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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.

SUBMITTER: Liu SL

PROVIDER: S-EPMC4432842 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Simultaneous in situ quantification of two cellular lipid pools using orthogonal fluorescent sensors.

Liu Shu-Lin SL Sheng Ren R O'Connor Matthew J MJ Cui Yang Y Yoon Youngdae Y Kurilova Svetlana S Lee Daesung D Cho Wonhwa W

Angewandte Chemie (International ed. in English) 20141024 52

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 sensit ...[more]

PMID: 25345859

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Project description:Treatment of aqueous zinc solutions with incremental additions of a ditopic fluorescent sensor of the Zinpyr family, based on pyridine/pyrazine-containing metal recognition units, affords a fluorescence titration curve with a sharp maximum at a sensor:Zn(2+) ratio of 0.5 (Zhang, X-a.; Hayes, D.; Smith, S. J.; Friedle, S.; Lippard, S. J. J. Am. Chem. Soc.2008, 130, 15788-15789). This fluorescence response enables the quantification of readily chelatable zinc in biological samples by a simple titration protocol. In the present work a new set of ditopic fluorescence zinc sensors functionalized with pyridine/pyrazine-containing metal chelating units is described, and through detailed studies the principles governing the characteristic OFF-ON-OFF fluorescence behavior and quantification capabilities of the family are delineated. Incorporation of carboxylate/ester groups in the 6 position of the fluorescein allows for control of the spatial distribution of the sensor for selective extra- or intracellular imaging of mobile zinc, without introducing significant changes in zinc-binding properties. A combination of spectrophotometric and potentiometric measurements provided a complete description of the H(+)- and Zn(2+)-binding properties of the compounds and their correlation with the observed fluorescence profile. The first zinc-binding event has an apparent affinity, K(1)', of 1.9 × 10(9)-3.1 × 10(9) M(-1), whereas for coordination of the second Zn(2+) ion, responsible for fluorescence turn-on, the apparent formation constant, K(2)', is 5.5 × 10(7)-6.9 × 10(7) M(-1). A detailed chemical and mathematical analysis of the system demonstrated that the difference in emission efficiencies of the dimetalated (LZn(2)) vs monometalated (LZn) and metal-free (L) forms, a consequence of the combined quenching effects of the two metal-chelating units, significantly influences the shape of the titration curve. The scope of the titration method was investigated mathematically, and a lower boundary for the range of concentrations that can be determined was established as a function of the magnitude of K(2)'. Our results suggest that the principles governing the response of the ZPP1 series are applicable to other analogues of the Zinpyr family. Moreover, they may guide the design of other ditopic sensors suitable for determining the concentrations of a wide range of mobile metal ions and other chemical signaling agents of relevance in biological systems.

Dataset Information

Simultaneous in situ quantification of two cellular lipid pools using orthogonal fluorescent sensors.

Publications

Simultaneous in situ quantification of two cellular lipid pools using orthogonal fluorescent sensors.

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