Does paranode formation and maintenance require partitioning of neurofascin 155 into lipid rafts? - PubMed (original) (raw)

Does paranode formation and maintenance require partitioning of neurofascin 155 into lipid rafts?

Dorothy P Schafer et al. J Neurosci. 2004.

Abstract

Paranodal axoglial junctions in myelinated nerve fibers are essential for efficient action potential conduction and ion channel clustering. We show here that, in the mature CNS, a fraction of the oligodendroglial 155 kDa isoform of neurofascin (NF-155), a major constituent of paranodal junctions, has key biochemical characteristics of a lipid raft-associated protein. However, despite its robust expression, NF-155 is detergent soluble before paranodes form and in purified oligodendrocyte cell cultures. Only during its progressive localization to paranodes is NF-155 (1) associated with detergent-insoluble complexes that float at increasingly lower densities of sucrose and (2) retained in situ after detergent treatment. Finally, mutant animals with disrupted paranodal junctions, including those lacking specific myelin lipids, have significantly reduced levels of raft-associated NF-155. Together, these results suggest that trans interactions between oligodendroglial NF-155 and axonal ligands result in cross-linking, stabilization, and formation of paranodal lipid raft assemblies.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

A subpopulation of NF-155 has key biochemical characteristics of a lipid raft-associated protein. A, B, Rat optic nerve sections double labeled for pan-NF and NF-155. C–E, Analysis of rat brain membranes (RBM). C, Immunoblot showing the fractionation of NF isoforms into the 1% TX-100 detergent-insoluble fraction (or pellet) or soluble fraction at either 4°C or 37°C. Each temperature condition shows the equivalent of 10 μg of protein split between soluble and pellet fractions. The RBM lane was loaded with 10 μg of protein. All immunoblots (C–F) were performed using pan-NF antibodies. D, Previous cholesterol extraction from brain membrane homogenate by 0.2% saponin at 4°C promotes the solubility of NF-155 in 1% TX-100 at 4°C. S, 1% TX-100-soluble fraction. P, 1% TX-100-insoluble pellet. E, Sucrose density gradient analysis of the detergent-insoluble fraction immunoblotted for NF isoforms. Scale bars, 5 μm.

Figure 7.

Figure 7.

Raft-associated NF-155 is reduced in rodents with disrupted paranodes. A, B, Nodes of Ranvier from sciatic nerves (A) and optic nerves (B) of WT and CST-null (CST) and CGT-null (CGT) mice double labeled for NF-155 (green) and Na+ channels (red). (Note that the axons run horizontally in A and vertically in B.) Rabbit polyclonal anti-NF-155 was used in all experiments shown (A–E). C, Immunoblots of sucrose gradients using detergent-insoluble pellets from P30 WT and CGT-null mouse brains assayed for NF-155, Caspr, contactin, and GM1. D, A comparison of the NF-155 protein levels in P30 WT and CGT-null mouse brains. Each lane was loaded with 40 μg of brain membrane proteins. E, Mouse oligodendrocyte cultures from WT and CGT-null mice. NF-155 and CNP immunoreactivity from cultures from four mouse pups, two of each genotype are shown. These experiments were repeated in two independent litters of animals with identical results. F, Immunoblot of brain membranes from WT and md rats showing (1% TX-100 at 4°C) soluble (S) and insoluble (P) fractions. Immunoblots were performed using anti-pan-NF antibodies (arrow indicates NF-155). Scale bars, 5 μm.

Figure 2.

Figure 2.

The density and detergent insolubility of NF-155 are developmentally regulated. A, Detergent extraction of NF-155 from P12, P18, and adult rat optic nerve membrane homogenates. Each temperature condition shows the equivalent of 10 μg of protein split between soluble (S) and pellet (P) fractions. Immunoblotting was performed using the pan-NF antibody_. B–E_, Detergent-insoluble pellets from P13, P18, P24, and adult rat optic nerve (ON) homogenates were floated on sucrose gradients. Equal volume fractions were collected from the gradients and immunoblotted using pan-NF (B), Caspr(C), or anti-CNP (D). Equal volumes from each fraction were loaded on the gels. The ganglioside GM1 was assayed by dot blot using peroxidase-conjugated cholera toxin (E). One microliter of each fraction was loaded. Pellets from the gradients (fraction 13) are not shown. F, The percentage of sucrose was measured for each fraction used in the experiments shown in B–E.

Figure 3.

Figure 3.

NF-155 is expressed at high levels in cultured oligodendrocytes and is soluble in 1% TX-100. A, Immunolabeling of purified oligodendrocytes using pan-NF and O4 or O1 antibodies. Oligodendrocytes expressed NF-155 in the cell body (arrow) and major processes (arrowheads), but NF-155 was excluded from the membrane sheets (asterisk). B, Homogenates from three separate populations of early progenitors (EP), late progenitors (LP), and oligodendrocytes (OL) immunoblotted using pan-NF; NF-186 was not detected. C, D, 1% TX-100 solubilization of oligodendrocytes and its progenitors at either 4°C or 37°C. E, Cultured oligodendrocytes stained for NF-155 and Hoechst with and without previous detergent extraction of live cells in 1% TX-100 at 4°C. Scale bars, 20 μm.

Figure 4.

Figure 4.

NF-155 expression and localization during myelination. A, B, pan-NF immunolabeling of P6 and P8 rat optic nerve. Pronounced NF-155 staining can be seen in the cell bodies of oligodendrocytes (arrowheads). C, Adult optic nerve labeled with pan-NF. Immunostaining is restricted to nodes and paranodes (arrowhead). D, Double labeling of rat optic nerves at P8 with antibodies against MBP (green) and pan-NF (red). NF-155 is localized at the ends of MBP-labeled oligodendrocyte processes (arrowheads). An oligodendrocyte cell body is identified by the asterisk. E, Double labeling of rat optic nerves at P9 with antibodies against MBP (green) and pan-NF (red). NF-155 is nested within MBP staining and is localized at the ends of the MBP-labeled oligodendrocyte processes (arrowheads). F, Double labeling of rat optic nerve at P8 using antibodies against Caspr (green) and pan-NF (red). Staining for NF-155 and Caspr is colocalized at forming paranodes (arrowheads). Scale bars: A, B, 100 μm; C, D, 10 μm; E, F, 5 μm.

Figure 5.

Figure 5.

NF-155 is readily extracted from optic nerves before paranode formation. Sections were double labeled using pan-NF (red) and antibodies against Caspr (green). At early stages of myelination, TX-100 penetrated completely through the nerve because of the fact that the tissue is much less dense than in adults and the nerves are much smaller. A, P7 optic nerve. B, P7 optic nerve after detergent treatment in 1% TX-100 at 4°C. C, P9 optic nerve. D, P9 optic nerve after detergent treatment in 1% TX-100 at 4°C. E, Detergent treatment of P9 rat optic nerve showed that NF-155 and Caspr immunoreactivity was retained at forming paranodes (arrowheads). Scale bars: A–D, 100 μm; E,10 μm.

Figure 6.

Figure 6.

Paranodal NF-155 and Caspr are resistant to detergent extraction from whole optic nerves. A, B, Detergent-treated rat optic nerves immunolabeled for voltage-dependent Na+ channels (red, Pan NaCh) or Kv1.2 K+ channels (red) and Caspr (green). Black arrowheads above the panels indicate the edge of the nerve. The line through each panel approximates the depth to which the detergent penetrated and the color of the fluorophore used. In regions in which detergent penetrated Na channels and Kv1.2 were efficiently extracted from nodes (A, arrow) and juxtaparanodes (B, arrow), respectively. Both were retained in regions in which detergent did not penetrate (A, B, arrowhead). C, Double labeling of detergent-treated optic nerves using antibodies against Caspr (green) and pan-NF (red). Paranodal NF-155 and Caspr are retained after detergent treatment (double arrowheads), but nodal NF-186 is extracted. In regions in which detergent did not penetrate, nodal NF-186 is retained (arrow). D, Detergent treatment at 37°C, followed by double labeling for neurofascin (red) and Caspr (green). Scale bars: A, B,10 μm; C, 5 μm; D, 20 μm.

Figure 8.

Figure 8.

Model for lipid raft-dependent paranode formation. A, Caspr, contactin, and NF-155 are initially unclustered, not present in rafts, and readily solubilized by 1% TX-100. B, During binding to its axonal ligand in trans, NF-155 and its associated lipid environment are stabilized and become detergent insoluble. Additional NF-155 is recruited through thermodynamically favorable inclusion into the lipid environment surrounding the trans stabilized NF-155. C, Binding of additional Caspr/contactin (or another axonal ligand) to NF-155 then results in a stable paranodal structure with distinct lipid environments in both the axon and oligodendrocyte. This paranodal structure may then be involved in cytoskeletal interactions through protein 4.1B and signaling through phosphorylation of NF-155 (pTyr). GalC, Galactocerebroside.

Similar articles

Cited by

References

    1. Arroyo EJ, Xu T, Grinspan J, Lambert S, Levinson SR, Brophy PJ, Peles E, Scherer SS (2002) Genetic dysmyelination alters the molecular architecture of the nodal region. J Neurosci 22: 1726–1737. - PMC - PubMed
    1. Bansal R, Kumar M, Murray K, Morrison RS, Pfeiffer SE (1996) Regulation of FGF receptors in the oligodendrocyte lineage. Mol Cell Neurosci 7: 263–275. - PubMed
    1. Bansal R, Winkler S, Bheddah S (1999) Negative regulation of oligodendrocyte differentiation by galactosphingolipids. J Neurosci 19: 7913–7924. - PMC - PubMed
    1. Baron W, Decker L, Colognato H, ffrench-Constant C (2003) Regulation of integrin growth factor interactions in oligodendrocytes by lipid raft microdomains. Curr Biol 13: 151–155. - PubMed
    1. Bhat MA, Rios JC, Lu Y, Garcia-Fresco GP, Ching W, St. Martin M, Li J, Einheber S, Chesler M, Rosenbluth J, Salzer JL, Bellen HJ (2001) Axonglia interactions and the domain organization of myelinated axons requires neurexin IV/Caspr/Paranodin. Neuron 30: 369–383. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources