Axo–glial interactions result in a highly segregated distribution of membrane proteins, defining distinct domains of the axolemma. The mechanisms leading to the enrichment of Na+
channels and associated proteins at the nodes of Ranvier, as well as those involved in the formation of paranodal axo–glial junctions, have been extensively investigated (for review see Girault and Peles, 2002
). In contrast, hardly anything is known about the basis for the accumulation of specific proteins, including potassium channels, at juxtaparanodes. The present work demonstrates the critical role of TAG-1 for the enrichment of axonal proteins Caspr2 and Kv1.1/Kv1.2 in juxtaparanodal regions, and points out unexpected molecular similarities in axo–glial interactions at paranodes and juxtaparanodes.
Despite the lack of major ultrastructural or functional alterations of myelinated fibers in TAG-1–deficient mice, a detailed analysis revealed that the normal distribution of the known molecular components of the juxtaparanodal region was selectively disturbed in the CNS and PNS of these animals. Although the localization of Na+
channels and paranodal proteins was normal in TAG-1 mutant mice, the normal accumulation of Caspr2 at juxtaparanodes was completely lost and the distribution of delayed rectifier K+
channels was severely altered. Thus, the phenotype of TAG-1–deficient mice is markedly different from that of other mutant strains described so far. For example, deletion of oligodendrocytes in transgenic mice during the first days after birth induces a virtually complete absence of organization of axonal proteins beyond the initial segments (Mathis et al., 2001
). Dysmyelination in jimpy
mice or md
rats, as well as targeted mutations in the galactolipid biosynthetic pathway, severely alter the organization of paranodal junctions without preventing the initial accumulation of K+
channels in direct contact with the nodes (Dupree et al., 1999
; Mathis et al., 2001
, Arroyo et al., 2002
). Targeted mutations of paranodal proteins prevent the formation of septate-like junctions and also result in a lack of separation between K+
channels and Na+
channels clusters (Bhat et al., 2001
; Boyle et al., 2001
). These observations support a role of fence for the paranodal junction (Pedraza et al., 2001
), separating the internode from the node. They also strongly indicate that the mechanisms leading to the accumulation of K+
channels and Caspr2 in the juxtaparanodal regions are relatively independent from those governing the formation of nodes and paranodal junctions. Thus, the phenotype of TAG-1 mutants provides novel insights into the organization of axonal domains.
Our results show that TAG-1 is closely associated with Caspr2 and is required for its accumulation at juxtaparanodes by recruiting and/or stabilizing it at this location. Co-IP experiments demonstrated that Caspr2 and TAG-1 form a complex in brain and in transfected cells. In addition, the two proteins were colocalized at the plasma membrane, and the presence of Caspr2 modified TAG-1 membrane distribution, which became more diffuse in intact cells and disappeared from the light fractions in sucrose gradients. These results indicate that the association of the two proteins alters significantly their membrane microenvironment and/or their interaction with other proteins.
Our findings in COS-7 cells demonstrate that TAG-1 can exchange cis interactions with Caspr2. This ability supports an association between the two proteins in the axolemma because TAG-1 is expressed in several types of neurons (Dodd et al., 1988
; Karagogeos et al., 1991
), including adult neurons of the dorsal root ganglia and their projections (unpublished data) and spinal motor neurons (Traka et al., 2002
). However, TAG-1 is also expressed in Schwann cells and oligodendrocytes and could be expected to exchange trans interactions with Caspr2. We tested this possibility using TAG-1-Fc chimeras and did not observe any binding, suggesting that in these conditions the two proteins interact directly only if they are present in the same membrane, in the same orientation. Yet, in these assays, TAG-1-Fc was readily capable to bind to membrane-bound TAG-1, in the absence or presence of cotransfected Caspr2. Thus, our results are compatible with a model in which TAG-1 interacts in cis with Caspr2 in the axolemma and in trans, through homophilic interaction, with another molecule of TAG-1 in the glial membrane (). A precedent for this type of interaction has been shown to occur between TAG-1 and L1 (Malhotra et al., 1998
). In that case, the trans homophilic interaction between TAG-1 molecules resulted in cis activation of L1, inducing its binding to ankyrin. Although the model depicted in is the simplest that accounts for all the presently available data, the possibility that additional components are part of this macromolecular complex cannot be excluded, as TAG-1 has been shown to interact with several other extracellular proteins (Milev et al., 1996
; Pavlou et al., 2002
). In addition, the association of Caspr2 with protein 4.1B (Denisenko-Nehrbass et al., 2003b
) suggests that the TAG-1–Caspr2 ternary complexes may be attached to the cytoskeleton through this protein that has the capability to interact with actin and spectrin (Gimm et al., 2002
). In TAG-1 knockout mice, we found that protein 4.1B was still present in juxtaparanodal regions although Caspr2-IR was not accumulated in these regions. This observation indicates that additional targeting mechanisms account for the localization of protein 4.1B to juxtaparanodes, and that the presence of protein 4.1B is not sufficient to induce the accumulation of Caspr2 in these regions. Therefore, we suggest that the combination of two complementary mechanisms may be required for the normal localization of the TAG-1–Caspr2 ternary complexes at juxtaparanodes: an axo–glial, TAG-1–mediated, homophilic interaction, and the anchoring of Caspr2 to cytoskeletal elements in the axon that may be found only in the vicinity of nodes of Ranvier. A prediction of this model is that TAG-1 localization should be altered in the absence of Caspr2 or of protein 4.1B.
Figure 8. Model of the molecular organization of juxtaparanodal regions. This model is the simplest that can account for the data from previous studies and the present work. Caspr2 is enriched in the axolemma, whereas TAG-1 is expressed in both neurons and myelinating (more ...)
An important conclusion of this paper is that TAG-1 is also essential for K+
channels enrichment at juxtaparanodes because a severely disrupted distribution of Kv1.1 and Kv1.2 was observed in mutant mice. It should be pointed out that the normal basic electrophysiological properties of TAG-1–deficient sciatic nerves is not surprising given the known insensitivity of adult sciatic nerves to K+
channel blockers (Vabnick et al., 1999
) and the absence of dysmyelination in TAG-1 mutants (this paper). Further analysis of TAG-1–deficient mice might be useful to address the function of the juxtaparanodal K+
channels. At any rate, this paper provides insights into the mechanisms of enrichment of these channels. These channels can coprecipitate with Caspr2 and there is indirect evidence that PDZ domain proteins are necessary for this association (Poliak et al., 1999
; Rasband et al., 2002
; ). Alternatively, the accumulation of K+
channels could originate from their interaction with TAG-1, a possibility that remains to be formally ruled out. Interestingly, some K+
channel accumulation was still observed in the vicinity of paranodes of 2-mo-old TAG-1–deficient mice in the absence of detectable Caspr2, indicating the minor contribution of additional targeting mechanism(s).
Paranodin/Caspr and contactin/F3, are essential for the formation of septate junctions where their glial partner is NF155, a transmembrane IgSF. Moreover, neurexin IV, the Drosophila
member of the NCP family is essential for septate junction formation in flies (Baumgartner et al., 1996
) and may form a tripartite complex with D-contactin and neuroglian, the orthologues of contactin/F3 and NF155, respectively (Faivre-Sarrailh, C., and M. Bhat, personal communication). Therefore, a core tripartite complex encompassing a NCP and a contactin-like protein in one membrane and an IgSF protein in the facing membrane appears to be a conserved molecular building block of intercellular contacts. The latter IgSF protein can be a transmembrane protein in the case of paranodal septate-like junctions and Drosophila
septate junctions, or a contactin-like molecule in the case of juxtaparanodes (this paper).
The molecular similarities between paranodal and juxtaparanodal protein complexes contrast with the striking ultrastructural differences between these two regions. At paranodes, the plasma membranes are separated by a narrow gap, interrupted by regularly spaced electron dense material that forms septa, in register with regularly organized intramembrane particles in glial and axonal membranes, as detected by freeze fracture (Wiley and Ellisman, 1980
). In contrast, at juxtaparanodes the membranes are more loosely apposed and do not display septate-like junctions. Freeze fracture has only revealed the presence of sparse particles in juxtaparanodal axonal and glial membranes (Stolinski et al., 1981
; Tao-Cheng and Rosenbluth, 1984
), which may correspond to ion channels and, possibly, to Cx 29 hemichannels (Stolinski et al., 1981
; Tao-Cheng and Rosenbluth, 1984
; Li et al., 2002
). Thus, the conserved NCP–IgSF ternary complexes appear to be involved in strikingly distinct types of cell–cell contacts. A noticeable difference is that NF155, the glial moiety of this complex at paranodes, is a transmembrane protein, presumably associated with the cytoskeleton, as in Drosophila
septate junctions, whereas at juxtaparanodes, TAG-1 is a GPI-anchored protein. Further experiments will be required to determine whether this difference accounts for the striking differences between paranodal and juxtaparanodal NCP-contactin–based intercellular complexes, or whether additional components are involved. In either case, the present work demonstrates the contribution of TAG-1 and associated Caspr2 in the organization of axonal domains at nodes of Ranvier.