Clathrin-Mediated Endoytosis

Ira Milosevic, Milosevic, Ira

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.