Lateral Segregation of Palmitoyl Ceramide-1-Phosphate in Simple and Complex Bilayers

Abstract

Ceramide-1-phosphate is a minor sphingolipid with important functions in cell signaling. In this study, we examined the propensity of palmitoyl ceramide-1-phosphate (Cer-1P) to segregate laterally into ordered domains in different bilayer compositions at 23 and 37 C and compared this with segregation of palmitoyl ceramide (PCer) and palmitoyl sphingomyelin (PSM). The ordered-domain formation in the fluid phosphatidylcholine bilayers was determined using the emission lifetime changes of trans-parinaric acid and from differential scanning calorimetry thermograms. The lateral segregation of Cer-1P was examined when hydrated to bilayers in Tris buffer (50 mM Tris, 140 mM NaCl (pH 7.4)). At this pH, Cer-1P was negatively charged. The lateral segregation propensity of Cer-1P in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers was intermediate between PCer and PSM. Based on differential scanning calorimetry analysis, we observed that the gel domains formed by Cer-1P in POPC bilayers (POPC:Cer-1P 70:30 by mol) were less stable (melting interval 16-37 C) than the corresponding POPC and PCer gel domains at equal composition (melting interval 20-55 C). The gel-phase melting enthalpy was also much lower in Cer-1P (1.5 kcal/mol) than in the PCer-containing POPC bilayers (9 kcal/mol). Cer-1P appeared to be at least partially miscible with PCer domains in POPC bilayers. Cer-1P domains were stabilized in the presence of PSM (POPC:PSM 85:15), similarly as seen with PCer-rich domains. In bilayers at 37 C, with an approximate outer-leaflet cell membrane composition (sphingomyelin and cholesterol enriched, aminophospholipid poor), Cer-1P segregation did not lead to the formation of ordered domains, at least when compared with PCer segregation. In bilayers with an approximate inner-leaflet composition (sphingomyelin poor, cholesterol and aminophospholipid enriched), Cer-1P also failed to form ordered domains. PCer segregated into ordered domains only after the PCer/cholesterol ratio exceeded an approximate equimolar ratio.

Investigation of nano- and microdomains formed by ceramide 1 phosphate in lipid bilayers

“…While phase behaviour of C1P18 remains unexplored in the literature, extrapolations from analogous studies on POPA 29 , suggest a probable L o /L d phase coexistence or a similar state in the membrane. Our investigation revealed significant findings for giant unilamellar vesicles (GUVs) incorporating saturated C1P species, notably C1P16, previously documented to induce gel domains 13,30 . The introduction of fluorescently tagged C1P16 has been shown to enhance rigidity and order within the lipid bilayer 31 .…”

Investigation of nano- and micro-domains formed by ceramide-1-phosphate in freestanding lipid bilayers

“…The most interesting results were obtained for GUVs with saturated species of C1P. C1P16:0 was reported to induce gel domains [15, 20]. Fluorescent modification of C1P16:0 was also reported to create more rigid and ordered membrane structure [21].…”