Retention of a cell adhesion complex at the paranodal junction requires the cytoplasmic region of Caspr - PubMed (original) (raw)

Retention of a cell adhesion complex at the paranodal junction requires the cytoplasmic region of Caspr

Leora Gollan et al. J Cell Biol. 2002.

Abstract

An axonal complex of cell adhesion molecules consisting of Caspr and contactin has been found to be essential for the generation of the paranodal axo-glial junctions flanking the nodes of Ranvier. Here we report that although the extracellular region of Caspr was sufficient for directing it to the paranodes in transgenic mice, retention of the Caspr-contactin complex at the junction depended on the presence of an intact cytoplasmic domain of Caspr. Using immunoelectron microscopy, we found that a Caspr mutant lacking its intracellular domain was often found within the axon instead of the junctional axolemma. We further show that a short sequence in the cytoplasmic domain of Caspr mediated its binding to the cytoskeleton-associated protein 4.1B. Clustering of contactin on the cell surface induced coclustering of Caspr and immobilized protein 4.1B at the plasma membrane. Furthermore, deletion of the protein 4.1B binding site accelerated the internalization of a Caspr-contactin chimera from the cell surface. These results suggest that Caspr serves as a "transmembrane scaffold" that stabilizes the Caspr/contactin adhesion complex at the paranodal junction by connecting it to cytoskeletal components within the axon.

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Figures

Figure 1.

Figure 1.

Structure and expression of Caspr constructs. (A) Schematic representation of the various Caspr constructs used in this study. In CSP–HA, an HA tag was added after the last amino acid of human Caspr, whereas in CSPdCT–HA, the HA tag replaced its cytoplasmic domain. Cyto, cytoplasmic region; Disc, discoidin-like domain; EGF, EGF repeat; fib, fibrinogen-like domain; LamG, laminin G domain; PGY, a proline/glycine/tyrosine-rich region; TM, transmembrane domain. (B) Expression of Caspr constructs in transfected cells. Sample of cell lysates prepared from parental HEK-293 (none), or cells transfected with CSP–HA (HA), CSPdCT–HA (dCT-HA), or human Caspr were used for immunoprecipitation and immunoblot analyses using anti-Caspr (top) or anti-HA antibody (bottom). Note that the anti-Caspr antibody used is directed against the cytoplasmic tail of the protein and therefore does not recognize CSPdCT–HA, which was detected by the HA antibody. Mol wt markers in kD are shown on the right. (C) Coimmunoprecipitation of contactin and Caspr mutants. HEK-293 cells were transfected with contactin alone (293), or together with CSP–HA (HA) or CSPdCT–HA (dCT) as indicated. Immunoprecipitation was performed with an antibody to HA tag (HA) or to contactin (Con) as indicated on the right of each panel (IP), followed by immunoblotting with anticontactin antibody. (D) Cell surface expression of Caspr constructs in transfected COS7 cells. Cells were transfected with contactin and CSP–HA or CSPdCT–HA as indicated on the top. Cells were fixed and stained with an antibody against the extracellular region (ECD) or an HA tag antibody found in the intracellular region of each Caspr construct (Cyto), with (+) or without (−) prior permeabilization (TX). The nuclei of the cells were labeled with DAPI (blue).

Figure 2.

Figure 2.

Expression of Caspr mutants in transgenic mice. (A) RT-PCR analyses. RNA was isolated from brains of adult transgenic animals expressing CSPdCT–HA (dCT–HA) or CSP–HA (HA) or control mice and used as a template for RT-PCR analysis using a primer set that recognizes CSP–HA and CSPdCT–HA. Plasmid templates containing the various mutants were used as specificity controls. (B) Protein expression of Caspr transgenes in adult mice brains. Brain membrane lysates from wild-type (WT) and the two Caspr transgenic mice were subjected to immunoprecipitation and immunoblotting using an antibody against Caspr or HA tag as indicated at the bottom of each panel. The immunoblots are shown along with mol wt markers in kD on the right. (C) Expression of Caspr and CSPdCT–HA during development. Brain membrane proteins were prepared from CSPdCT–HA mice at different postnatal days as indicated and subjected to immunoblot analyses with antibody to Caspr (top) or HA tag (bottom). (D) Interaction of Caspr transgenes with contactin in mouse brain. Brain lysates of wild-type (WT), CSP–HA (HA), or CSPdCT–HA (dCT) mice were subjected to immunoprecipitation with an antibody to HA tag (HA), Caspr, or contactin (Con) as indicated on the left of each panel. The resulted complexes were immunoblotted with an anticontactin antibody.

Figure 3.

Figure 3.

Localization of Caspr transgenes in peripheral nerve. Teased sciatic nerves from adult wild-type (WT) or the two transgenic mice (as indicated on top of the figure) were double labeled with an antibody against Na+ channel (red) and with either Caspr (green, top) or HA tag (green, bottom). Paranodal localization of the transgenes was detected in all the axons that expressed the transgenes. Bar, 10 μm.

Figure 4.

Figure 4.

Immunolocalization of Caspr transgenes in CNS white matter. Sections of adult optic nerves from wild-type (A, D, and G), CSP–HA (B, E, and H), and CSPdCT–HA mice (C, F, and I) were double labeled with an antibody to Na+ channel (red) and HA tag (green; A–C), Na+ channel (red) and Caspr (green; D–F), or HA tag (red) and Caspr (green; G–I). Bar, 10 μm. Note that Caspr transgenes were not expressed in all retinal ganglion cells and thus were only found in 30–40% of the paranodes as detected by a Caspr antibody (D–F, yellow).

Figure 5.

Figure 5.

Immunoelectron microscopy localization of Caspr transgenes and contactin in optic nerve. Immunogold labeling of Caspr in cross sections at the paranodal level of optic nerves from adult wild type (A) or CSPdCT–HA (B). Similar sections were labeled with HA tag antibody in optic nerve from CSP–HA (C) or CSPdCT–HA (D) animals. Longitudinal sections at nodal regions of optic nerve from wild type labeled for Caspr (E) or from CSPdCT–HA mice labeled for HA tag (F and G). Note that in both cross and longitudinal sections, whereas the endogenous Caspr or HA tag in CSP–HA animals was confined to the inner surface of the axonal membrane in the paranodes, HA tag labeling in CSPdCT–HA mice was observed in the axonal cytoplasm at the paranodes. At a higher magnification, HA tag staining in optic nerves from CSPdCT–HA mice was clearly detected in vesicular structures (F, inset). Labeling of contactin in cross sections of optic nerve from wild-type (H), CSP–HA (I), or CSPdCT–HA mice (J–K). Bars, 0.2 μm (except for inset, which is 0.02 μm).

Figure 6.

Figure 6.

Interaction of Caspr with protein 4.1B. (A) Pulldown of protein 4.1B by the cytoplasmic domain of Caspr. Lysates of HEK-293 cells expressing protein 4.1B were mixed with agarose-bound GST or GST fusion protein containing the cytoplasmic domain of Caspr as indicated. Bound proteins were immunoblotted with an antibody to protein 4.1B. Immunoprecipitation with an antibody to protein 4.1B (4.1B) was used as a control. (B) Coimmunoprecipitation of protein 4.1B with Caspr from rat brain. Adult rat brain lysates were subjected to immunoprecipitation with antibodies to protein 4.1B (4.1B) or the cytoplasmic (Caspr/CT) or extracellular (Caspr/ECD) domains of Caspr as indicated. Preimmune serum (CS) or protein A beads (beads) were used as controls. (C) Association of protein 4.1B with CSP–HA, but not with CSPdCT–HA. HEK-293 cells were transfected with CSP–HA or CSPdCT–HA, with (+m4.1B) or without myc-tagged protein 4.1B as indicated on the top. Cell lysates were subjected to immunoprecipitation with an antibody against the extracellular domain of Caspr. Washed immune complexes were separated on SDS gel and immunoblotted with an antibody to myc (right) or HA tag (left). The sizes of mol wt markers are shown on the right in kD. (D) Binding of βC–Fc fusion protein under nonclustering conditions. Hela cells expressing CSPdCT–HA, contactin, and protein 4.1B were stained with the indicated antibodies or with βC–Fc fusion protein. The second and fourth panels show the same cell stained for contactin and the βC–Fc fusion protein. (E) Clustering of CSP–HA, but not CSPdCT–HA, induces aggregation of protein 4.1B. Hela cells expressing contactin, protein 4.1B, and CSP–HA (HA) or CSPdCT–HA (dCT), as indicted on the bottom of each column, were allowed to bind βC–Fc followed by clustering with Cy3-conjugated anti–human Fc antibody. Cells were stained with an antibody to Caspr, contactin, or protein 4.1B as indicated in the upper row. The distribution of the clustered βC–Fc fusion protein and the merged images (βC–Fc, red; antibody staining, green) are shown in the middle and lower rows, respectively. Note that although βC-Fc induced clustering of contactin, CSP–HA, and CSPdCT-HA, immobilization of protein 4.1B into these clusters occurred in CSP–HA- but not in CSPdCT–HA-expressing cells.

Figure 7.

Figure 7.

Protein 4.1B binds a short sequence at the transmembrane domain of Caspr. (A) Schematic representation of contactin–Caspr chimeras. ConCT contains the extracellular region of contactin fused to the transmembrane and cytoplasmic domain of Caspr. ConJXCT lacks nine amino acids from the juxtamembrane domain of Caspr (sequence shown in the bottom). Both chimeras contain an HA tag at their carboxy terminus. (B) Cell surface localization of the chimeras. HEK-293 cells expressing ConCT or ConJXCT, as indicated on the top, were labeled with biotin, lysed, and subjected to immunoprecipitation with an antibody to HA tag. Western blotting was performed with an antibody to HA (HA) or HRP-conjugated streptavidin to detect cell surface expression of the chimeras. (C) Association of ConCT, but not ConJXCT, with protein 4.1B. HEK-293 cells were transfected with ConCT or ConJXCT with (+m4.1B) or without a myc-tagged protein 4.1B, as indicated on the top. Cells expressing 4.1B alone (4.1B) served as an additional control. Immunoprecipitation (IP) and immunoblotting (blot) were performed with the different antibody combinations indicated on the left of each panel. The sizes of mol wt markers are shown on the right in kD. (D) Clustering of protein 4.1B by ConCT but not ConJXCT. HeLa cells expressing protein 4.1B and ConCT or ConJXCT were incubated with βC–Fc, with (+) or without (−) further clustering by anti–human Fc antibody as indicted on the left. Staining of the cells with an antibody to protein 4.1B (green) and βC–Fc (red) are shown along with the merged images on the right. Protein 4.1B was not incorporated into clusters in cells expressing ConJXCT. (E) Internalization of ConCT and ConJXCT from the cell surface during time in culture. HEK-293 cells expressing equal amounts of protein 4.1B and ConCT (top) or ConJXCT (bottom) were biotinylated using sulfo-NHS-_S_-_S_-biotin. Cells were then incubated with βC–Fc-containing medium at 37°C for the indicated times to allow internalization of biotinylated surface proteins. Cells were placed on ice to stop trafficking and, subsequently, were either treated with glutathione to remove remaining labeled proteins on the cell surface (internalized) or were left untreated (total). Biotinylated proteins were precipitated from cell lysates using agarose–streptavidin followed by immunoblotting with anti–HA tag antibody.

Figure 8.

Figure 8.

Abnormal distribution of protein 4.1B in peripheral nerves of contactin-deficient mice. Teased sciatic fibers from 8-d-old wild-type animals (WT) or mice lacking contactin (Con−/−) were double labeled with antibodies to Na+ channel (red) and protein 4.1B (green) as indicated in each panel. The merged images are shown in the right panels. Note that in contactin−/− nerves, protein 4.1B is diffusely distributed along the axon.

Figure 9.

Figure 9.

Proposed mechanisms involved in the localization of Caspr and contactin at the paranodal junction. Localization of Caspr at the paranodes involves several molecular interactions. During development of myelinated nerves, the interaction with contactin is required for a proper transport of Caspr out of the neuronal cell body. This complex is then accumulated at the paranodes probably as a result of its interactions with NF155 present on the glial loops. Once at the paranodal region, Caspr recruits protein 4.1B, which helps to stabilize the Caspr–contactin complex at the paranodal junction by linking it to the underlying axonal cytoskeleton.

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