Phosphatidylinositol-4,5-bisphosphate Affects Ceramide 1-phosphate Phase Behavior

Abstract

Ceramide is a well-characterized sphingolipid metabolite and second messenger that participates in numerous biological processes. When ceramide is phosphorylated by ceramide kinase (CERK), ceramide-1-phosphate (Cer1P) is obtained. It has recently been proposed that Cer1P is involved in cell survival, cell proliferation, inflammation and phagocytosis. It has been observed that the CERK activity is dependent on the interaction with phosphatidyl inositol-4,5-bisphosphate (PI(4,5)P2). In turn, Cer1P was found to affect the PI3K/AKT pathway. This suggests that PI(4,5)P2 and Cer1P might co-localize and interact. To address this issue, giant unilamellar vesicles (GUVs) composed of POPC and 10% Cer1P were made with different concentrations of brain PI(4,5)P2 (from 2%, 5%, 10% to 20 mol%), labeled with fluorescent lipids and analyzed by fluorescence microscopy. GUVs composed of POPC and 10% Cer1P showed irregular branch-shaped domains, which are characteristic for the Cer1P gel phase. In the presence of 2% brain PI(4,5)P2, the irregularly branched domains took on a shape of beads on strings, i.e., the domains were round indicating increased fluidity. With increasing amount of PI(4,5)P2 added, the bead region became larger and larger. In the presence of 10% PI(4,5)P2; the gel type string regions can barely be seen. For 20% PI(4,5)P2 only one fluid type region can be seen. Since the portion of Cer1P in the GUVs was fixed, (10% of the total lipids) the increasingly larger domains have to be the result of the incorporation of brain PI(4,5)P2 in the Cer1P phase. This incorporation of PI(4,5)P2 in the Cer1P phase leads to an increasing fluidity and increasing size of the domain. In conclusion, Cer1P and PI(4,5)P2 co-localize into a domain when mixed with POPC and this domain exhibits fluid like properties at high PI(4,5)P2 concentrations.