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Phosphoinositides pp 91–104Cite as

Imaging the Nanoscale Distribution of Phosphoinositides in the Cell Plasma Membrane with Single-Molecule Localization Super-Resolution Microscopy

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2251))

Abstract

Phosphoinositides make up only a small fraction of cellular phospholipids yet control cell function in a fundamental manner. Through protein interactions, phosphoinositides define cellular organelle identity and regulate protein function and organization and recruitment at the cytosol–membrane interface. As a result, perturbations on phosphoinositide metabolism alter cell physiology and lead to a wide range of human diseases, including cancer and diabetes. Among seven phosphoinositide members, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2, also known as PI(4,5)P2 or PIP2) is abundant in the plasma membrane. Besides its role in the second messenger pathway of phospholipase C that cleaves PtdIns(4,5)P2 to form diacylglycerol and inositol-1,4,5-trisphosphate (IP3), PtdIns(4,5)P2 regulates membrane trafficking and the function of the cytoskeleton, ion channels, and transporters. The nanoscale organization of PtdIns(4,5)P2 in the plasma membrane becomes essential to understand cellular signaling specificity in time and space. Here, we describe a single-molecule method to visualize the nanoscale distribution of PtdIns(4,5)P2 in the plasma membrane by using super-resolution microscopy and the dual-color fluorescent probes based on the PLCδ1 pleckstrin homology (PH) domain. This approach can be extended to image other phosphoinositides by changing the specific probes.

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Acknowledgments

This work is supported partially by the National Institutes of Health (NIH) (R01DK093953 and R21NS101584) and the grant AAB1425-135-A5362. We thank Tamas Balla at NIH and Pietro De Camilli at Yale for the PH-domain constructs and Dr. Christopher Newgard at Duke for INS-1 832/13 cells.

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  1. Chen Ji

    Present address: National Institute of Health, Bethesda, MD, USA

Authors and Affiliations

  1. Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA

    Fan Fan, Chen Ji & Xuelin Lou

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  1. Fan Fan
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  2. Chen Ji
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Correspondence to Xuelin Lou .

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Editors and Affiliations

  1. Department of Chemistry and Biology and the Graduate Program in Molecular Science, Ryerson University, Toronto, ON, Canada

    Roberto J. Botelho

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Fan, F., Ji, C., Lou, X. (2021). Imaging the Nanoscale Distribution of Phosphoinositides in the Cell Plasma Membrane with Single-Molecule Localization Super-Resolution Microscopy. In: Botelho, R.J. (eds) Phosphoinositides. Methods in Molecular Biology, vol 2251. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1142-5_6

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  • DOI: https://doi.org/10.1007/978-1-0716-1142-5_6

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  • Publisher Name: Humana, New York, NY

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