Expression and interactions of cytoskeletal elements and sphingolipids in the differentiation of myelin-forming cells in the vertebrate nervous system

Abstract

A tissue culture system was developed and characterized, which permitted the biochemical and immunocytochemical analysis of the differentiation of oligodendrocytes from their progenitors Three facets of this differentiation process were investigated. 1 The microtubule associated protein MAP1B was shown to be expressed immediately before terminal differentiation. Since inhibition of MAP1B synthesis caused the thinning and retraction oligodendrocyte processes. MAP1B appears to stabilize the extension of myelin processes during myelination. 2 Galactocerebroside (GalC) is a characteristic lipid of differentiated oligodendrocytes and it abundant in the myelin membrane, therefore the biosynthesis and trafficking of GalC was studied using fluorescently-labeled ceramides and Brefeldin A (BFA). The metabolic rate of labeled ceramides was related to their sites of accumulation in the Golgi network and was shown to be influenced by the reorganization of the secretory pathway in response to BFA. 3. The possible role of sphingolipid and protein interaction with the cytoskelton in myelination during development was investigated in cultured cells and purified myelin Sphingolipids are enriched in cytoskeletons copurifying with CNS and PNS myelin and GalC codistributes with oligodendrocyte cytoskeletal proteins during development incubations with fluorescent sphingolipids confirmed the ability of sphingolipids to interact with the cytoskeleton, but suggested that association probably depends on acyl chain composition. The importance of sphingolipids in myelin assembly was emphasized by their marked reduction in the hypomyelinating mutant rumpshaker (rah) Myelin associated glycoprotein (MAG) associates with mature oligodendrocyte cytoskeltons. colocalzing with microtubles MAG interacts with microtubles and early isoforms of myelin basic protein (MBP) in the myelin cytoskelton at early stages of myelination, microfilaments and 2',3’-cylic nucleotide 3 phosphohydrolase (CNP) become assiociated with the cytoskelton as myelination proceeds. Cytoskeletal myelin proteins (except CNP2) are underphosphorylated compared to detergent soluble forms, indicating that cytoskeletal association may either restrain phosphorylation or characterize specific subsets of myelin proteins