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Association of TAG-1 with Caspr2 is essential for the molecular organization of juxtaparanodal regions of myelinated fibers.

Traka M, Goutebroze L, Denisenko N, Bessa M, Nifli A, Havaki S, Iwakura Y, Fukamauchi F, Watanabe K, Soliven B, Girault JA, Karagogeos D - J. Cell Biol. (2003)

Bottom Line: Myelination results in a highly segregated distribution of axonal membrane proteins at nodes of Ranvier.In the absence of TAG-1, axonal Caspr2 did not accumulate at juxtaparanodes, and the normal enrichment of shaker-type K+ channels in these regions was severely disrupted, in the central and peripheral nervous systems.This complex is analogous to that described previously at paranodes, suggesting that similar molecules are crucial for different types of axo-glial interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Science, University of Crete Medical School, Heraklion 71110, Crete, Greece.

ABSTRACT
Myelination results in a highly segregated distribution of axonal membrane proteins at nodes of Ranvier. Here, we show the role in this process of TAG-1, a glycosyl-phosphatidyl-inositol-anchored cell adhesion molecule. In the absence of TAG-1, axonal Caspr2 did not accumulate at juxtaparanodes, and the normal enrichment of shaker-type K+ channels in these regions was severely disrupted, in the central and peripheral nervous systems. In contrast, the localization of protein 4.1B, an axoplasmic partner of Caspr2, was only moderately altered. TAG-1, which is expressed in both neurons and glia, was able to associate in cis with Caspr2 and in trans with itself. Thus, a tripartite intercellular protein complex, comprised of these two proteins, appears critical for axo-glial contacts at juxtaparanodes. This complex is analogous to that described previously at paranodes, suggesting that similar molecules are crucial for different types of axo-glial interactions.

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Sucrose density gradient of TAG-1 and Caspr2 in transfected COS-7 cells. COS-7 cells overexpressing either (A, top, COS_TAG-1) TAG-1 alone or (B, top, COS_Caspr2) Caspr2 alone, or (A and B, bottom, COS_Caspr2/TAG-1) both proteins, were lysed in a Triton X-100 containing buffer and lysates were submitted to discontinuous sucrose gradients as described in Materials and methods and IB with antibodies against (A) TAG-1 and (B) Caspr2. Aliquots of crude protein extracts (Lysate) were also analyzed to verify the expression of the proteins. TAG-1 expressed alone was present in the 10 and 25% sucrose fractions (A, top). In contrast, Caspr2 was recovered in the heavier 40% fraction and in the pellet (B, bottom). When coexpressed with Caspr2, TAG-1 was no longer present in the light fractions but was found in the pellet with Caspr2 (A and B, bottom).
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fig7: Sucrose density gradient of TAG-1 and Caspr2 in transfected COS-7 cells. COS-7 cells overexpressing either (A, top, COS_TAG-1) TAG-1 alone or (B, top, COS_Caspr2) Caspr2 alone, or (A and B, bottom, COS_Caspr2/TAG-1) both proteins, were lysed in a Triton X-100 containing buffer and lysates were submitted to discontinuous sucrose gradients as described in Materials and methods and IB with antibodies against (A) TAG-1 and (B) Caspr2. Aliquots of crude protein extracts (Lysate) were also analyzed to verify the expression of the proteins. TAG-1 expressed alone was present in the 10 and 25% sucrose fractions (A, top). In contrast, Caspr2 was recovered in the heavier 40% fraction and in the pellet (B, bottom). When coexpressed with Caspr2, TAG-1 was no longer present in the light fractions but was found in the pellet with Caspr2 (A and B, bottom).

Mentions: TAG-1 is a GPI-anchored protein found in cholesterol-rich, Triton X-100–insoluble membrane fractions (Buttiglione et al., 1998; Kasahara et al., 2000; Prinetti et al., 2001). In transfected COS-7 cells, TAG-1 was Triton X-100 insoluble at 4°C, whereas Caspr2, a type 1 transmembrane protein, was soluble (unpublished data). We examined the membrane compartments in which Caspr2 and TAG-1 were distributed and whether their coexpression altered this distribution by loading Triton X-100 cell lysates on discontinuous sucrose gradients (Fig. 7). Analysis of the proteins in the fractions revealed that TAG-1 was present in the 10 and 25% sucrose fractions (Fig. 7 A, top). In contrast, Caspr2 expressed alone was recovered in the heavier 40% fraction and in the pellet (Fig. 7 B, top). When coexpressed with Caspr2, TAG-1 was no longer present in the light fractions but was only found in the pellet with Caspr2 (Fig. 7, A and B, bottom). These experiments indicate that when Caspr2 and TAG-1 are coexpressed, their interaction alters the distribution of TAG-1 by changing its microenvironment and/or by linking the complexes to cytoplasmic components. Altogether our results reveal that Caspr2 is able to reach the plasma membrane by itself, and that Caspr2 and TAG-1 are colocalized and associated in cis at the plasma membrane in transfected cells.


Association of TAG-1 with Caspr2 is essential for the molecular organization of juxtaparanodal regions of myelinated fibers.

Traka M, Goutebroze L, Denisenko N, Bessa M, Nifli A, Havaki S, Iwakura Y, Fukamauchi F, Watanabe K, Soliven B, Girault JA, Karagogeos D - J. Cell Biol. (2003)

Sucrose density gradient of TAG-1 and Caspr2 in transfected COS-7 cells. COS-7 cells overexpressing either (A, top, COS_TAG-1) TAG-1 alone or (B, top, COS_Caspr2) Caspr2 alone, or (A and B, bottom, COS_Caspr2/TAG-1) both proteins, were lysed in a Triton X-100 containing buffer and lysates were submitted to discontinuous sucrose gradients as described in Materials and methods and IB with antibodies against (A) TAG-1 and (B) Caspr2. Aliquots of crude protein extracts (Lysate) were also analyzed to verify the expression of the proteins. TAG-1 expressed alone was present in the 10 and 25% sucrose fractions (A, top). In contrast, Caspr2 was recovered in the heavier 40% fraction and in the pellet (B, bottom). When coexpressed with Caspr2, TAG-1 was no longer present in the light fractions but was found in the pellet with Caspr2 (A and B, bottom).
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Related In: Results  -  Collection

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fig7: Sucrose density gradient of TAG-1 and Caspr2 in transfected COS-7 cells. COS-7 cells overexpressing either (A, top, COS_TAG-1) TAG-1 alone or (B, top, COS_Caspr2) Caspr2 alone, or (A and B, bottom, COS_Caspr2/TAG-1) both proteins, were lysed in a Triton X-100 containing buffer and lysates were submitted to discontinuous sucrose gradients as described in Materials and methods and IB with antibodies against (A) TAG-1 and (B) Caspr2. Aliquots of crude protein extracts (Lysate) were also analyzed to verify the expression of the proteins. TAG-1 expressed alone was present in the 10 and 25% sucrose fractions (A, top). In contrast, Caspr2 was recovered in the heavier 40% fraction and in the pellet (B, bottom). When coexpressed with Caspr2, TAG-1 was no longer present in the light fractions but was found in the pellet with Caspr2 (A and B, bottom).
Mentions: TAG-1 is a GPI-anchored protein found in cholesterol-rich, Triton X-100–insoluble membrane fractions (Buttiglione et al., 1998; Kasahara et al., 2000; Prinetti et al., 2001). In transfected COS-7 cells, TAG-1 was Triton X-100 insoluble at 4°C, whereas Caspr2, a type 1 transmembrane protein, was soluble (unpublished data). We examined the membrane compartments in which Caspr2 and TAG-1 were distributed and whether their coexpression altered this distribution by loading Triton X-100 cell lysates on discontinuous sucrose gradients (Fig. 7). Analysis of the proteins in the fractions revealed that TAG-1 was present in the 10 and 25% sucrose fractions (Fig. 7 A, top). In contrast, Caspr2 expressed alone was recovered in the heavier 40% fraction and in the pellet (Fig. 7 B, top). When coexpressed with Caspr2, TAG-1 was no longer present in the light fractions but was only found in the pellet with Caspr2 (Fig. 7, A and B, bottom). These experiments indicate that when Caspr2 and TAG-1 are coexpressed, their interaction alters the distribution of TAG-1 by changing its microenvironment and/or by linking the complexes to cytoplasmic components. Altogether our results reveal that Caspr2 is able to reach the plasma membrane by itself, and that Caspr2 and TAG-1 are colocalized and associated in cis at the plasma membrane in transfected cells.

Bottom Line: Myelination results in a highly segregated distribution of axonal membrane proteins at nodes of Ranvier.In the absence of TAG-1, axonal Caspr2 did not accumulate at juxtaparanodes, and the normal enrichment of shaker-type K+ channels in these regions was severely disrupted, in the central and peripheral nervous systems.This complex is analogous to that described previously at paranodes, suggesting that similar molecules are crucial for different types of axo-glial interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Science, University of Crete Medical School, Heraklion 71110, Crete, Greece.

ABSTRACT
Myelination results in a highly segregated distribution of axonal membrane proteins at nodes of Ranvier. Here, we show the role in this process of TAG-1, a glycosyl-phosphatidyl-inositol-anchored cell adhesion molecule. In the absence of TAG-1, axonal Caspr2 did not accumulate at juxtaparanodes, and the normal enrichment of shaker-type K+ channels in these regions was severely disrupted, in the central and peripheral nervous systems. In contrast, the localization of protein 4.1B, an axoplasmic partner of Caspr2, was only moderately altered. TAG-1, which is expressed in both neurons and glia, was able to associate in cis with Caspr2 and in trans with itself. Thus, a tripartite intercellular protein complex, comprised of these two proteins, appears critical for axo-glial contacts at juxtaparanodes. This complex is analogous to that described previously at paranodes, suggesting that similar molecules are crucial for different types of axo-glial interactions.

Show MeSH
Related in: MedlinePlus