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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.
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 Rose 1992; Harder and Simons 1997; Simons and Ikonen 1997; Melkonian et al. 1999; London and Brown 2000). Glycosphingolipid (GSL)/cholesterol-enriched membrane domains can be isolated on the basis of their resistance to either high pH or non-ionic detergents, such as Triton X-100 (TX-100), and their low density (Parton and Simons 1995).
The myelin sheath is a unique tissue as it contains a high lipid to protein ratio and is enriched in the GSLs, galactosylceramide (GalC) and its sulfated form cerebroside sulfate (CBS), and cholesterol (Norton 1977). It has a complex structure as it contains several substructures with different molecular compositions (compact myelin and cytosol-containing paranodal loops, outer loop and periaxonal inner loop). Compact myelin also contains cytoskeletal proteins (Wilson and Brophy 1987; Pereyra et al. 1988; Gillespie et al. 1989; Cabrera et al. 2000; Marta et al. 2002) and a series of tight junctions called radial component which passes through many layers of compact myelin (Karthigasan et al. 1994). The potential presence of different plasma membrane microdomains is an added level of complexity to the organization of myelin membranes.
The low density and high content of GSLs and cholesterol in myelin are similar to those of DIGs from other membranes. Nevertheless, it can be fractionated further into DIGs of even lower density and increased GSL/phospholipid ratio by detergent extraction. Early detergent solubilization studies of myelin and/or oligodendrocytes (OLs) reported the isolation of a detergent-insoluble cytoskeletal fraction and/or radial component containing actin, tubulin, GalC/CBS, cholesterol, exon II+ isoforms of myelin basic protein (MBP), 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP), myelin/OL basic protein and the tight junction protein claudin-11 (formerly called OSP) (Pereyra et al. 1988; Gillespie et al. 1989; Wilson and Brophy 1989; Karthigasan et al. 1994; Morita et al. 1999; Yamamoto et al. 1999). However, this insoluble material was not fractionated further. In more recent detergent solubilization studies of myelin and/or OLs, low-density DIGs were isolated by sucrose density centrifugation (Kramer et al. 1997, 1999; Kim and Pfeiffer 1999; Simons et al. 2000; Taylor et al. 2002). The presence of actin and tubulin in DIGs isolated from myelin has not been reported, but those isolated from OLs contained some actin and tubulin (Klein et al. 2002; Marta et al. 2003).
Our efforts to define specialized membrane compartments and protein–protein complexes within the myelin sheath began with characterization of the TX-100-soluble fraction of bovine brain myelin (Arvanitis et al. 2002). Here we report the identification and characterization of two buoyant fractions of different density that resulted from fractionating the TX-100-insoluble residue obtained by the extraction method of (Karthigasan et al. 1994) by sucrose density gradient centrifugation. Both fractions, in accordance with their detergent insolubility, densities and lipid composition, had characteristics of GSL/cholesterol-enriched membrane domains, and contained distinctive protein residents. The higher-density DIGs contained most of the cytoskeletal elements but were enriched in GSLs and were of much lower density than cytoskeletal proteins. They also contained most of the caveolin, which we recently reported was present in myelin (Arvanitis et al. 2004).
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- Materials and methods
This is the first report that two different DIG fractions can be isolated from myelin by TX-100 extraction with sodium phosphate buffer lacking EDTA/EGTA, at room temperature. Both of these fractions have characteristics of GSL/cholesterol-enriched membrane domains based on their TX-100 insolubility, high ratios of cholesterol and GalC to phospholipid, enrichment in the GPI-linked protein NCAM120, and buoyancy on a sucrose density gradient. However, B2 also contained the cytoskeletal proteins actin and tubulin, and the raft markers flotillin-1, caveolin and GM1, and thus may consist of caveolae or other types of GSL/cholesterol-enriched membrane domain associated with cytoskeletal proteins. It does not contain non-membrane-associated cytoskeleton, which would sediment faster to a much higher density.
Although B1 did not contain the raft marker GM1, it contained more of the raft marker NCAM120 than B2. (Schnitzer et al. 1995b) similarly found that caveolae from endothelial cells contained GM1 but were not rich in GPI-linked proteins, whereas non-caveolar DIGs were enriched in GPI-linked proteins but lacked GM1. Other cell types have also been found to have several different microdomains of different composition (Madore et al. 1999; Roper et al. 2000). He and Meiri (2002) observed two visible bands on a sucrose density gradient after centrifuging a detergent extract of growth cones. The lower-density fraction contained a population enriched in NCAM120 that lacked caveolin and the higher-density one contained a population that was enriched in caveolin but lacked NCAM120. Two different sphingolipid/cholesterol-enriched DIG fractions from melanoma cells could be separated by immunoprecipitation (Iwabuchi et al. 1998). One was enriched in GM3 and lacked caveolin whereas the other lacked GM3 but contained caveolin.
Our ability to detect two distinct DIG fractions, and the lower solubility of several myelin proteins in TX-100 in contrast to other studies on myelin using extraction procedure 2, may be due to the absence of EDTA and/or higher extraction temperature. Although raft solubility in detergents usually increases with temperature, several studies have shown that some types of rafts persist in Brij98 and TX-100 at 37°C (Drevot et al. 2002; Braccia et al. 2003). Extraction by procedure 1 allowed retention of all of the tubulin and half of the myelin actin in B2. Microtubules are usually unstable at 4°C (Pirollet et al. 1992). Although we do not know whether the actin and tubulin in myelin are still polymerized or form a network, similar extraction procedures have been shown by microscopy to cause retention of a cytoskeletal network in OLs (Wilson and Brophy 1989) and of intact radial component in myelin (Karthigasan et al. 1994). Use of buffer containing EDTA and extraction at a low temperature resulted in dispersal of the tubulin and actin throughout the gradient and less association of these cytoskeletal proteins with a lipid-protein fraction (Fig. 4). More tubulin, but less actin was also found in the supernatant after procedure 2 extraction. Intact cytoskeletal elements are also Triton insoluble and would sediment faster to a high density if not associated with lipid. Most of the actin, but no tubulin, was found at high density after procedure 2 extraction. However, most of the tubulin was found in fractions 1 and 2, not associated with the opaque lipid-protein band. This probably represented tubulin monomers, which would have a slow sedimentation rate, as a result of tubulin depolymerization or dissociation from other proteins in the DIGs while on the gradient or during manipulation of the TX-100-insoluble pellet. The tubulin must have remained associated with the DIGs after the extraction or it would have gone into the soluble phase.
Association of cytoskeletal proteins with DIGs could also be affected by dissociation of Ca2+-binding proteins. EGTA was found to cause dissociation of the Ca2+-binding proteins annexins II and VI from higher-density DIGs from lung membranes, resulting in the conversion of two low-density DIGs to one (Parkin et al. 1996). Gillespie et al. (1989) extracted myelin at room temperature but included EGTA in their buffer and found only a single broad band on sucrose density centrifugation at 30–38% sucrose, a considerably higher density than B2. Our observation of two bands after extraction of myelin with buffer lacking EDTA is consistent with the report of Olive et al. (1995) of two types of DIGs isolated from cerebellar tissue by a procedure similar to that of procedure 2 but using buffer lacking EDTA. However, they did not examine the cytoskeletal protein content of their fractions.
The question of whether myelin proteins such as PLP, MAG and MBP are constituents of DIGs is important. We found more of these proteins in TX-100 DIGs by procedure 1 than has been found by procedure 2 (Kramer et al. 1997, 1999; van der Haar et al. 1998; Kim and Pfeiffer 1999; Simons et al. 2000; Taylor et al. 2002). Their solubility depends on the type of detergent and the salt concentrations used for extraction. They are solubilized more by TX-100 if 300 mm KCl or 25 mm MgCl2 is added to the buffer (Pereyra et al. 1988; Arvanitis 2004). However, PLP interacts with CHAPS-insoluble DIGs from myelin and OLs, although not with the DIGs isolated from PLP-transfected BHK cells (Simons et al. 2000). MAG is located in low-density buoyant Lubrol-insoluble membrane fractions isolated from whole brain and primary OLs (Vinson et al. 2003). On the other hand, MOG from myelin is partially TX-100 insoluble by either procedure 1 or 2 (Taylor et al. 2002). Thus PLP, MAG and MBP may be more loosely associated with GSL/cholesterol-enriched domains than other myelin proteins such as MOG.
The differences in protein and GSL composition of B1 and B2 suggest distinct functions of these two myelin microdomains. The compartmentalization of particular protein and lipid components may be important for cell and membrane functions such as polarization and signal transduction. Of the proteins in the Triton-insoluble fraction, B1 contained most of the p44 MAPK, two-thirds of the MAG (predominantly L-MAG), NCAM120, MBP and most of the CNP, whereas B2 contained most of the p42 MAPK in addition to the MAPK activator MEK. The MAG present in B2 was predominantly S-MAG. Fyn has been found in the single type of DIGs isolated from myelin using procedure 2 (Kramer et al. 1997). We also detected fyn in B2 but not Akt or GSK.
In addition to selectivity for size isoforms of MAG and MAPK, B1 and B2 also showed selectivity for specific size isoforms of PLP; the DM20 isoform was the major PLP isoform present in B1 whereas a greater amount of the larger PLP isoform was present in B2. The two isoforms of PLP and of MAG differ in their cytosolic domains, and differences in their interactions with cytosolic and membrane proteins or negatively charged lipids, and differences in membrane trafficking have been reported (Boggs et al. 1977; Horvath et al. 1990; Minuk and Braun 1996; Kursula et al. 2001; Arvanitis et al. 2002; Gudz et al. 2002). Differences in association of these size isoforms with different membrane domains may also be due to post-translational modifications such as cysteine palmitoylation of PLP (Messier and Bizzozero 2000) and MAG (Pedraza et al. 1990) and differential phosphorylation of S- and L-MAG (Bambrick and Braun 1991; Jaramillo et al. 1994). Previous studies have demonstrated that DM20 cannot functionally replace PLP in compact myelin, demonstrating that the PLP-specific peptide confers critical properties for the long-term stability and normal function of myelin (Stecca et al. 2000). L-MAG and S-MAG also appear to have different functions in myelin (Schachner and Bartsch 2000).
The higher content of CNP, MOG and NCAM120 in B1 compared with B2 supports its identification as a GSL/cholesterol-enriched membrane domain. CNP and MOG were previously characterized as constituents of myelin DIGs by Kim and Pfeiffer (1999) and GPI-linked NCAM120 is enriched in myelin and OL DIGs isolated by procedure 2 (Kramer et al. 1997). CNP undergoes phosphorylation and acylation (De Angelis and Braun 1994; O'Neill and Braun 2000) and contains consensus sequences similar to those found in G proteins (Morell and Quarles 1999). These intrinsic properties suggest an involvement in signaling. NCAM120 and MAG are associated with fyn in DIGs from OLs and/or myelin (Umemori et al. 1994; Kramer et al. 1999) and so are probably also involved in signaling, as for NCAM120 in neurons (Niethammer et al. 2002). Although fyn could not be detected in B1, other kinases were present, which might be involved in signaling. MAG and MOG are transmembrane proteins localized in non-compact membrane regions of myelin, i.e. adaxonal myelin and the outer surface respectively. MOG may transmit extracellular information to the myelin sheath (reviewed in Johns and Bernard 1999) and MAG may be involved in myelin–axon communication (Quarles 2002). Therefore, the enrichment of GalC, cholesterol, CNP, L-MAG, p44 MAPK and NCAM120, and presence of MOG in B1 is consistent with non-caveolar membrane domains that may be involved in signaling events in the myelin sheath.
Unlike B1, the low-density fraction B2 resembles a membrane domain associated with a cytoskeletal network or radial component of myelin. The cytoskeleton can control membrane domain organization in some cell types (Foger et al. 2000; Holowka et al. 2000; Gomez-Mouton et al. 2001). It appears to participate in the redistribution of MOG to detergent-insoluble microdomains in OLs upon ligation with anti-MOG antibodies (Marta et al. 2003). However, little is known about the role of cytoskeletal proteins found in myelin, or whether they are part of or distinct from the radial component in myelin. The higher content of GM1 and CBS in B2 compared with B1 is also distinctive. The enrichment of CBS in B2 relative to B1 may be a result of electrostatic interactions with proteins or a specific sorting mechanism. PLP and CBS, but not GalC, are transported together to the OL plasma membrane (Brown et al. 1993).
The presence of some kinases in B1 and B2, and some phosphorylated proteins in B2, further suggests that both membrane domains could be involved in signaling. The fyn in adult myelin has not been found to be active, although it is active in myelin from young animals and in OLs (Kramer et al. 1999). However, we showed for the first time that a kinase of similar Mr to the p42 MAPK isoform in myelin was active on MBP. In a previous study of kinases in myelin, no p42 or p44 MAPK activity was detected, although they were active in OLs (Bhat and Zhang 1996). However, Bhat and Zhang (1996) showed that some kinases of higher Mr in myelin were active on MBP. In contrast to B1, which contained primarily the p44 isoform of MAPK, B2 contained almost exclusively the p42 isoform along with MEK. The p42 isoform was active in both B1 and B2. Other higher molecular weight kinases in B1 and B2 were also active on MBP. Although p44 MAPK predominated in B1, no activity on MBP was detected. Lower molecular weight kinase activity on MBP, which might be p38 MAPK, was also detected in B2. The p38 isoform has been detected in myelin sheaths in situ in the brain (Maruyama et al. 2000).
The p44 and p42 isoforms of MAPK are thought to be functionally redundant as they share 90% amino acid identity in their catalytic domain (Boulton et al. 1991). In several studies, however, it has been observed that a selective activation of p42 MAPK can occur (Bading and Greenberg 1991). The p42 form was found to predominate and be activated by PMA and receptor agonists in OL progenitors (Bhat and Zhang 1996; Larocca and Almazan 1997; Liu et al. 1999), whereas the p44 form predominated in more mature OLs and was also activated by PMA (Stariha and Kim 2001). These results have led to the suggestion that p42 MAPK may be important for mitogenesis of progenitor OLs whereas p44 MAPK may be important for differentiation of OLs (Stariha and Kim 2001). Their role in myelin is not known, but MBP, a MAPK substrate, is phosphorylated in vivo in myelin at the same site phosphorylated by MAPK in vitro (Martenson et al. 1983; Erickson et al. 1990). Microtubule-associated protein MAP1B is also a substrate for MAPK and has been found in myelin (Cabrera et al. 2000).
We previously demonstrated the presence of a mER and caveolin-1 in the myelin sheath (Arvanitis et al. 2004) and found that they were mostly in B2. The mER has been identified in numerous cells and shown to be responsible for rapid, non-genomic, signaling responses initiated at the plasma membrane of these cells (reviewed in Levin 2002). The mER appears to localize partially to caveolae (Kim et al. 1999), but the mechanisms by which this small pool of estrogen receptor translocates to this site are currently unknown. Although caveolin was detected in B2, we do not know if caveolae structures are present. Other prominent protein spots, including some phosphorylated proteins, were detected in the two-dimensional gel of B2, some of which became enriched in this fraction compared to myelin or the TX-100-insoluble extract.
Although myelin rafts have been of interest in recent years, much is still unknown about their molecular organization, formation and function. The isolation of two low-density DIGs from myelin indicates that the myelin membrane is heterogeneous, as found for plasma membranes from other cell types (Madore et al. 1999; Oliferenko et al. 1999; Roper et al. 2000; Nebl et al. 2002). Advancement towards a more detailed model of myelin organization should help us to understand the mechanisms that regulate its biological processes.