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Detection of Plasma Membrane Phosphoinositide Dynamics Using Genetically Encoded Fluorescent Protein Probes

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Phosphoinositides

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

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Abstract

The dynamic phosphorylation of phosphatidylinositol produces seven distinct but interconvertible phosphatidylinositol phosphates (PIPs). Each PIP exhibits specific enrichment in a subset of membrane compartments as a result of dynamic phosphorylation and dephosphorylation by lipid kinases and phosphatases, and/or by vesicle-mediated transport. Several PIPs are found within the plasma membrane, such as phosphatidylinositol-4-phosphate [PI(4)P], phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2], and phosphatidylinositol-3,4,5-trisphosphate (PIP3), and these control many aspects of cell physiology, including receptor signaling and membrane traffic. As a result, measurement of the cell surface abundance of these PIPs is a valuable resource to allow understanding of the regulation and function of these cell surface lipids. Here, we describe methods based on quantification of the localization of genetically encoded fluorescent PIP probes to the cell surface by either spinning disc confocal microscopy or total internal reflection fluorescence microscopy that allow detection of changes in cell surface levels of PI(4,5)P2, PI(3,4)P2, and PIP3. These methods can also be applied to the measurement of other PIPs or lipid species at the cell surface, and thus represent a useful resource for the study of the cell biology of PIPs.

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Authors and Affiliations

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

    Rebecca Cabral-Dias, Yasmin Awadeh, Roberto J. Botelho & Costin N. Antonescu

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  1. Rebecca Cabral-Dias
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  2. Yasmin Awadeh
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  3. Roberto J. Botelho
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  4. Costin N. Antonescu
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Corresponding author

Correspondence to Costin N. Antonescu .

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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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Cabral-Dias, R., Awadeh, Y., Botelho, R.J., Antonescu, C.N. (2021). Detection of Plasma Membrane Phosphoinositide Dynamics Using Genetically Encoded Fluorescent Protein Probes. 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_5

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  • DOI: https://doi.org/10.1007/978-1-0716-1142-5_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1141-8

  • Online ISBN: 978-1-0716-1142-5

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