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In situ quantitative imaging of cellular lipids using molecular sensors

Abstract

Membrane lipids are dynamic molecules that play important roles in cell signalling and regulation, but an in situ imaging method for quantitatively tracking lipids in living cells is lacking at present. Here, we report a new chemical method of quantitative lipid imaging using sensors engineered by labelling proteins with an environmentally sensitive fluorophore. A prototype sensor for phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2)—a key signalling lipid in diverse cellular processes—was generated by covalently attaching a single 2-dimethylamino-6-acyl-naphthalene group to the N-terminal α-helix of the engineered epsin1 ENTH domain, a protein that selectively binds PtdIns(4,5)P2. The sensor allows robust and sensitive in situ quantitative imaging in mammalian cells, providing new insight into the spatiotemporal dynamics and fluctuation of this key signalling lipid. Application of the sensor to immune cells reveals the presence of a local threshold PtdIns(4,5)P2 concentration required for triggering phagocytosis. This sensor strategy is generally applicable to in situ quantification of other cellular lipids.

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Figure 1: Structure and spectral properties of the PtdIns(4,5)P2 sensor, DAN–eENTH.
Figure 2: In vitro PtdIns(4,5)P2 calibration curves of DAN–eENTH.
Figure 3: In situ quantification of PtdIns(4,5)P2 in a representative NIH 3T3 cell by DAN–eENTH.
Figure 4: Monitoring PtdIns(4,5)P2 decrease in NIH 3T3 cells using a rapamycin-induced PtdIns(4,5)P2 depletion system.
Figure 5: In situ quantification of PtdIns(4,5)P2 in immune cells, macrophages, during phagocytosis (cell engulfing).

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Acknowledgements

This study was supported by the National Institutes of Health (grant no. GM68849). It was also supported in part by the Chicago Biomedical Consortium with support from the Searl Funds at the Chicago Community Trust. The authors thank I. Kim, S.-Y. Kim, S. Bhattacharjee, Y. Kanaho and L.-W. Gong for technical assistance in phagocytosis and membrane trafficking experiments, and T. Balla for the generous gift of the PtdIns(4,5)-depletion system.

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W.C. and Y.Y. conceived the lipid sensor strategy. W.C. supervised the project and Y.Y. designed and prepared the sensor. P.J.L. and S.K. performed all microscopy imaging and image analysis. Y.Y. and P.J.L. contributed equally to this work.

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Correspondence to Wonhwa Cho.

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The authors declare no competing financial interests.

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Yoon, Y., Lee, P., Kurilova, S. et al. In situ quantitative imaging of cellular lipids using molecular sensors. Nature Chem 3, 868–874 (2011). https://doi.org/10.1038/nchem.1163

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