Abstract Schwann cells and oligodendrocytes make themyelin sheaths of the PNS and CNS, respectively. Theirmyelin sheaths are structurally similar, consisting of mul-tiple layers of specialized cell membrane that spiralaround axons, but there are several differences. (1) CNSmyelin has a “radial component” composed of a tightjunction protein, claudin-11/oligodendrocyte-specific pro-tein. (2) Schwann cells have a basal lamina and microvilli.(3) Although both CNS and PNS myelin sheaths have in-cisures, those in the CNS lack the structural as well as themolecular components of “reflexive” adherens junctionsand gap junctions. In spite of their structural differences,the axonal membranes of the PNS and CNS are similarlyorganized. The nodal axolemma contains high concentra-tions of voltage-dependent sodium channels that arelinked to the axonal cytoskeleton by ankyrinG. The paran-odal membrane contains Caspr/paranodin, which may par-ticipate in the formation of axoglial junctions. The juxta-paranodal axonal membrane contains the potassium chan-nels Kv1.1 and Kv1.2, their associated β2 subunit, as wellas Caspr2, which is closely related to Caspr. The myelinsheath probably organizes these axonal membrane-relatedproteins via trans interactions.

Introduction

Myelin is a multilamellar spiral of specialized membranethat ensheathes axons larger than 1 µm in diameter. It isone of the fundamental adaptations of vertebrates, compa-rable to the outer segments of rods and cones as a func-tional macromolecular structure mainly comprised of cellmembrane. The periodicity of the mature myelin sheath isabout 14 nm following conventional preparation for elec-

tron microscopy (Fig. 1) and slightly greater (18 nm) infresh nerves by X-ray diffraction. The apposed mem-branes form the intraperiod line; the thin space that sepa-rates the membranes is contiguous with the extracellularspace. The major dense line is contiguous with the cyto-plasm. Although the myelin sheaths of the CNS and PNSdiffer in their cellular origins, anatomical details, and mo-lecular constituents, they are thought to function similarly(Berthold and Rydmark 1995; Peters et al. 1991; Ritchie1995; Sandri et al. 1982; Thomas et al. 1993).

Myelinated axons are completely covered by myelinsheaths except at nodes of Ranvier, the small gaps (lessthan 1 µm in length) directly exposed to the extracellularmilieu. By reducing the capacitance and/or increasing theresistance, myelin reduces current flow across the inter-nodal axonal membrane, thereby facilitating saltatory con-duction at nodes. As shown in Fig. 2, owing to their differ-ential staining, Ramon y Cajal (1928) deduced that nodes,paranodes, and incisures contained different molecularcomponents. His observations presaged the later ultrastruc-tural studies of the myelin sheath, which, together with re-cent investigations of the organization of its molecular con-stituents, provide new insights on the molecular architec-ture of myelinated fibers – the subject of this review.

The PNS myelin sheath

Figure 3A shows a schematic PNS myelinated fiber. Itdepicts two internodes, one of which has been unrolledto reveal its trapezoidal shape. Most of the cytoplasm isexternal to the myelin sheath. As in other cell types, therough endoplasmic reticulum and Golgi apparatus arefound in a perinuclear distribution, and newly synthe-sized proteins largely travel in cytoplasmic channels onthe outside of the myelin sheath (Gould and Mattingly1990). The lateral borders of the Schwann cell cytoplasmare tipped with microvilli. These contain F-actin (Trappet al. 1989b), and their membranes appear similar tothose of internodal Schwann cells by freeze-fracture andhave similar numbers of voltage-sensitive sodium chan-

E.J. Arroyo · S.S. Scherer (✉)Department of Neurology, Room 460 Stemmler Hall, 36th Street and Hamilton Walk, The University of Pennsylvania Medical Center, Philadelphia, PA 19104, USAe-mail: scherer@mail.med.upenn.eduTel.: +1-215-5733198, Fax: +1-215-573-2029

Histochem Cell Biol (2000) 113:1–18 © Springer-Verlag 2000

R E V I E W

Edgardo J. Arroyo · Steven S. Scherer

On the molecular architecture of myelinated fibers

Accepted: 25 November 1999

nels (Blanchard et al. 1985; Devor et al. 1993; Ritchie et al. 1990; Waxman and Black 1987). The tips of thesemicrovilli contact the nodal axolemma (Ichimura and Ellisman 1991; Raine 1982), and the microvilli have in-wardly rectifying K+ channels IRK1 and IRK3, whichmay allow them to accumulate K+ during axonal activity(Mi et al. 1996). In the CNS, the processes of “perinodalastrocytes” have been postulated to have a similar func-tion as these microvilli (Black and Waxman 1988).

The myelin sheath itself can be divided into two do-mains, compact and non-compact myelin, each of whichcontains a non-overlapping set of proteins (Fig. 3B).Compact myelin forms the bulk of the myelin sheath;non-compact myelin is found in paranodes (the lateralborders of the myelin sheath) and in Schmidt-Lantermanincisures (the funnel-shaped interruptions in the compactmyelin; Fig. 2B). By analogy to epithelial cells, the bas-al/abaxonal surface apposes the basal lamina (Fig. 1A).The Schwann cell basal lamina contains laminin 2 (com-prised of α2/merosin, β1, and γ1 laminin chains), typeIV collagens, entactin/nidogen, fibronectin, N-syndecan,

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Fig. 1A, B Ultrastructure of a myelinated fiber in the PNS (from a4-day-old mouse). A This electron micrograph shows an axon, itsmyelin sheath (m), and basal lamina (arrowheads), as well as theinner (i) and outer (o) mesaxons. B This image shows that thecompact myelin sheath is composed of alternating major dense(arrows) and intraperiod lines. Note the “double nature” of the in-traperiod line (Revel and Hamilton 1969)

Fig. 2 Ramon y Cajal’s (1928) depiction of the nodal region (A)and incisures (B).With permission of Oxford University Press

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and glypican (Bunge 1993; Scherer 1996). The basal/adaxonal Schwann cell membrane contains the integrinα6β4 and dystroglycan, both of which probably bind to laminin 2 (Einheber et al. 1993; Feltri et al. 1994;Rambukkana et al. 1998; Saito et al. 1999). The api-cal/adaxonal surface apposes the axon and is highly en-riched in myelin-associated glycoprotein (MAG), whichmay bind to molecules on the axonal surface (Sawada et

al. 1999; Yang et al. 1996). The lateral borders of thetrapezoid define the paranodes; they taper slightly, sothat the apical/adaxonal surface is furthest from thenode. The inner and outer edges of the Schwann cellmembrane, which contact the adjacent layer of the myelin sheath, are called the inner and outer mesaxon,respectively (Figs. 1A, 3A).

Adherens junctions (commonly referred to as “des-mosome-like” junctions in the older literature) are foundin both the inner and outer mesaxons as well as in paran-odes and incisures (Fannon et al. 1995). Owing to the peculiar geometry of the myelin sheath, the adherensjunctions in the paranodes and incisures form a series ofradially arranged junctions; these typically span manylayers and are most prominent in the outer layers of themyelin sheath (Fig. 4). Adherens junctions contain E-cadherin, a Ca2+-dependent cell adhesion molecule thatforms “strand dimers”, which bind homophilically intrans with those on the apposing membrane (Shapiro etal. 1995). The cytoplasmic domain of E-cadherin bindsα-catenin and β-catenin (Fannon et al. 1995), which linkE-cadherin to the actin cytoskeleton (Nagafuchi et al.1993). Thus, the junctions of incisures and paranodes, aswell as inner and outer mesaxons, although tentativelyidentified as tight junctions by freeze-fracture electronmicroscopy (Sandri et al. 1982), are probably adherensjunctions.

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Fig. 3A, B Schematic view of a myelinated axon in the PNS andthe proteins of CNS and PNS myelin sheaths. A One myelinatingSchwann cell has been unrolled revealing the regions that formnon-compact myelin, the incisures and paranodes. Adherens junc-tions are depicted as two continuous (purple) lines; these form acircumferential belt and are also found in incisures. Gap junctionsare depicted as (orange) ovals; these are found between the rowsof adherens junctions. The nodal, paranodal, and juxtaparanodalregions of the axonal membrane are colored blue, red, and green,respectively. From Arroyo et al. (1999), with permission ofKluwer Academic Press. B CNS and PNS myelin sheaths containdistinct sets of proteins. In the PNS, compact myelin contains pro-tein zero (P0), peripheral myelin protein 22 kDa (PMP22), andmyelin basic protein (MBP); in the CNS, it contains proteolipidprotein (PLP), oligodendrocyte-specific protein (OSP), myelin-oli-godendrocyte basic protein, and MBP. In the PNS, the non-com-pact myelin contains E-cadherin, myelin-associated glycoprotein(MAG), and connexin32 (Cx32). Note P0 and MAG have extracel-lular immunoglobulin-like domains (semicircles), and that OSP,PLP, PMP-22, and Cx32 all have four transmembrane domains

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Fig. 4A–E Adherens junctionsin the PNS myelin sheath.A Teased fibers from an adultmouse sciatic nerve stainedwith a monoclonal antibodyagainst E-cadherin. At the node(double arrowheads), the twobands of E-cadherin staining of adjacent paranodes is soclose that they are difficult toseparate at this magnification.E-cadherin is also found in incisures (arrowheads), and in mesaxons (arrows).B–D Immunoelectron micros-copy of paranodal E-cadherinin 7-day-old rats or mice. Scalebars 0.1 µm. From Fannon etal. (1995), with permission ofRockefeller University Press.E A schematic drawing of ad-herens junctions in incisures.From Hall and Williams(1970), with permission of The Company of Biologists

Gap junctions were occasionally noted between therows of adherens junctions by freeze-fracture electronmicroscopy (Sandri et al. 1982). One gap junction protein, connexin32 (Cx32, also called GJβ1), is local-ized to the same places (Fig. 5; Bergoffen et al. 1993;Chandross et al. 1996; Scherer et al. 1995). Dye transferstudies on acutely isolated, living myelinated fibers frommice, rats, and frogs demonstrate a radial pathway of dyediffusion across incisures, from the outer/abaxonal to theinner/adaxonal cytoplasm (Balice-Gordon et al. 1998).Small molecular mass dyes, such as 5,6-carboxyfluores-cein, create a pattern of labeling similar to a pair of“train tracks.” Each track consisted of a double line offluorescence, separated by an unstained space that corre-sponded precisely to the location of compact myelin(Fig. 6). In contrast, the dye remained confined to theouter/abaxonal collar of cytoplasm following injection ofa high molecular mass dye, such as 3000-Da rhodaminedextran, or injection of 5,6-carboxyfluorescein in thepresence of a pharmacological blocker of gap junctions,such as α-glycerrhetinic acid.

These results provide functional evidence that gapjunctions mediate a radial pathway of diffusion acrossincisures, and by extension, across paranodes, too. Theadaptive significance of a radial pathway is that it pro-vides a much shorter pathway, owing to the geometry ofthe myelin sheath. For example, an unrolled myelinsheath surrounding a 7-µm-diameter axon would be ex-pected to be about 4 mm long, but only 2.3 µm thick(Friede and Bischhausen 1980). Thus, the radial length is1700 times shorter, and, since diffusion in a plane is pro-portional to the square of the distance (Balice-Gordon etal. 1998), a radial pathway for diffusion could be about 3 million times faster than diffusion through the cyto-plasm. The actual length of pathway through the inci-sures and paranodes may be many times longer than thethickness of the myelin sheath itself, but this correctiondoes not alter the fundamental economy of this pathway.Disruption of this radial pathway may be the reason thatmutations in Cx32 cause X-linked Charcot-Marie-Toothdisease (CMTX), an inherited demyelinating neuropathy(Bergoffen et al. 1993). Because the pathway and therate of 5,6-carboxyfluorescein diffusion in cx32-nullmice did not appear to be different than in wild-typemice (Balice-Gordon et al. 1998), there appear to be functional gap junctions in the myelin sheaths formedby another connexin(s), perhaps Cx26 and/or Cx43(Mambetisaeva et al. 1999; Nagaoka et al. 1999;Yoshimura et al. 1996; Zhao and Spray 1998). The exis-

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Fig. 5A, B Cx32 is localized to incisures and inner mesaxons. Theseare photomicrographs of a teased myelinated fiber immunostainedwith a combination of a mouse monoclonal antibody against rat Cx32(A; fluorescein) and a rabbit antiserum against MAG (B; rhodamine).Cx32 and MAG immunoreactivity colocalize at incisures (arrows).MAG also surrounds the axon at the adaxonal Schwann cell surface(between arrowheads in B), whereas only a thin line of Cx32 stainingis found at the adaxonal surface, probably at the inner mesaxon (ar-rows in panel A). Scale bar 10 µm. From Scherer et al. (1995), withpermission of The Society for Neuroscience

tence of a radial pathway demonstrates that myelin doesnot provide a high resistance insulation around axons, inkeeping with the experimental evidence that CNS myelinhas a low resistance (Funch and Faber 1984).

Compact myelin in the PNS is largely composed oflipids, mainly cholesterol and sphingolipids, includinggalactocerebroside and sulfatide (Hudson 1990; Suterand Snipes 1995). The main myelin proteins are proteinzero (P0), peripheral myelin protein 22 kDa (PMP22),and myelin basic protein (MBP). P0 is by far the mostabundant protein and is the main adhesive molecule incompact myelin. It has an IgG-like extracellular domain

that forms tetramers in cis (in the plane of the mem-brane) (Shapiro et al. 1996). As shown in Fig. 7, thesetetramers interact with each other in trans. The analysisof P0-null mice confirms the importance of P0, as inthese mice, Schwann cells form a multilamellar spiral ofmembrane around axons, but the myelin does not com-pact (Giese et al. 1992). P0 heterozygous mice also de-velop a late-onset demyelinating neuropathy, indicatingthat even a modest reduction in the amount of P0 causesinstability of compact myelin (Martini et al. 1995b; Shyet al. 1997; Zielasek et al. 1996). Thus, one of the waysthat mutations in the human P0 gene could cause demy-elination (CMT type 1B) is by a dose-related decrease inthe amount of P0 protein (Murakami et al. 1996; Scherer1997; Suter and Snipes 1995).

PMP22 is a hydrophobic intrinsic membrane proteinof unknown function and is much less abundant than P0.Nevertheless, the amount of PMP22 in compact myelin iscritical (Murakami et al. 1996; Suter and Snipes 1995).

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Fig. 6A–G Dye diffusion results in labeling of adaxonal and abax-onal cytoplasm. Shown are images taken following pressure injec-tion (40 psi, 250 ms pulses, 2 Hz for 1–3 min) of 5,6-carboxyfluo-rescein into a myelinating Schwann cell. A Schwann cell perinucle-ar region and incisures visualized with polarized light. B Followinginjection, the fiber was immunostained for MAG, which is localizedto incisures and colocalizes with incisures identified with polarizedlight. C Image taken about midway through depth of the cell showsthat dye occupies the outer and inner collar of Schwann cell cyto-plasm creating a double train track pattern indicative of the radialdiffusion of dye through incisures. Bracket indicates region enlargedin E. D Image taken about 5 µm above plane shown in G revealsfingers of cytoplasm on the surface of the cell; these are easily dis-tinguished from the double train track pattern. E Enlargement of re-gion indicated by brackets in C; arrowheads indicate position of theline across which intensity was mapped. F Histogram of intensityacross line perpendicular to the long axis of the fiber at location in-dicated by arrowhead; scale is the same as in image shown in E.Doublet of peaks on either end of the histogram is the quantitativerepresentation of the double train track pattern evident in the imageshown in E. Vertical scale is 0–255 intensity levels. Scale bars10 µm. From Balice-Gordon et al. (1998), with permission of TheRockefeller University Press

A

B

Fig. 7A, B Tetramers of P0 form a lattice. A The extracellular do-mains of three P0 tetramers viewed from the side, as they mightemanate from their respective membrane surfaces (dotted lines;the blue-colored tetramer protrudes from the blue surface; the yel-low tetramers from the yellow surface). The Trp28 side chains,which may intercalate into the opposite membrane bilayer, areshown in red. B Perpendicular view of this layer of crystal lattice.From Shapiro et al. (1996), with permission of Cell Press

Three copies of the PMP22 gene lead to CMT1A, themost common form, probably because of a modest in-crease in PMP22 in compact myelin (Vallat et al. 1996;cf. Hanemann and Muller 1998). Further, one copy of thePMP22 gene leads in a different kind of inherited demy-elinating neuropathy, hereditary neuropathy with liabilityto pressure palsies (HNPP), probably owing to a modestdecrease of PMP22 (Vallat et al. 1996). The complete ab-sence of PMP22, which has been created in Pmp22-nullmice but has not yet been described in humans, leads to asevere demyelinating neuropathy in mice (Adlkofer et al.1995). Heterozygous mice (Pmp22+/-) have a mild demy-elinating neuropathy with the structural abnormalitiesfound in HNPP (Adlkofer et al. 1997).

MBP is a cytoplasmic protein and comprises the bulkof the major dense line of compact myelin (Figs. 1, 3B),as shown by its absence in shiverer mice, which com-pletely lack MBP because of a mutation in the MBPgene (Kirschner and Ganser 1980; Rosenbluth 1980).The reason that the PNS is not affected in shiverer micemay owe to the basic cytoplasmic domain of P0, whichmay effectively substitute for MBP (Ding and Brunden1995; Martini et al. 1995a).

The targeted disruption of UDP-galactose ceramidegalactosyltransferase gene (cgt) provided the unprece-dented opportunity to perturb the lipid components ofmyelin (Bosio et al. 1996; Coetzee et al. 1996, 1998).Because CGT is necessary for the synthesis of galacto-cerebroside and sulfatide, these glycolipids are com-pletely absent in cgt-/- mice, although the levels of glu-coceramides are elevated. cgt-/- mice develop neurologi-cal signs with the onset of myelination, and most die be-tween 18 and 30 days. Surprisingly, except for the split-ting of some CNS myelin sheaths, most CNS and PNSmyelin sheaths have a normal ultrastructure. In spite ofthis normal appearance, axonal conduction velocity isdramatically slowed, which may be related to abnormalparanodes (Bosio et al. 1998; Dupree et al. 1998).

Thus, the myelin sheath has been likened to a liquidcrystal, as perturbations in the stoichiometry of any onecomponent can alter the entire structure. In the PNS, thelayers of this liquid crystal are largely held together byP0, which has been recently shown to interact withPMP22 (D’Urso et al. 1999). How lipids, PMP22, andthe other minor intrinsic membrane proteins of compactmyelin such as MAL (Frank et al. 1998; Schaeren-Wiemers et al. 1995) “fit” into the lattice of P0 tetramersremains to be determined.

The CNS myelin sheath

Myelination in the CNS differs from that in the PNS inseveral ways. Each oligodendrocyte makes multiple my-elin sheaths; the number varies from tract to tract and appears to relate to the caliber of the axons. Oligoden-drocytes make fewer sheaths in tracts containing largemyelinated fibers; the result of axon–oligodendrocyte in-teractions rather than an intrinsic trait of the oligoden-

drocytes themselves (Fanarraga et al. 1998). Oligoden-drocytes do not have a basal lamina or microvilli, andtheir “incisures” (Peters et al. 1991) do not have any dis-tinguishing molecular markers such as Cx32, MAG, orE-cadherin. “Perinodal astrocytes” contact CNS nodes(Black and Waxman 1988), and are thought to have ananalogous function to Schwann cell microvilli, althoughthese may be glial progenitors rather than astrocytes(Butt et al. 1999).

The molecular components of the CNS myelinsheaths partially overlap with those of the PNS (Hudson1990). Both contain high amounts of lipids, especiallycholesterol and sphingolipids, including galactocerebro-side and sulfatide (Kirschning et al. 1998; Schiff andRosenbluth 1995). Similarly, in both the CNS and thePNS, compact myelin contains MBP and the adaxonalsurface contains MAG (Trapp et al. 1989a). Like my-elinating Schwann cells, oligodendrocytes also expressCx32, but mainly on their cell bodies and proximal pro-cesses; whether there is Cx32 in the paranodal myelin isnot settled (Kunzelmann et al. 1997; Li et al. 1997;Scherer et al. 1995). Unlike myelinating Schwann cells,oligodendrocytes express Cx45 and are coupled to otheroligodendrocytes as well as astrocytes (Dermietzel et al.1997; Kunzelmann et al. 1997; Pastor et al. 1998; Rashet al. 1999; Robinson et al. 1993).

Proteolipid protein (PLP) is the main protein in CNScompact myelin. Like PMP22, PLP is a highly hydro-phobic, intrinsic membrane protein (Fig. 3B) of un-known function. Further, either deletion or duplication ofthe human PLP gene results in CNS dysmyelination (Inoue et al. 1999; Nave and Boespflug-Tangu, 1996),demonstrating again the importance of gene dosage formyelin proteins. Remarkably, CNS myelin appears large-ly normal in Plp-null mice; a finding that underscoresour lack of knowledge about the function of this protein.Although PLP is expressed at low levels by myelinatingSchwann cells, mainly localized to non-compact myelin,PNS abnormalities appear to be uncommon in patientsand mice with PLP mutations (Anderson et al. 1997;Garbern et al. 1997).

CNS myelin has a distinctive structural feature that isnot seen in PNS myelin, the so-called “radial component”(Peters et al. 1991). The radial component is a series of ra-dially arranged intralamellar strands that span the myelinsheath, usually in a single sector (Fig. 9). These intrala-mellar strands look like tight junctions, and recently havebeen shown to contain claudin-11/oligodendrocyte-specif-ic protein (OSP), a member of a large family of distantlyrelated tight junction proteins (Bronstein et al. 1997;Morita et al. 1999). The functional significance of the ra-dial component is unknown, so that the analysis of OSP-null mice should be highly informative.

Oligodendrocytes express two proteins that are notexpressed by Schwann cells, myelin-oligodendrocyteglycoprotein (MOG) on their outer cell membrane(Brunner et al. 1989; Johns and Bernard 1999) and myelin-oligodendrocyte basic protein (MOBP), in themajor dense line of compact myelin (Holz et al. 1996;

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Yamamoto et al. 1999). CNS myelin appears to formnormally in Mobp-null mice, although it is more suscep-tible to the effects of chlorohexaphene, a “myelin toxin”(Yamamoto et al. 1999). The ultrastructure of the radialcomponent is altered in Mobp-null mice. P0 and PMP22are not present in CNS myelin.

Specializations at nodes

In spite of the differences between myelinating Schwanncells and oligodendrocytes and their myelin sheaths, theorganization of the axon itself is quite similar in the PNSand CNS. Electron microscopy of the nodal axolemmareveals an electron-dense undercoating and a high densi-ty of large intramembranous particles (Peters et al. 1991;Quick and Waxman 1977; Sandri et al. 1982). Ritchieand Rogart (1977) first deduced that voltage-dependentNa+ channels were highly concentrated in the nodal axolemma as intact and homogenized nerve had thesame amount of 3H-saxitonin binding; this conclusionwas subsequently confirmed by immunohistochemistry(Fig. 11; Ellisman and Levinson 1982; Elmer et al. 1990;Haimovich et al. 1984). Voltage-dependent Na+ channelsbelong to a multigene family, but the Sca8a/PN4 channelappears to be the main one expressed at nodes (Novakovic et al. 1999). The gene encoding this channelis mutated in mice with motor endplate disease (med), arecessively inherited disease (Burgess et al. 1995), andtheir nodes have been reported to have an altered ultra-structure (Rieger et al. 1984). Sca8a is probably not theonly voltage-dependent Na+ channel at nodes, becausenerve conduction velocity is only minimally slowed inmed mice (Duchen and Stefani 1971; Rieger et al. 1984).An isoform of Na+/K+-ATPase was localized to the nodalaxolemma of CNS axons in goldfish, and subsequentlyto the nodal axolemma in peripheral nerve (Ariyasu et al.1985; Mata et al. 1991; Schwartz et al. 1981; Vorbrodt etal. 1982; Wood et al. 1977). The high concentrations ofNa+ channels and Na+/K+-ATPase is in keeping with thephysiological function of the nodal membrane.

Other molecules have subsequently been localized tonodal axolemma, including ankyrinG 480/270 kDa, aswell as neurofascin and Nr-cell adhesion molecule(CAM), two members of the L1 family of CAMs. Bothneurofascin and Nr-CAM have complex alternativelyspliced isoforms, and only neurofascin isoforms contain-ing a mucin-like domain and lacking one of the fibronec-tin domains are localized to nodes. Nodes contain two al-ternatively spliced isoforms of the ankyrinG gene, anky-rinG 480 and 270 kDa (Kordeli et al. 1990, 1995); theseisoforms are distinguished by their membrane-bindingdomain composed of ANK repeats, a spectrin-bindingdomain, and a serine/threonine-rich domain (Zhang andBennett 1996). As depicted in Fig. 8A, ankyrin interactswith the cytoplasmic domains of neurofascin, Nr-CAM,and voltage-dependent Na+ channels (Davis and Bennett1994; Davis et al. 1993, 1996; Srinivasan et al. 1988).The binding of ankyrinG 480/270 kDa to neurofascin can

be modulated, as phosphorylation of a highly conservedtyrosine in neurofascin abolishes binding and increasesits mobility in the membrane (Garver et al. 1997). In oth-er cell types, ankyrinG has been shown to interact withspectrin, which appears to be concentrated at nodes andparanodes (Koenig and Repasky 1985; Trapp et al.1989b).

These data indicate that ankyrinG is a multivalent pro-tein and links voltage-dependent Na+ channels, neurofas-cin, and Nr-CAM, to the spectrin cytoskeleton. In keep-ing with this idea, inactivation of the ankyrinG gene in thecerebellum reduces the amount of voltage-dependent Na+

channels and neurofascin in the initial segments of gran-ule cells and Purkinje cells, respectively (Zhou et al.1998a). Although these workers were unable to visualizea reduced number of Na+ channels in Purkinje cell initialsegments, the diminished ability of these cells to initiateaxon potentials is in keeping with this idea. These micealso revealed that ankyrinG appears to be necessary forrestricting the localization of neurofascin to initial seg-ments of Purkinje cells. Since initial segments and nodesshare many molecular characteristics, one expects that thenodal membranes of Purkinje cells are similarly affected.

The roles of neurofascin and Nr-CAM at nodes haveyet to be defined. They may establish the location ofnodes in developing nerves, as clusters of neurofascinand Nr-CAM appear before those of ankyrin and voltage-dependent Na+ channels (Lambert et al. 1997).Bennett and colleagues (Bennett et al. 1997; Davis et al.1996; Lambert et al. 1997) have proposed that neurofas-cin and Nr-CAM have heterophilic interactions with oth-er CAMs on the microvilli as depicted in Fig. 8B, in ac-cord with the ultrastructural data showing tethering ofthe microvilli to the nodal axolemma (Ichimura and Ellisman 1991; Raine 1982). Other molecular interac-tions that may serve to localize Na+ channels to nodeshave been proposed: the extracellular domain of the Na+

channel β2 subunit may interact in with tenascinC ortenascinR (Xiao et al. 1999), extracellular matrix mole-cules that have been reported to be localized to the nodalregion in the PNS and CNS (Bartsch et al. 1993; ffrench-Constant et al. 1986; Rieger et al. 1986).

Specializations at paranodes

At the paranode, the lateral edge of the myelin sheath spi-rals around the axon, forming the axoglial junction (Ichimura and Ellisman 1991; Sandri et al. 1982; Thomas etal. 1993). As shown in Fig. 10A, the paranodal loops of themyelin sheath contain rows of large particles that are in reg-ister with a double row of smaller particles on the axolem-ma; these particles are thought to connect the terminal loopsto the axon. These axoglial junctions have also been calledseptate-like junctions, as they resemble invertebrate septatejunctions (Einheber et al. 1997). Septate junctions mayfunction similarly to vertebrate tight junctions, forming in-tercellular junctions that prevent the diffusion of small mol-ecules and ions. Septate-like axoglial junctions, however,

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ton. This potential linkage of Caspr to the cytoskeletonmay relate to the finding that filaments appear to insertinto the paranodal axolemma (Fig. 10A). The cytoplas-mic domain of Caspr also contains an SH3 binding site,which could allow Caspr to participate in signaling path-ways within the axon.

Caspr belongs to the neurexin superfamily, which inmammals currently consists of the neurexins I, II, and IIIas well as Caspr2 (Bellen et al. 1998; Missler et al. 1998;

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Fig. 8 A Schematic depict onof the node, paranode, and jux-taparanode. B Schematic draw-ing of possible cis and trans in-teractions between the molecu-lar components of nodes, par-anodes, and juxtaparanodes

do not prevent the diffusion of lanthanum into the periax-onal space (Fig. 10B); hence are not “tight” in the conven-tional sense (Hirano et al. 1969; MacKenzie et al. 1984).

As shown in Fig. 12, an intrinsic membrane glycopro-tein called Caspr or paranodin is localized to the paran-odal axolemma in myelinated fibers of the PNS and CNS(Einheber et al. 1997; Menegoz et al. 1997). The cyto-plasmic domain of Caspr contains a binding site for pro-tein 4.1, which, in turn, can bind to the actin cytoskele-

Poliak et al 1999). A homologue of the neurexin super-family in Drosophila, called neurexin IV (even though ithas turned out to be more closely related to Caspr2 thanto neurexins I–III), and gliotactin are both localized toseptate junctions, along with coracle, a homologue ofprotein 4.1 (Bellen et al. 1998). Flies lacking eitherneurexin IV or gliotactin fail to form the glial septatejunctions that constitute the blood–nerve barrier (Auld etal. 1995; Baumgartner et al. 1996). In mammals, somealternatively spliced forms of neurexins bind to a familyof ligands known as the neuroligins, which are related togliotactin (Ichtchenko et al. 1996). Although it remainsto be shown whether neurexin IV and gliotactin interactheterophilically to form septate junctions, this kind of in-teraction has been postulated to occur between Caspr anda neuroligin family member expressed in the paranodalloops (Einheber et al. 1997; Menegoz et al. 1997), as de-picted in Fig. 8B.

Specializations at juxtaparanodes

By freeze-fracture electron microscopy, the axolemma inthe region extending 10–15 µm from the paranode con-tains clusters of 5 or 6 particles (Miller and Da Silva1977; Rosenbluth 1976; Stolinski et al. 1981, 1985; Tao-Cheng and Rosenbluth 1984). The distribution of thesejuxtaparanodal particles corresponds to the distributionof delayed rectifying K+ channels (Chiu and Ritchie1980), likely comprised of Kv1.1 and Kv1.2 and their as-sociated β2 subunit (Mi et al. 1995; Rasband et al. 1998;Vabnick and Shrager 1998; Wang et al. 1993; Zhou et al.1998b). The size of the particles (10 nm in diameter)compares well to the expected size of a Shaker-type K+

channel (Kreusch et al. 1998). Kv1.1 and Kv1.2 subunitscan freely mix in varying proportions to form tetramers,the functional channels (Hopkins et al. 1994). EachKv1.1 or 1.2 subunit is associated with a Kvβ2 subunit(Gulbis et al. 1999). Although Kv1.1 and Kv1.2 channelsappear to be concealed under the myelin sheath (Hilde-brand et al. 1994; Kocsis et al. 1983), juxtaparanodal K+

channels are thought to have an important physiologicalfunction, dampening the excitability of myelinated fi-bers. The finding that Kv1.1-null mice have abnormalimpulse generators near the neuromuscular junctionssupports this idea (Smart et al. 1998; Zhou et al. 1998b).Similarly, mutations in the human Kv1.1 gene cause aform of familial episodic ataxia that is associated withectopic impulse generators somewhere within the periph-eral nerve (Adelman et al. 1995; Browne et al. 1994;Brunt and Van Weerden 1990; Van Dyke et al. 1975;Zerr et al. 1998).

Interestingly, freeze-fracture electron microscopy hasalso revealed 12-nm diameter juxtaparanodal particles inthe adaxonal Schwann cell membrane (Stolinski andBreathnach 1982; Stolinski et al. 1981). Perhaps theseare the inwardly rectifying and delayed rectifying “par-anodal” K+ channels that have been identified electro-physiologically (Chiu 1991), but whose molecular iden-

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Fig. 9A–C OSP/claudin-11 is localized to the radial component ofcompact myelin. A Freeze-fracture electron microscopy of amouse brain. A series of interlamellar strands (arrows, and en-larged in the inset) are found between each layer of the compactmyelin sheath. B, C Immunoelectron microscopy localizes OSP/claudin-11 (arrows) to the interlamellar strands of freeze-fracturedmouse brain (B) and optic nerve (C). The asterisks mark axons.From Morita et al. (1999), with permission of The RockefellerUniversity Press

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Fig. 10 A An artistic view of the node and paranode. The nodalaxolemma has many large particles, and the paranodal axolemmahas rows of particles in register with the rows of particles in theparanodal loops. Transcellular bridges connect the Schwann cellmicrovilli, the node, and the paranodal loops to the paranodal axo-

lemma. From Ichimura and Ellisman (1991), with permission ofKluwer Academic Press. B Lanthanum diffuses between the sep-tate-like junctions of CNS axons. From Hirano and Dembitzer(1982), with permission of Kluwer Academic Press

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Fig. 12A–H Laser scanningconfocal analysis of Caspr andKv1.1 in the paranodal region.This myelinated fiber wasteased from an adult mouse sciatic nerve and labeled with arabbit antiserum against Caspr(rhodamine), a mouse mono-clonal antibody against Kv1.1(fluorescein), and a rat mono-clonal antibody against NF-H(cyanine 5; blue). For clarity,the labeling from each antibodyis shown separately in panelsA–E; panels F, G show themerged images; panels B, D,G, H are enlargements. Notethe separation of Caspr andKv1.1 staining at the paranode(large arrows) and juxtaparan-ode (large arrowheads), andthat the spiral of Caspr stainingin the juxtaparanodal regionfills a void in the Kv1.1 stain-ing. In the internodal region,the double line of Kv1.1 stain-ing flanks the single line ofCaspr staining. NF-H stainingis diminished at the node (dou-ble arrowheads). Scale bars 5µm. From Arroyo et al. (1999),with permission of KluwerAcademic Press

Fig. 11 Laser scanning confocal micrograph of Na+ and K+ chan-nels. A myelinated fiber teased from rat sciatic nerve was labeledwith a rabbit antiserum against voltage-gated Na+ channels (rho-damine) and a monoclonal antibody against Kv1.2 (fluorescein).

Na+ channels are restricted to the node (double arrowheads),where Kv1.2 channels are found in the juxtaparanodal region (ar-rowheads) and apposed to incisures (arrowhead) and the innermesaxon (arrow)

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Fig. 13A–F Laser scanningconfocal analysis of Caspr inthe internodal region. Thismyelinated fiber was teasedfrom an adult mouse sciaticnerve and was labeled with arabbit antiserum against Caspr(rhodamine), a mouse mono-clonal antibody against Kv1.1(fluorescein), and a rat mono-clonal antibody against NF-H(cyanine 5; blue). For clarity,the labeling from each antibodyis shown separately in panelsA–C; panels D–F show themerged images. Note the dou-ble line of Kv1.1 stainingflanking the single line ofCaspr staining, and the dimin-ished NF-H staining at an inci-sure (small arrowhead). Scalebars 5 µm. From Arroyo et al.(1999), with permission ofKluwer Academic Press

tity remains to be established. These juxtaparanodal K+

channels have been posited to siphon K+ that accu-mulates in the periaxonal space during neural activity(Konishi 1990). Once inside the adaxonal cytoplasm ofmyelinating Schwann cells, the K+ could diffuse throughgap junctions in the paranodes and incisures (Balice-Gordon et al. 1998; Konishi 1990). Just as the spatialbuffering of the K+ has been considered an importantfunction of astrocytes in the CNS (Orkand et al. 1966), itmay be an essential role of gap junctions in the PNS my-elin sheath.

A homologue of Caspr, Caspr2, was discovered in hu-man and mouse brain cDNA libraries (Nagase et al.1998; Poliak et al. 1999). It has a similar domain struc-ture to Caspr, especially in the extracellular region, butthe most carboxy terminal aspect of its intracellular do-main contains a PDZ domain, like neurexins. Caspr2 islocalized to the juxtaparanodes of myelinated fibers inboth the CNS and the PNS, colocalizing withKv1.1/1.2/β2. Furthermore, Caspr2 appears to be com-plexed with Kv1.1 and Kv1.2, probably mediated by aPDZ protein. Although transcellular connections be-tween the juxtaparanodal axonal membrane and the my-elin sheath have not been described, it is possible thatCaspr2 has a binding partner, as has been posited forCaspr.

The internodal region

The internodal axonal membrane lacks the conspicuousspecializations of the nodal region. Nevertheless, in thePNS, intramembranous particles similar to those of thejuxtaparanodal region are found apposing the internalmesaxon and incisures of the myelin sheath (Stolinski etal. 1985). In accord with these findings, we observedthat the internodal membranes of PNS axons have a tri-partite strand, consisting of a central strand of Casprstaining, flanked on either side by a strand ofKv1.1/1.2/β2 immunoreactivity (Fig. 13). Furthermore,there is a circumferential ring composed of the same tri-partite elements apposed to the innermost aspect of inci-sures (Arroyo et al. 1999). Whether Caspr2 is also foundalong with Kv1.1/1.2/β2 at these locations remains to bedetermined. The tripartite organization of Caspr andKv1.1/Kv1.2/β2 suggests the possibility that Caspr (orCaspr2) could be localized to the internodal membraneby a trans interaction with a protein expressed by themyelinating Schwann cell, and recruit the other proteinsby cis interactions. Finally, the size and distribution of intramembranous particles of the adaxonal Schwanncell membrane, at both the incisures and the inner me-saxon, are similar to those of the juxtaparanodal region(Stolinski and Breathnach 1982; Stolinski et al. 1981,1985), indicating that the trans interactions that occur atjuxtaparanodes extend along these specialized portionsof the internodal region, too.

In our initial analysis of myelinated CNS axons, it ap-pears that Caspr may appose the inner mesaxon as notedby Menegoz et al. (1997), whereas Kv1.1, Kv1.2, andKvβ2 are not focally localized in the internodal region(Arroyo and Scherer unpublished observations).

Conclusion

In summary, the intricate localization of numerous ax-onal proteins is highly related to the structure of theoverlying myelin sheath. The most parsimonious expla-nation for their precise localization is that the myelinsheath organizes them; the converse seems unlikely.These data provide strong evidence that the myelinsheath organizes the underlying axonal membrane andprovide further evidence for a central role of Schwanncells in the development of axonal specializations.

Acknowledgements Our work was supported by the NIH(NS37100, NS34528, and NS08075) and the MDA. I thank Drs.David Colman, Mark Ellisman, Susan Hall, and Shoichiro Tsukita,for contributing figures, and especially our colleagues and collab-orators, Drs. Rita Balice-Gordon, Linda Bone, Bill Chiu, SuzanneDeschênes, Kurt Fischbeck, Albee Messing, David Paul, OriPeles, and Lei Zhou.

References

Adelman JP, Bond CT, Pessia M, Maylie J (1995) Episodic ataxiaresults from voltage-dependent potassium channels with al-tered functions. Neuron 15:1449–1454

Adlkofer K, Martini R, Aguzzi A, Zielasek J, Toyka KV, Suter U(1995) Hypermyelination and demyelinating peripheral neu-ropathy in pmp22-deficient mice. Nat Genet 11:274–280

Adlkofer K, Frei R, Neuberg DH-H, Zielasek J, Toyka KV, SuterU (1997) Heterozygous peripheral myelin protein 22-deficientmice are affected by a progressive demyelinating peripheralneuropathy. J Neurosci 17:4662–4671

Anderson TJ, Montague P, Nadon N, Nave KA, Griffiths IR(1997) Modification of Schwann cell phenotype with Plptransgenes: evidence that the PLP and DM 20 isoproteins aretargeted to different cellular domains. J Neurosci Res 50:13–22

Ariyasu RG, Nichol JA, Ellisman MH (1985) Localization of so-dium/potassium adenosine triphosphatase in multiple celltypes of the murine nervous system with antibodies raisedagainst the enzyme from kidney. J Neurosci 5:2581–2596

Arroyo EJ, Xu Y-T, Zhou L, Messing A, Peles E, Chiu SY, Scherer SS (1999) Myelinating Schwann cells determine theinternodal localization of Kv1.1, Kv1.2, Kvβ2, and Caspr. JNeurocytol 28:333–347

Auld VJ, Fetter RD, Broadie K, Goodman CS (1995) Gliotactin, anovel transmembrane protein on peripheral glia, is required toform the blood-nerve barrier in Drosophia. Cell 81:757–767

Balice-Gordon RJ, Bone LJ, Scherer SS (1998) Functional gapjunctions in the Schwann cell myelin sheath. J Cell Biol142:1095–1104

Bartsch U, Pesheva P, Raff M, Schachner M (1993) Expression ofjanusin (J1–160/180) in the retina and optic nerve of the de-veloping and adult mouse. Glia 9:57–69

Baumgartner S, Littleton JT, Broadie K, Bhat MA, Harbecke R,Lengyel JA, Chiquet-Ehrismann R, Prokop A, Bellen HJ(1996) A Drosophila neurexin is required for septate junctionand blood-nerve barrier formation and function. Cell 87:1059–1068

14

Bellen HJ, Lu Y, Beckstead R, Bhat MA (1998) Neurexin IV,Caspr and paranodin – novel members of the neurexin family:encounters of axons and glia. Trends Neurosci 21:444–449

Bennett V, Lambert S, Davis JQ, Zhang X (1997) Molecular archi-tecture of the specialized axonal membrane at the node ofRanvier. Soc Gen Physiol 52:107–120

Bergoffen J, Scherer SS, Wang S, Oronzi-Scott M, Bone L, PaulDL, Chen K, Lensch MW, Chance P, Fischbeck K (1993) Con-nexin mutations in X-linked Charcot-Marie-Tooth disease.Science 262:2039–2042

Berthold C-H, Rydmark M (1995) Morphology of normal periph-eral axons. In: Waxman SG, Kocsis JD, Stys PK (eds) The ax-on. Oxford University Press, New York pp 13–48

Black JA, Waxman SG (1988) The perinodal astrocyte. Glia1:169–183

Blanchard CE, Mackenzie ML, Sikri K, Allt G (1985) Filipin-ste-rol complexes at nodes of Ranvier. J Neurocytol 14:1053–1062

Bosio A, Binczek E, Stoffel W (1996) Functional breakdown ofthe lipid bilayer of the myelin membrane in central and pe-ripheral nervous system myelin by disrupted galactocerebro-side synthesis. Proc Natl Acad Sci USA 93:13280–13285

Bosio A, Bussow H, Adam J, Stoffel W (1998) Galactosphingolip-ids and axono-glial interaction in myelin of the central nervoussystem. Cell Tissue Res 292:199–210

Bronstein JM, Micevych PE, Chen K (1997) Oligodendrocyte-specific protein (OSP) is a major component of CNS myelin. JNeurosci Res 50:713–720

Browne DL, Gancher ST, Nutt JG, Brunt ERP, Smith EA, KramerP, Litt M (1994) Episodic ataxia/myokymia syndrome is asso-ciated with point mutations in the human potassium channelgene, KCNA1. Nat Genet 8:136–140

Brunner C, Lassmann H, Waehneldt TV, Matthieu J-M, LiningtonC (1989) Differential ultrastructural localization of myelin ba-sic protein, myelin/oligodendrocyte glycoprotein, and 2’,3’-cyclic nucleotide 3’-phosphodiesterase in the CNS of adultrats. J Neurochem 52:296–304

Brunt ERP, Van Weerden TW (1990) Familial paroxysmal kinesi-genic ataxia and continuous myokymia. Brain 113:1361–1382

Bunge MB (1993) Schwann cell regulation of extracellular matrixbiosynthesis and assembly. In: Dyck PJ, Thomas PK, Low PA,Poduslo JF (eds) Peripheral neuropathy, 3rd edn, vol 1. Saun-ders, Philadelphia, pp 299–316

Burgess DL, Kohrman DC, Galt J, Plummer NW, Jones JM, SpearB, Meisler MH (1995) Mutation of a new sodium channelgene, Scn8a, in the mouse mutant ‘motor endplate disease’.Nat Genet 10:461–465

Butt AM, Duncan A, Hornby MF, Kirvell SL, Hunter A, LevineJM, Berry M (1999) Cells expressing the NG2 antigen contactnodes of Ranvier in adult CNS white matter. Glia 26:84–91

Chandross KJ, Kessler JA, Cohen RI, Simburger E, Spray DC,Bieri P, Dermietzel R (1996) Altered connexin expression af-ter peripheral nerve injury. Mol Cell Neurosci 7:501–518

Chiu SY (1991) Functions and distribution of voltage-gated sodi-um and potassium channels in mammalian Schwann cells.Glia 4:541–558

Chiu SY, Ritchie JM (1980) Potassium channels in nodal and in-ternodal axonal membrane of mammalian myelinated fibres.Nature 284:170–171

Coetzee T, Fujita N, Dupree J, Shi R, Blight A, Suzuki K, SuzukiK, Popko B (1996) Myelination in the absence of galactocere-broside and sulfatide: normal structure with abnormal functionand regional instability. Cell 86:209–219

Coetzee T, Dupree JL, Popko B (1998) Demyelination and alteredexpression of myelin-associated glycoprotein isoforms in thecentral nervous system of galactolipid-deficient mice. J Neu-rosci Res 54:613–622

Davis JQ, Bennett V (1994) Ankyrin binding activity shared bythe neurofascin/L1/NrCAM family of nervous system cell ad-hesion molecules. J Biol Chem 269:27163–27166

Davis JQ, McLaughlin T, Bennett V (1993) Ankyrin-binding pro-teins related to nervous system cell adhesion molecules: candi-

dates to provide transmembrane and intercellular connectionsin adult brain. J Cell Biol 121:121–133

Davis JQ, Lambert S, Bennett V (1996) Molecular composition ofthe node of Ranvier: identification of ankyrin-binding cell ad-hesion molecules neurofascin (mucin+ third FNIII domain-)and NrCAM at nodal axon segments. J Cell Biol 135:1355–1367

Dermietzel R, Farooq M, Kessler JA, Althaus H, Hertzberg EL,Spray DC (1997) Oligodendrocytes express gap junction pro-teins connexin32 and connexin45. Glia 20:101–114

Devor M, Govrin-Lippmann R, Angelides K (1993) Na+ channelimmunolocalization in peripheral mammalian axons andchanges following nerve injury and neuroma formation. JNeurosci 13:1976–1992

Ding Y, Brunden KR (1995) The cytoplasmic domain of myelinglycoprotein P0 interacts with negatively charged phospholipidbilayers. J Biol Chem 269:10764–10770

Duchen LW, Stefani E (1971) Electrophysiological studies of neu-romuscular transmission hereditary “motor end-plate disease”of the mouse. J Physiol 212:535–548

Dupree JL, Coetzee T, Blight A, Suzuki K, Popko B (1998) Myelin galactolipids are essential for proper node of Ranvierformation in the CNS. J Neurosci 18:1642–1649

D’Urso D, Ehrhardt P, Muller HW (1999) Peripheral myelin pro-tein 22 and protein zero: a novel association in peripheral ner-vous system myelin. J Neurosci 19:3396–3403

Einheber S, Milner T, Giancotti F, Salzer J (1993) Axonal regula-tion of Schwann cell integrin expression suggests a role forα6β4 in myelination. J Cell Biol 123:625–638

Einheber S, Zanazzi G, Ching W, Scherer SS, Milner TA, Peles E,Salzer JL (1997) The axonal membrane protein Caspr/neure-xin IV is a component of the septate-like paranodal junctionsthat assemble during myelination. J Cell Biol 139:1495–1506

Ellisman MH, Levinson SR (1982) Immunocytochemical localiza-tion of sodium channel distributions in the excitable mem-branes of Electrophorus electricus. Proc Natl Acad Sci USA79:6707–6711

Elmer LW, Black JA, Waxman SG, Angelides KJ (1990) The volt-age-dependent sodium channel in mammalian CNS and PNS:antibody characterization and immunocytochemical localiza-tion. Brain Res 532:222–231

Fanarraga ML, Griffiths IR, Zhao M, Duncan ID (1998) Oligoden-drocytes are not inherently programmed to myelinate a specif-ic size of axon. J Comp Neurol 399:94–100

Fannon AM, Sherman DL, Ilyina-Gragerova G, Brophy PJ, Friedrich VL, Colman DR (1995) Novel E-cadherin mediatedadhesion in peripheral nerve: Schwann cell architecture is sta-bilized by autotypic adherens junctions. J Cell Biol 129:189–202

Feltri ML, Scherer SS, Nemni R, Kamholz J, Vogelbacker H, Oro-nzi-Scott M, Canal N, Quaranta V, Wrabetz L (1994) β4 inte-grin expression in myelinating Schwann cells is ab-axonallypolarized, developmentally-regulated, and axonally-depen-dent. Development 120:1287–1301

ffrench-Constant C, Miller RH, Kruse J, Schachner M, Raff MC(1986) Molecular specialization of astrocyte processes atnodes of Ranvier in rat optic nerve. J Cell Biol 102:844–852

Frank M, Haar ME van der, Schaeren-Wiemers N, Schwab ME(1998) rMAL is a glycosphingolipid-associated protein of my-elin and apical membranes of epithelial cells in kidney andstomach. J Neurosci 18:4901–4913

Friede RL, Bischhausen R (1980) The precise geometry of largeinternodes. J Neurol Sci 48:367–381

Funch PG, Faber DS (1984) Measurement of myelin sheath resis-tances: implications for axonal conduction and pathophysiolo-gy. Science 225:538–540

Garbern JY, Cambi F, Tang XM, Sima AAF, Vallat JM, BoschEP, Lewis R, Shy M, Sohi J, Kraft G, Chen KL, Joshi I,Leonard DGB, Johnson W, Raskind W, Dlouhy SR, Pratt V,Hodes ME, Bird T, Kamholz J (1997) Proteolipid protein isnecessary in peripheral as well as central myelin. Neuron 19:205–218

15

Garver TD, Ren Q, Tuvia S, Bennett V (1997) Tyrosine phosphor-ylation at a site highly conserved in the L1 family of cell adhe-sion molecules abolishes ankyrin binding and increases lateralmobility of neurofascin. J Cell Biol 137:703–714

Giese KP, Martini R, Lemke G, Soriano P, Schachner M (1992)Mouse P0 gene disruption leads to hypomyelination, abnormalexpression of recognition molecules, and degeneration of my-elin and axons. Cell 71:565–576

Gould RM, Mattingly G (1990) Regional localization of RNA andprotein metabolism in Schwann cells in vivo. J Neurocytol19:285–301

Gulbis JM, Mann S, MacKinnon R (1999) Structure of a voltage-dependent K+ channel beta subunit. Cell 97:943–952

Haimovich B, Bonilla E, Casadei J, Barchi RL (1984) Immuno-cytochemical localization of the mammalian voltage-depen-dent sodium channel using polyclonal antibodies against thepurified protein. J Neurosci 4:2259–2268

Hall SM, Williams PL (1970) Studies on the ‘incisures’ ofSchmidt and Lanterman. J Cell Sci 6:767–791

Hanemann CO, Muller HW (1998) Pathogenesis of Charcot-Marie-Tooth IA (CMTIA) neuropathy. Trends Neurosci 21:282–286

Hildebrand C, Bowe CM, Remahl IN (1994) Myelination and my-elin sheath remodelling in normal and pathological PNS nervefibres. Prog Neurobiol 43:85–141

Hirano A, Dembitzer HM (1982) Further studies on the transversebands. J Neurocytol 11:861–866

Hirano A, Becker NH, Zimmerman HM (1969) Isolation of theperiaxonal space of the central myelinated nerve fiber with re-gard to the diffusion of peroxidase. J Histochem Cytochem17:512–516

Holz A, Schaeren N, Schaefer C, Pott U, Colello RJ, Schwab ME(1996) Molecular and developmental characterization of novelcDNAs of the myelin-associated/oligodendrocytic basic pro-tein. J Neurosci 16:467–477

Hopkins WF, Allen ML, Mouamed KM, Tempel BL (1994) Prop-erties of voltage-gated K+ currents expressed in Xenopus oo-cytes by mKv1.1, mKv1.2 and their heteromultimers as re-vealed by mutagenesis of the dendrotoxin-binding site inKv1.1. Pflügers Arch 428:382–390

Hudson L (1990) Molecular biology of myelin proteins in the cen-tral and peripheral nervous systems. Semin Neurosci 2:487–496

Ichimura T, Ellisman MH (1991) Three-dimensional fine structureof cytoskeletal-membrane interactions at nodes of Ranvier. JNeurocytol 20:667–681

Ichtchenko K, Nguyen T, Sudhof TC (1996) Structures, alternativesplicing, and neurexin binding of multiple neuroligins. J BiolChem 271:2676–2682

Inoue K, Osaka H, Imaizumi K, Nezu A, Takanashi J, Arii J, Murayama K, Ono J, Kikawa Y, Mito T, Shaffer LG, LupskiJR (1999) Proteolipid protein gene duplications causing Pelizaeus-Merzbacher disease: molecular mechanism and phe-notypic manifestations. Ann Neurol 45:624–632

Johns TG, Bernard CCA (1999) The structure and function of my-elin oligodendrocyte glycoprotein. J Neurochem 72:1–9

Kirschner DA, Ganser AL (1980) Compact myelin exists in theabsence of basic protein in the shiverer mutant mouse. Nature283:207–210

Kirschning E, Rutter G, Hohenberg H (1998) High-pressure freez-ing and freeze-substitution of native rat brain: suitability forpreservation and immunoelectron microscopic localization ofmyelin glycolipids. J Neurosci Res 53:465–474

Kocsis JD, Ruiz JA, Waxman SG (1983) Maturation of mammali-an myelinated fibers: changes in action-potential characteris-tics following 4-aminopyridine application. J Neurophysiol50:449–463

Koenig E, Repasky E (1985) A regional analysis of α-spectrin inthe isolated Mauthner neuron and in isolated axons of thegoldfish and rabbit. J Neurosci 5:705–714

Konishi T (1990) Voltage-gated potassium currents in myelinatingSchwann cells in the mouse. J Physiol 431:123–139

Kordeli E, Davis J, Trapp B, Bennett V (1990) An isoform of an-kyrin is localized at nodes of Ranvier in myelinated axons ofcentral and peripheral nerves. J Cell Biol 110:1341–1352

Kordeli E, Lambert S, Bennett V (1995) AnkyrinG: a new ankyringene with neural-specific isoforms localized at the axonal ini-tial segment and node of Ranvier. J Biol Chem 270:2352–2359

Kreusch A, Pfaffinger PJ, Stevens CF, Choe S (1998) Crystalstructure of the tetramerization domain of the Shaker potassi-um channel. Nature 392:945–948

Kunzelmann P, Blumcke I, Traub O, Dermietzel R, Willecke K(1997) Coexpression of connexin45 and -32 in oligodendro-cytes of rat brain. J Neurocytol 26:17–22

Lambert S, Davis JQ, Bennett V (1997) Morphogenesis of thenode of Ranvier: co-clusters of ankyrin and ankyrin-bindingintegral proteins define early developmental intermediates. JNeurosci 17:7025–7036

Li J, Hertzberg EL, Nagy JI (1997) Connexin32 in oligodendro-cytes and association with myelinated fibers in mouse and ratbrain. J Comp Neurol 379:571–591

MacKenzie ML, Ghabriel MN, Allt G (1984) Nodes of Ranvierand Schmidt-Lanterman incisures: an in vivo lanthanum tracerstudy. J Neurocytol 13:1043–1055

Mambetisaeva ET, Gire V, Evans WH (1999) Multiple connexinexpression in peripheral nerve, Schwann cells, and Schwanno-ma cells. J Neurosci Res 57:166–175

Martini R, Mohajeri MH, Kasper S, Giese KP, Schachner M(1995a) Mice doubly deficient in the genes for P0 and myelinbasic protein show that both proteins contribute to the forma-tion of the major dense line in peripheral nerve myelin. J Neu-rosci 15:4488–4495

Martini R, Zielasek J, Toyka KV, Giese KP, Schachner M (1995b)Protein zero (P0)-deficient mice show myelin degeneration inperipheral nerves characteristic of inherited human neuropat-hies. Nat Genet 11:281–285

Mata M, Fink DJ, Ernst SA, Segal GJ (1991) Immunocytochemi-cal demonstration of Na+, K+-ATPase in internodal axolemmaof myelinated fibers of rat sciatic and optic nerves. J Neuro-chem 57:184–192

Menegoz M, Gaspar P, Le Bert M, Galvez T, Burgaya F, PalfreyC, Ezan P, Arnos F, Girault J-A (1997) Paranodin, a glycopro-tein of neuronal paranodal membranes. Neuron 19:319–331

Mi HY, Deerinck TJ, Ellisman MH, Schwarz TL (1995) Differen-tial distribution of closely related potassium channels in ratSchwann cells. J Neurosci 15:3761–3774

Mi HY, Deerinck TJ, Jones M, Ellisman MH, Schwarz TL (1996)Inwardly rectifying K+ channels that may participate in K+

buffering are localized in microvilli of Schwann cells. J Neu-rosci 16:2421–2429

Miller RG, Da Silva PP (1977) Particle rosettes in the periaxonalSchwann cell membrane and particle clusters in the axolemmaof rat sciatic nerve. Brain Res 130:135–141

Missler M, Fernandez-Chacon R, Sudhof TC (1998) The makingof neurexins. J Neurochem 71:1339–1347

Morita K, Sasaki H, Fujimoto K, Furuse M, Tsukita S (1999)Claudin-11/OSP-based tight junctions of myelin sheaths inbrain and Sertoli cells in testis. J Cell Biol 145:579–588

Murakami T, Garcia CA, Reiter LT, Lupski JR (1996) Charcot-Marie-Tooth disease and related inherited neuropathies. Medi-cine 75:233–250

Nagafuchi A, Tsukita S, Takeichi M (1993) Transmembrane con-trol of cadherin-mediated cell–cell adhesion. Semin Cell Biol4:175–181

Nagaoka T, Oyamada M, Okajima S, Takamatsu T (1999) Differ-ential expression of gap junction proteins connexin26, 32, and43 in normal and crush-injured rat sciatic nerves: close rela-tionship between connexin43 and occludin in the perineurium.J Histochem Cytochem 47:937–948

Nagase T, Ishikawa K, Suyama M, Kikuno R, Hirosawa M, Miyajima N, Tanaka A, Kotani H, Nomura N, Ohara O (1998)Prediction of the coding sequences of unidentified human genesXII. The complete sequences of 100 new cDNA clones frombrain which code for large proteins in vitro. DNA Res 5:355–364

16

Nave K-A, Boespflug-Tanguy O (1996) Developmental defects ofmyelin formation: from X-linked mutations to human dysmy-elinating diseases. Neuroscientist 2:33–43

Novakovic S, Tzoumaka E, Tischler A, Sangameswaran L, EglenR, Hunter J (1999) Sodium channel isoform localization in theperipheral nerve and changes following injury. Soc NeurosciAbstr 25:1732

Orkand PM, Nicholls JG, Kuffler SW (1966) Effect of nerve im-pulses on the membrane potential of glial cells in the centralnervous system of amphibia. J Neurophysiol 29:788–806

Pastor A, Kremer M, Moller T, Kettenmann H, Dermietzel R(1998) Dye coupling between spinal cord oligodendrocytes:differences in coupling efficiency between gray and whitematter. Glia 24:108–120

Peters A, Palay SL, Webster HD (1991) The fine structure of thenervous system, 3rd edn. Oxford University Press, New York,pp 494

Poliak S, Gollan L, Martinez R, Custer A, Einheber S, Salzer JL,Trimmer J, Shrager P, Peles E (1999) Caspr2, a new memberof the neurexin superfamily is localized at the juxtaparanodesof myelinated axons and associates with K+ channels. Neuron24:1037–1047

Quick DC, Waxman SG (1977) Ferric ion, ferrocyanide, and inor-ganic phosphate as cytochemical reactants at peripheral nodesof Ranvier. J Neurocytol 6:555–570

Raine CS (1982) Differences between the nodes of Ranvier oflarge and small diameter fibres in the P.N.S. J Neurocytol 11:935–947

Rambukkana A, Yamada H, Zanazzi G, Mathus T, Salzer JL, Yurchenco PD, Campbell KP, Fischetti VA (1998) Role of α-dystroglycan as a Schwann cell receptor for Mycobacteriumleprae. Science 282:2076–2079

Ramon y Cajal S (1928) Degeneration and regeneration of the ner-vous system. May RM (ed) Oxford University Press, Oxford

Rasband M, Trimmer JS, Schwarz TL, Levinson SR, EllismanMH, Schachner M, Shrager P (1998) Potassium channel distri-bution, clustering, and function in remyelinating rat axons. JNeurosci 18:36–47

Rash JE, Yasumura T, Dudek FE, Nagy JI (1999) Oligodendro-cytes have gap junctions almost exclusively with astrocytes:direct demonstration by freeze-fracture immunogold labelingof connexins Cx43, Cx30, Cx32, and Cx45. Soc NeurosciAbstr 25:1506

Revel J-P, Hamilton DW (1969) The double nature of the intermedi-ate dense line in peripheral nerve myelin. Anat Rec 163:7–16

Rieger F, Pincon-Raymond M, Lombet A, Ponzio G, LazdunskiM, Sidman RL (1984) Paranodal dysmyelination and increasein tetrodotoxin binding sites in the sciatic nerve of the motorend-plate disease (med/med) mouse during postnatal develop-ment. Dev Biol 101:401–409

Rieger F, Daniloff JK, Pincon-Raymond M, Crossin KC, GrumetM, Edelman GM (1986) Neuronal cell adhesion molecules andcytotactin are colocalized at the node of Ranvier. J Cell Biol103:379–391

Ritchie JM (1995) Physiology of axons. In: Waxman SG, KocsisJD, Stys PK (eds) The axon. Oxford University Press, NewYork, pp 68–96

Ritchie JM, Rogart RP (1977) Density of sodium channels inmammalian myelinated nerve fibers and nature of the axonalmembrane under the myelin sheath. Proc Natl Acad Sci USA74:211–215

Ritchie JM, Black JA, Waxman SG, Angelides KJ (1990) Sodiumchannels in the cytoplasm of Schwann cells. Proc Natl AcadSci USA 87:9290–9294

Robinson, SR, Hampson ECGM, Munro MN, Vaney DI (1993)Unidirectional coupling of gap junctions between neuroglia.Science 262:1072–1074

Rosenbluth J (1976) Intramembranous particle distribution at thenode of Ranvier and adjacent axolemma in myelinated axonsof the frog brain. J Neurocytol 5: 731–745

Rosenbluth J (1980) Peripheral myelin in the mouse mutant shiv-erer. J Comp Neurol 193:729–739

Saito F, Masaki T, Kamakura K, Anderson LVB, Fujita S, FukutaOhi H, Sunada Y, Shimizu T, Matsumura K (1999)Characterization of the transmembrane molecular architectureof the dystroglycan complex in Schwann cells. J Biol Chem274:8240–8246

Sandri C, Van Buren JM, Akert K (1982) Membrane morphologyof the vertebrate nervous system. Prog Brain Res 46:201–265

Sawada N, Ishida H, Collins BE, Schnaar RL, Kiso M (1999)Ganglioside GD1 alpha analogues as high-affinity ligands formyelin-associated glycoprotein (MAG). Carbohydr Res 316:1–5

Schaeren-Wiemers N, Valenzuela DM, Frank M, Schwab ME(1995) Characterization of a rat gene, rMAL, encoding a pro-tein with four hydrophobic domains in central and peripheralmyelin. J Neurosci 15:5753–5764

Scherer SS (1996) Molecular specializations at nodes and paran-odes in peripheral nerve. Microsc Res Technique 34:452–461

Scherer SS (1997) Molecular genetics of demyelination: newwrinkles on an old membrane. Neuron 18:13–16

Scherer SS, Deschênes SM, Xu Y-T, Grinspan JB, Fischbeck KH,Paul DL (1995) Connexin32 is a myelin-related protein in thePNS and CNS. J Neurosci 15:8281–8294

Schiff R, Rosenbluth J (1995) Distribution of myelin lipid anti-gens in adult and developing rat spinal cord. Brain Res 686:143–149

Schwartz M, Ernst SA, Siegel GJ, Agranoff B (1981) Immuno-cytochemical localization of (Na+, K+)-ATPase in the goldfishoptic nerve. J Neurochem 36:107–115

Shapiro L, Fannon AM, Kwong PD, Thompson A, Lehmann MS,Grubel G, Legrand J-F, Als-Nielsen J, Colman DR, Hendrick-son WA (1995) Structural basis of cell–cell adhesion by cad-herins. Nature 374:327–337

Shapiro L, Doyle JP, Hensley P, Colman DR, Hendrickson WA(1996) Crystal structure of the extracellular domain from P0,the major structural protein of peripheral nerve myelin. Neu-ron 17:435–449

Shy ME, Arroyo E, Sladky J, Menichella D, Jiang H, Kamholz J,Scherer SS (1997) Heterozygous P0 knockout mice develop aperipheral neuropathy that resembles chronic inflammatorydemyelinating polyneuropathy (CIDP). J Neuropathol ExpNeurol 56:811–821

Smart SL, Lopantsev V, Zhang CL, Robbins CA, Wang H, ChiuSY, Schwartzkroin PA, Messing A, Tempel BL (1998) Dele-tion of the Kv1.1 potassium channel causes epilepsy in mice.Neuron 20:809–819

Srinivasan Y, Elmer L, Davis H, Bennett V, Angelides K (1988)Ankyrin and spectrin associate with voltage-dependent sodiumchannels in brain. Nature 333:177–180

Stolinski C, Breathnach AS (1982) Freeze-fracture replication ofmammalian peripheral nerve – a review. J Neurol Sci 57:1–28

Stolinski C, Breathnach AS, Martin B, Thomas PK, King RMH,Gabriel G (1981) Associated particle aggregates in juxtaparan-odal axolemma and adaxonal Schwann cell membrane of ratperipheral nerve. J Neurocytol 10:679–691

Stolinski C, Breathnach AS, Thomas PK, Gabriel G, King RMH(1985) Distribution of particle aggregates in the internodalaxolemma and adaxonal Schwann cell membrane of rodent pe-ripheral nerve. J Neurol Sci 67:213–222

Suter U, Snipes GJ (1995) Biology and genetics of hereditary mo-tor and sensory neuropathies. Annu Rev Neurosci 18:45–75

Tao-Cheng J-H, Rosenbluth J (1984) Extranodal particle accumu-lations in the axolemma of myelinated frog optic axons. BrainRes 308: 289–300

Thomas PK, Berthold C-H, Ochoa J (1993) Microscopic anatomyof the peripheral nervous system. In: Dyck PJ, Thomas PK,Griffin JW, Low PA, Poduslo JF (eds) Peripheral neuropathy,3rd edn. Saunders, Philadelphia, pp 28–91

Trapp BD, Andrew SB, Cootauco C, Quarles R (1989a) The my-elin-associated glycoprotein is enriched in multivesicular bod-ies and periaxonal membrane of actively myelinating oligo-dendrocytes. J Cell Biol 109:2417–2425

17

Trapp BD, Andrews SB, Wong A, O’Connell M, Griffin JW(1989b) Co-localization of the myelin-associated glycoproteinand the microfilament components, F-actin and spectrin, inSchwann cells of myelinated nerve fibers. J Neurocytol 18:47–60

Vabnick I, Shrager P (1998) Ion channel redistribution and func-tion during development of the myelinated axon. J Neurobiol37:80–96

Vallat JM, Sindou P, Preux PM, Tabaraud F, Milor AM, CouratierP, Leguern E, Brice A (1996) Ultrastructural PMP22 expres-sion in inherited demyelinating neuropathies. Ann Neurol39:813–817

Van Dyke DH, Griggs RC, Murphy MJ, Goldstein MN (1975) He-reditary myokymia and periodic ataxia. J Neurol Sci 25:109–118

Vorbrodt AW, Lossinsky AS, Wisneiwski HM (1982) Cytochemi-cal localization of ouabain-sensitive, K+-dependent p-nitro-phenylphosphatase (transport ATPase) in the mouse centraland peripheral nervous systems. Brain Res 243:225–234

Wang H, Kunkel DD, Martin TM, Schwartkroin PA, Tempel BL(1993) Heteromultimeric K+ channels in terminal and juxta-paranodal regions of neurons. Nature 365:75–79

Waxman SG, Black JA (1987) Macromolecular structure of theSchwann cell membrane. Perinodal microvilli. J Neurol Sci77:23–34

Wood JG, Jean DH, Whitaker JN, McLaughlin BJ, Albers RW(1977) Immunocytochemical localization of the sodium, po-tassium activated ATPase in knifefish brain. J Neurocytol6:571–581

Xiao ZC, Ragsdale DS, Malhotra JD, Mattei LN, Braun PE,Schachner M, Isom LL (1999) Tenascin-R is a functionalmodulator of sodium channel beta subunits. J Biol Chem274:26511–26517

Yamamoto Y, Yoshikawa H, Nagano S, Kondoh G, Sadahiro S,Gotow T, Yanagihara T, Sakoda S (1999) Myelin-associatedoligodendrocytic basic protein is essential for normal arrange-ment of the radial component in central nervous system my-elin. Eur J Neurosci 11:847–855

Yang LJS, Zeller CB, Shaper NL, Kiso M, Hasegawa A, ShapiroRE, Schnaar RL (1996) Gangliosides are neuronal ligands formyelin-associated glycoprotein. Proc Natl Acad Sci USA93:814–818

Yoshimura T, Satake M, Kobayashi T (1996) Connexin43 is an-other gap junction protein in the peripheral nervous system. JNeurochem 67:1252–1258

Zerr P, Adelman JP, Maylie J (1998) Episodic ataxia mutations inKv1.1 alter potassium channel function by dominant negativeeffects or haploinsufficiency. J Neurosci 18:2842–2848

Zhang X, Bennett V (1996) Identification of O-linked N-acetyl-glucosamine modification of ankyrinG isoforms targeted tonodes of Ranvier. J Biol Chem 271:31391–31398

Zhao S, Spray DC (1998) Localization of Cx26, Cx32 and Cx43in myelinating Schwann cells of mouse sciatic nerve duringpostnatal development. In: Werner R (ed) Gap junctions. IOSPress, Amsterdam, pp 198–202

Zhou DX, Lambert S, Malen PL, Carpenter S, Boland LM, Ben-nett V (1998a) AnkyrinG is required for clustering of voltage-gated Na channels at axon initial segments and for normal ac-tion potential firing. J Cell Biol 143:1295–1304

Zhou L, Zhang CL, Messing A, Chiu SY (1998b) Temperature-sensitive neuromuscular transmission in Kv1.1 null mice: roleof potassium channels under the myelin sheath in youngnerves. J Neurosci 18:7200–7215

Zielasek J, Martini R, Toyka KV (1996) Functional abnormalitiesin P0-deficient mice resemble human hereditary neuropathieslinked to P0 gene mutations. Muscle Nerve 19:946–952

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