Abstract
Ceramide phosphoethanolamine (CPE), a major sphingolipid in invertebrates, is crucial for axonal ensheathment in Drosophila. Darkfield microscopy revealed that an equimolar mixture of bovine buttermilk CPE (milk CPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (diC18:1 PC) tends to form tubules and helical ribbons, while pure milk CPE mainly exhibits amorphous aggregates and, at low frequency, straight needles. Negative staining electron microscopy indicated that helices and tubules were composed of multilayered 5–10 nm thick slab-like structures. Using different molecular species of PC and CPE, we demonstrated that the acyl chain length of CPE but not of PC is crucial for the formation of tubules and helices in equimolar mixtures. Incubation of the lipid suspensions at the respective phase transition temperature of CPE facilitated the formation of both tubules and helices, suggesting a dynamic lipid rearrangement during formation. Substituting diC18:1 PC with diC18:1 PE or diC18:1 PS failed to form tubules and helices. As hydrated galactosylceramide (GalCer), a major lipid in mammalian myelin, has been reported to spontaneously form tubules and helices, it is believed that the ensheathment of axons in mammals and Drosophila is based on similar physical processes with different lipids.
Highlights
The vertebrate nervous system is characterized by ensheathment of axons with myelin, a multilamellar membrane enriched in galactosylceramide (GalCer, Fig. 1)
We examined the morphology of ceramide phosphoethanolamine (CPE)-containing membranes using commercially available CPE obtained from bovine buttermilk (Fig. 1), N-acyl-sphingosine-1-phosphoethanolamine (Matreya, Pleasant Gap, PA)
In case of equimolar milk CPE:diC18:1 PC mixtures, helical ribbon assemblies were observed by darkfield microscopy (Fig. 3A)
Summary
The vertebrate nervous system is characterized by ensheathment of axons with myelin, a multilamellar membrane enriched in galactosylceramide (GalCer, Fig. 1). GalCer is one of the few natural lipids that forms helical ribbons in aqueous solution[1,2,3,4] These helical ribbon structures have been postulated to be stabilized by intermolecular hydrogen bonds between GalCer molecules[3]. The detailed molecular mechanism is not fully understood, it has been speculated that planar lipid sheets roll up into cochleate cylinders to form myelin. Unlike vertebrates, invertebrates such as Drosophila do not have myelin, but specialized glial cells that ensheath individual axons and fascicles of axons[5]. The small size of the CPE headgroup allows stronger intermolecular hydrogen bonding between CPE molecules compared to SM, as highlighted by its elevated phase transition temperature[8,18]
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