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Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol

Nature Chemical Biology volume 13, pages 268–274 (2017)Cite this article

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Abstract

Controlled distribution of lipids across various cell membranes is crucial for cell homeostasis and regulation. We developed an imaging method that allows simultaneous in situ quantification of cholesterol in two leaflets of the plasma membrane (PM) using tunable orthogonal cholesterol sensors. Our imaging revealed marked transbilayer asymmetry of PM cholesterol (TAPMC) in various mammalian cells, with the concentration in the inner leaflet (IPM) being ∼12-fold lower than that in the outer leaflet (OPM). The asymmetry was maintained by active transport of cholesterol from IPM to OPM and its chemical retention at OPM. Furthermore, the increase in the IPM cholesterol level was triggered in a stimulus-specific manner, allowing cholesterol to serve as a signaling lipid. We found excellent correlation between the IPM cholesterol level and cellular Wnt signaling activity, suggesting that TAPMC and stimulus-induced PM cholesterol redistribution are crucial for tight regulation of cellular processes under physiological conditions.

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Figure 1: Simultaneous quantification of OPM ([Chol]o) and IPM cholesterol ([Chol]i) of HeLa cells by orthogonal cholesterol sensors.
Figure 2: Stimulus-induced increases in [Chol]i (Δ[Chol]i) in HeLa cells.
Figure 3: Mechanisms for PM transbilayer asymmetry of cholesterol and its physiological relevance.

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Acknowledgements

We thank A. Heuck (University of Massachusetts) for a kind gift of the D4 domain construct and P. Subbaiah, T. Steck and Y. Lange for helpful discussion. This work was supported by the grants from the US National Institutes of Health (GM68849 and GM110128 to W.C. and HL-073965 and HL-083298 to I.L.) and from the Japan Society for the Promotion of Science (25221203 to K.U.).

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Author notes

  1. Shu-Lin Liu and Ren Sheng: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois, USA

    Shu-Lin Liu, Ren Sheng, Li Wang, Ewa Stec, Matthew J O'Connor, Seohyoen Song, Daesung Lee & Wonhwa Cho

  2. Department of Applied Chemistry, Kyung Hee University, Yongin, Korea

    Jae Hun Jung & Kwang-Pyo Kim

  3. Department of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA

    Rama Kamesh Bikkavilli, Robert A Winn & Irena Levitan

  4. Department of Genetic Engineering, Kyung Hee University, Yongin, Korea

    Kwanghee Baek & Wonhwa Cho

  5. and Division of Applied Life Sciences, Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, Japan

    Kazumitsu Ueda

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  1. Shu-Lin Liu
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Contributions

R.S. contributed to sensor development and other biochemical studies and S.-L.L. performed all imaging work. L.W., S.S., R.K.B., R.A.W. and I.L. contributed to cell studies. J.H.J., K.B. and K.-P.K. performed MS analysis and K.U. contributed to lipid transporter studies. M.J.O'C. and D.L. prepared fluorophores. E.S. participated in sensor preparation. W.C. conceived the work and wrote the paper.

Corresponding author

Correspondence to Wonhwa Cho.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–3, and Supplementary Figures 1–6. (PDF 1908 kb)

Supplementary Video 1

A 300x time-lapse video of spatiotemporal [Chol]o fluctuation. (MOV 163 kb)

Supplementary Video 2

A 300x time-lapse video of spatiotemporal [Chol]i fluctuation. (MOV 103 kb)

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Liu, SL., Sheng, R., Jung, J. et al. Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol. Nat Chem Biol 13, 268–274 (2017). https://doi.org/10.1038/nchembio.2268

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