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

Abstract

Phosphatidylinositol 4-phosphate (PtdIns(4)P) is the immediate precursor of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂), which is located on the cytoplasmic leaflet of the plasma membrane and has been reported to possess multiple cellular functions. Although PtdIns(4)P and PtdIns(4,5)P₂ 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 PtdIns(4)P and PtdIns(4,5)P₂ distributions, at a nanoscale, we employed an electron microscopy technique that specifically labels PtdIns(4)P and PtdIns(4,5)P₂ 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 PtdIns(4)P was localized to the cytoplasmic leaflet of Golgi apparatus and vesicular-shaped structures. The PtdIns(4,5)P₂ labeling was observed in the cytoplasmic leaflet of the mitochondrial inner membrane and vesicular-shaped structures. Double labeling of PtdIns(4)P and PtdIns(4,5)P₂ with endosome markers illustrated that PtdIns(4)P and PtdIns(4,5)P₂ were mainly localized to the late endosome/lysosome and early endosome, respectively. PtdIns(4)P and PtdIns(4,5)P₂ 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 PtdIns(4)P- and PtdIns(4,5)P₂-enriched microdomains in the endosome, of likely importance for endosome functionality.

Keywords: Electron microscopy; Endosome; Freeze-fracture; Lipid; Mitochondria; Nanometer scale.