors.affiliation: Department of Chemistry, University of Illinois at Chicago, 845 W. Taylor St., Chicago, IL (USA).
authors.forename: Shu-Lin
authors.initials: SL
authors.lastname: Liu
authors.forename: Ren
authors.initials: R
authors.lastname: Sheng
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: O'Connor
authors.forename: Yang
authors.initials: Y
authors.lastname: Cui
authors.forename: Youngdae
authors.initials: Y
authors.lastname: Yoon
authors.forename: Svetlana
authors.initials: S
authors.lastname: Kurilova
authors.forename: Daesung
authors.initials: D
authors.lastname: Lee
authors.forename: Wonhwa
authors.initials: W
authors.lastname: Cho
mesh_terms: D000818:Animals; D002235:Carbofuran / Q000031:analogs & derivatives / Q000138:chemical synthesis; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D008055:Lipids / Q000737:chemistry; D051379:Mice; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D010078:Oxazines / Q000138:chemical synthesis / Q000737:chemistry; D051059:PTEN Phosphohydrolase / Q000737:chemistry / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D008055:Lipids; D010078:Oxazines; D019869:Phosphatidylinositol 3-Kinases; D051059:PTEN Phosphohydrolase; C044808:nile red; D002235:Carbofuran
keywords: imaging; lipids; membranes; sensors