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Spatial and Temporal Aspects of Phosphoinositides in Endocytosis Studied in the Isolated Plasma Membranes

  • Ira MilosevicEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1847)

Abstract

Endocytosis is a well-orchestrated cascade of lipid–protein and protein–protein interactions resulting in formation and internalization of vesicles. Membrane phospholipids have key regulatory functions in endocytosis and membrane traffic. I have previously described an in vitro assay based on the isolated, substrate-attached plasma membrane to study the spatial distribution and levels of phosphoinositides, in particular phosphatidylinositol-4,5-bisphospate [PI(4,5)P2]. This assay utilizes cultured cells subjected to a brief ultrasonic pulse, resulting in the formation of thin, flat inside-out plasma membrane sheets with elements of cell cytoskeleton. Here, I describe an experimental procedure for “on-stage” and “off-stage” detection of PI(4,5)P2 spatial distribution, and semi-quantification of PI(4,5)P2 levels in the plasma membrane using fluorescence microscopy. Depending on the probe selected for lipid detection, this simple assay can be modified to study other plasmalemmal phospholipids and/or proteins.

Key words

Endocytosis Phospholipids Plasma membrane Cell-free assay Isolated membrane sheets Phosphoinositides PI(4,5)P2 Lipid probe 

Notes

Acknowledgment

I thank Dr. A. Milosevic and Dr. M. Barszczewski for kind help with the figures, and Dr. N. Raimundo for comments. The author declares no competing financial interests. This work is supported by the grants of the German Research Foundation (DFG) through the collaborative research center SFB-889 (project A8) and SFB-1190 (project P02), and the Emmy Noether Young Investigator Award (1702/1).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.European Neuroscience Institute (ENI) and University Medical Center Göttingen (UMG)GöttingenGermany

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