Segregation of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate into distinct microdomains on the endosome membrane

Phosphatidylinositol 4-phosphate (PtdIns(4)P) is the immediate precursor of phosphatidylinositol 4,5-bisphosphate (Ptdlns(4,5)P-2), which is located on the cytoplasmic leaflet of the plasma membrane and has been reported to possess multiple cellular functions. Although Ptdlns(4)P and Ptdlns(4,5)P-2 have been reported to localize to multiple intracellular compartments and to each function as regulatory molecules, their generation, regulation and functions in most intracellular compartments are not well-defined. To analyze Ptdlns(4)P and Ptdlns(4,5)P-2 distributions, at a nanoscale, we employed an electron microscopy technique that specifically labels Ptdlns(4)P and Ptdlns(4,5)P-2 on the freeze-fracture replica of intracellular biological membranes. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilized in situ. Using this technique, we found that Ptdlns(4)P was localized to the cytoplasmic leaflet of Golgi apparatus and vesicular-shaped structures. The Ptdlns(4,5)P-2 labeling was observed in the cytoplasmic leaflet of the mitochondrial inner membrane and vesicular-shaped structures. Double labeling of Ptdlns(4)P and Ptdlns(4,5)P-2 with endosome markers illustrated that Ptdlns(4)P and Ptdlns(4,5)P-2 were mainly localized to the late endosome/lysosome and early endosome, respectively. Ptdlns(4)P and Ptdlns(4,5)P-2 were colocalized in some endosomes, with the two phospholipids separated into distinct microdomains on the same endosomes. This is the first report showing, at a nanoscale, segregation of Ptdlns(4)P- and Ptdlns(4,5)P-2-enriched microdomains in the endosome, of likely importance for endosome functionality. (C) 2017 Elsevier B.V. All rights reserved.