Limits...
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.

Show MeSH

Related in: MedlinePlus

Association of TAG-1 and Caspr2 in brain and transfected COS-7 cells. (A and B) Association of TAG-1 and Caspr2 in brain. Rat brain proteins were extracted and subjected to IP with (A) αCaspr2 or (B) αTAG-1. The presence of specific proteins in the precipitates was examined by IB with the indicated antibodies. Aliquots of crude protein extracts (Lysate, 1/60 of protein amount used for each coIP) were also subjected to IB to verify the expression of the proteins. (A) Caspr2 was detected in immune precipitates with TAG-1 antibodies but not with antibodies against other IgSF proteins (αNrCAM, αL1, and αF3). (B) TAG-1 was detected in immune precipitates with Caspr2 antibodies but not nonimmune serum (αNI). (C and D) Association of TAG-1 and Caspr2 in transfected COS-7 cells. Lysates from COS-7 cells overexpressing either Caspr2 or TAG-1 alone, or both, were prepared as described in Materials and methods and subjected to IP either with (C) αTAG-1 or (D) αCaspr2. Precipitates were resolved by SDS-PAGE, analyzed by IBs with antibodies against Caspr2 and TAG-1, to detect the presence of specific proteins (C and D, top and middle). Aliquots of crude protein extracts (Lysates) were also subjected to immunoblotting to verify the expression of the proteins (C and D, lower panels). TAG-1 antibodies coIP Caspr2 only in doubly transfected cells (C), whereas Caspr2 antibodies pulled-down TAG-1 only in cotransfected cells (D). Note the slight shift of migration of Caspr2 in the presence of TAG-1, which results in a doublet with a predominant lower band in cotransfected cells (C, Lysates). The position of molecular mass markers (kD) is indicated.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172849&req=5

fig5: Association of TAG-1 and Caspr2 in brain and transfected COS-7 cells. (A and B) Association of TAG-1 and Caspr2 in brain. Rat brain proteins were extracted and subjected to IP with (A) αCaspr2 or (B) αTAG-1. The presence of specific proteins in the precipitates was examined by IB with the indicated antibodies. Aliquots of crude protein extracts (Lysate, 1/60 of protein amount used for each coIP) were also subjected to IB to verify the expression of the proteins. (A) Caspr2 was detected in immune precipitates with TAG-1 antibodies but not with antibodies against other IgSF proteins (αNrCAM, αL1, and αF3). (B) TAG-1 was detected in immune precipitates with Caspr2 antibodies but not nonimmune serum (αNI). (C and D) Association of TAG-1 and Caspr2 in transfected COS-7 cells. Lysates from COS-7 cells overexpressing either Caspr2 or TAG-1 alone, or both, were prepared as described in Materials and methods and subjected to IP either with (C) αTAG-1 or (D) αCaspr2. Precipitates were resolved by SDS-PAGE, analyzed by IBs with antibodies against Caspr2 and TAG-1, to detect the presence of specific proteins (C and D, top and middle). Aliquots of crude protein extracts (Lysates) were also subjected to immunoblotting to verify the expression of the proteins (C and D, lower panels). TAG-1 antibodies coIP Caspr2 only in doubly transfected cells (C), whereas Caspr2 antibodies pulled-down TAG-1 only in cotransfected cells (D). Note the slight shift of migration of Caspr2 in the presence of TAG-1, which results in a doublet with a predominant lower band in cotransfected cells (C, Lysates). The position of molecular mass markers (kD) is indicated.

Mentions: The colocalization of TAG-1 and Caspr2 in mice and rats, together with the mislocalization of Caspr2 in TAG-1–deficient mice prompted us to examine the possibility that these proteins form a complex at juxtaparanodes by performing coimmunoprecipitation experiments from brain extracts. Caspr2 was detected in TAG-1 immune precipitates but not in immunoprecipitation (IP) performed with antibodies against other IgSF proteins (Fig. 5 A). Conversely, TAG-1 was specifically detected in Caspr2 immune precipitates (Fig. 5 B). These results indicate the existence of a specific association between TAG-1 and Caspr2 in vivo. We examined further the association between TAG-1 and Caspr2 using COS-7 cells transfected with expression plasmids for either of these proteins, alone or in combination (Fig. 5, C and D). IP with TAG-1 antibodies pulled down Caspr2 in cells doubly transfected with TAG-1 and Caspr2 but not in cells expressing only Caspr2 (Fig. 5 C). On the other hand, Caspr2 antibodies coprecipitated TAG-1 only in doubly transfected cells (Fig. 5 D).


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)

Association of TAG-1 and Caspr2 in brain and transfected COS-7 cells. (A and B) Association of TAG-1 and Caspr2 in brain. Rat brain proteins were extracted and subjected to IP with (A) αCaspr2 or (B) αTAG-1. The presence of specific proteins in the precipitates was examined by IB with the indicated antibodies. Aliquots of crude protein extracts (Lysate, 1/60 of protein amount used for each coIP) were also subjected to IB to verify the expression of the proteins. (A) Caspr2 was detected in immune precipitates with TAG-1 antibodies but not with antibodies against other IgSF proteins (αNrCAM, αL1, and αF3). (B) TAG-1 was detected in immune precipitates with Caspr2 antibodies but not nonimmune serum (αNI). (C and D) Association of TAG-1 and Caspr2 in transfected COS-7 cells. Lysates from COS-7 cells overexpressing either Caspr2 or TAG-1 alone, or both, were prepared as described in Materials and methods and subjected to IP either with (C) αTAG-1 or (D) αCaspr2. Precipitates were resolved by SDS-PAGE, analyzed by IBs with antibodies against Caspr2 and TAG-1, to detect the presence of specific proteins (C and D, top and middle). Aliquots of crude protein extracts (Lysates) were also subjected to immunoblotting to verify the expression of the proteins (C and D, lower panels). TAG-1 antibodies coIP Caspr2 only in doubly transfected cells (C), whereas Caspr2 antibodies pulled-down TAG-1 only in cotransfected cells (D). Note the slight shift of migration of Caspr2 in the presence of TAG-1, which results in a doublet with a predominant lower band in cotransfected cells (C, Lysates). The position of molecular mass markers (kD) is indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172849&req=5

fig5: Association of TAG-1 and Caspr2 in brain and transfected COS-7 cells. (A and B) Association of TAG-1 and Caspr2 in brain. Rat brain proteins were extracted and subjected to IP with (A) αCaspr2 or (B) αTAG-1. The presence of specific proteins in the precipitates was examined by IB with the indicated antibodies. Aliquots of crude protein extracts (Lysate, 1/60 of protein amount used for each coIP) were also subjected to IB to verify the expression of the proteins. (A) Caspr2 was detected in immune precipitates with TAG-1 antibodies but not with antibodies against other IgSF proteins (αNrCAM, αL1, and αF3). (B) TAG-1 was detected in immune precipitates with Caspr2 antibodies but not nonimmune serum (αNI). (C and D) Association of TAG-1 and Caspr2 in transfected COS-7 cells. Lysates from COS-7 cells overexpressing either Caspr2 or TAG-1 alone, or both, were prepared as described in Materials and methods and subjected to IP either with (C) αTAG-1 or (D) αCaspr2. Precipitates were resolved by SDS-PAGE, analyzed by IBs with antibodies against Caspr2 and TAG-1, to detect the presence of specific proteins (C and D, top and middle). Aliquots of crude protein extracts (Lysates) were also subjected to immunoblotting to verify the expression of the proteins (C and D, lower panels). TAG-1 antibodies coIP Caspr2 only in doubly transfected cells (C), whereas Caspr2 antibodies pulled-down TAG-1 only in cotransfected cells (D). Note the slight shift of migration of Caspr2 in the presence of TAG-1, which results in a doublet with a predominant lower band in cotransfected cells (C, Lysates). The position of molecular mass markers (kD) is indicated.
Mentions: The colocalization of TAG-1 and Caspr2 in mice and rats, together with the mislocalization of Caspr2 in TAG-1–deficient mice prompted us to examine the possibility that these proteins form a complex at juxtaparanodes by performing coimmunoprecipitation experiments from brain extracts. Caspr2 was detected in TAG-1 immune precipitates but not in immunoprecipitation (IP) performed with antibodies against other IgSF proteins (Fig. 5 A). Conversely, TAG-1 was specifically detected in Caspr2 immune precipitates (Fig. 5 B). These results indicate the existence of a specific association between TAG-1 and Caspr2 in vivo. We examined further the association between TAG-1 and Caspr2 using COS-7 cells transfected with expression plasmids for either of these proteins, alone or in combination (Fig. 5, C and D). IP with TAG-1 antibodies pulled down Caspr2 in cells doubly transfected with TAG-1 and Caspr2 but not in cells expressing only Caspr2 (Fig. 5 C). On the other hand, Caspr2 antibodies coprecipitated TAG-1 only in doubly transfected cells (Fig. 5 D).

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