Two types of detergent‐insoluble, glycosphingolipid/cholesterol‐rich membrane domains from isolated myelin

Abstract

Two different types of low-density detergent-insoluble glycosphingolipid-enriched membrane domain (DIG) fractions were isolated from myelin by extraction with Triton X-100 (TX-100) in 50 mM sodium phosphate buffer at room temperature (20°C) (procedure 1), in contrast to a single low-density fraction obtained by extraction with TX-100 in Tris buffer containing 150 mM NaCl and 5 mM EDTA at 4°C (procedure 2). Procedure 1 has been used in the past by others for myelin extraction to preserve the cytoskeleton and/or radial component of oligodendrocytes and myelin, whereas procedure 2 is now more commonly used to isolate myelin DIG fractions. The two DIG fractions obtained by procedure 1 gave opaque bands, B1 and B2, at somewhat lower and higher sucrose density respectively than myelin itself. The single DIG fraction obtained by procedure 2 gave a single opaque band at a similar sucrose density to B1. Both B1 and B2 had characteristics of lipid rafts, i.e. high galactosylceramide and cholesterol content and enrichment in GPI-linked 120-kDa neural cell adhesion molecule (NCAM)120, as found by others for the single low-density DIG fraction obtained by procedure 2. However, B2 had most of the myelin GM1 and more of the sulfatide than B1, and they differed significantly in their protein composition. B2 contained 41% of the actin, 100% of the tubulin, and most of the flotillin-1 and caveolin in myelin, whereas B1 contained more NCAM120 and other proteins than B2. The single low-density DIG fraction obtained by procedure 2 contained only low amounts of actin and tubulin. B1 and B2 also had size-isoform selectivity for some proteins, suggesting specific interactions and different functions of the two membrane domains. We propose that B1 may come from non-caveolar raft domains whereas B2 may derive from caveolin-containing raft domains associated with cytoskeletal proteins. Some kinases present were active on myelin basic protein suggesting that the DIGs may come from signaling domains. Keywords: caveolin, cerebroside sulfate, cytoskeleton, galactosylceramide, rafts, oligodendrocytes. Extensive studies have demonstrated the existence of lateral membrane domains that contain a specific repertoire of lipids and proteins. These lipid-rich microdomains, or rafts, in the plasma membrane are thought to be formed by the tight packing of the long and highly saturated fatty acids of the sphingolipids and cholesterol and contain GPI-linked and acylated proteins, and certain integral membrane proteins (Brown and

Immobilization of the Type XIV Myosin Complex inToxoplasma gondii

“…In this regard, the IMC membrane seems to be similar to membrane domains found in myelin. These domains were also found to be insoluble in TX100 in phosphate buffer, but they were solubilized efficiently by TX100 in a Tris-based buffer (Arvanitis et al, 2005), and, like we observed for the IMC membrane, the TX100/ phosphate-resistant myelin fraction was found to have a higher density than typical DRMs. It is interesting to note in this context that both the Toxoplasma IMC and myelin are composed of closely apposed membranes, suggesting that phosphate may play a role in their association.…”

The localization and non‐genomic function of the membrane‐associated estrogen receptor in oligodendrocytes

“…Immunoprecipitation with anti-ER and probing for caveolin-l confirmed an association of ER with caveolin-l in OLs (data not shown). The mER and caveolin in myelin partition to a low density detergent-insoluble glycosphingolipid/cholesterol-enriched fraction containing actin and tubulin (Arvanitis et al, 2005). Simoncini et al (2006) revealed a novel nongenomic function of mERa in EC, triggered by its interaction with the G protein Ga 13, resulting in remodeling of the actin cytoskeleton.…”

Classic 18.5- and 21.5-kDa Myelin Basic Protein Isoforms Associate with Cytoskeletal and SH3-Domain Proteins in the Immortalized N19-Oligodendroglial Cell Line Stimulated by Phorbol Ester and IGF-1

“…Numerous previous studies have shown in vitro that MBP can polymerize both actin and tubulin, and can crosslink actin filaments and microtubules to each other and tether them to a membrane surface [23,24,26,27,69,70]. Post-translational modifications (deimination, deamidation, phosphorylation, etc.)…”

“…Classic MBP isoforms also have a PXXP motif which is a ligand for SH3-domains, and have been shown to bind to several proteins with SH3-domains and tether the SH3-domain of Fyn to a membrane surface in vitro [16,[34][35][36]. The phosphorylated form of MBP is enriched along with cytoskeletal proteins and kinases including MAPK, Fyn, and Lyn within insoluble Triton-X100 membrane domains when extracted from myelin [67,69,70]. These detergent-insoluble membrane domains from myelin also contain the radial component consisting of a series of tight junctions between myelin layers ( [71,72], see also [73]).…”