Spatial and temporal aspects of phosphoinositides in endocytosis studied in the isolated plasma membranes

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)P 2 ]. 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)P 2 spatial distribution, and semi-quantification of PI(4,5)P 2 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.