Axonal targeting of Caspr2 in hippocampal neurons via selective somatodendritic endocytosis.

Contactin-associated protein 2 (Caspr2) is a neuronal membrane protein that is mutated in autism and related disorders. Although it is highly enriched at juxtaparanodes of Ranvier where it is essential for Shaker-type K(+) channel clustering, little is known about its function and regulation. In the present study, we examined the polarized expression of Caspr2 in hippocampal neurons using extracellular hemagglutinin (HA)-tagged Caspr2 constructs. We found that Caspr2 was targeted to the axonal surface, but colocalized with early endosomes in the somatodendritic compartment. The inhibition of endocytosis using a Dynamin-1 mutant or treatment with Dynasore prevented Caspr2 internalization from the dendrites and cell body. We identified a short sequence included into the 4.1B-binding domain that is required for the endocytosis of Caspr2. This sequence contains a protein kinase C (PKC) substrate motif on Thr1292, and point mutation of this residue or treatment with a PKC inhibitor prevented the somatodendritic internalization of Caspr2. Thus, the PKC-dependent trafficking of Caspr2 underlies its polarized expression in hippocampal neurons.

The lateral mobility of cell adhesion molecules is highly restricted at septate junctions in Drosophila.

BACKGROUND: A complex of three cell adhesion molecules (CAMs) Neurexin IV(Nrx IV), Contactin (Cont) and Neuroglian (Nrg) is implicated in the formation of septate junctions between epithelial cells in Drosophila. These CAMs are interdependent for their localization at septate junctions and e.g. null mutation of nrx IV or cont induces the mislocalization of Nrg to the baso-lateral membrane. These mutations also result in ultrastructural alteration of the strands of septate junctions and breakdown of the paracellular barrier. Varicose (Vari) and Coracle (Cora), that both interact with the cytoplasmic tail of Nrx IV, are scaffolding molecules required for the formation of septate junctions. RESULTS: We conducted photobleaching experiments on whole living Drosophila embryos to analyze the membrane mobility of CAMs at septate junctions between epithelial cells. We show that GFP-tagged Nrg and Nrx IV molecules exhibit very stable association with septate junctions in wild-type embryos. Nrg-GFP is mislocalized to the baso-lateral membrane in nrx IV or cont null mutant embryos, and displays increased mobile fraction. Similarly, Nrx IV-GFP becomes distributed to the baso-lateral membrane in null mutants of vari and cora, and its mobile fraction is strongly increased. The loss of Vari, a MAGUK protein that interacts with the cytoplasmic tail of Nrx IV, has a stronger effect than the null mutation of nrx IV on the lateral mobility of Nrg-GFP. CONCLUSION: The strands of septate junctions display a stable behavior in vivo that may be correlated with their role of paracellular barrier. The membrane mobility of CAMs is strongly limited when they take part to the multimolecular complex forming septate junctions. This restricted lateral diffusion of CAMs depends on both adhesive interactions and clustering by scaffolding molecules. The lateral mobility of CAMs is strongly increased in embryos presenting alteration of septate junctions. The stronger effect of vari by comparison with nrx IV null mutation supports the hypothesis that this scaffolding molecule may cross-link different types of CAMs and play a crucial role in stabilizing the strands of septate junctions.

PGY repeats and N-glycans govern the trafficking of paranodin and its selective association with contactin and neurofascin-155.

Formation of nodes of Ranvier requires contact of axons with myelinating glial cells, generating specialized axo-glial subdomains. Caspr/paranodin is required for the formation of septate-like junctions at paranodes, whereas the related caspr2 is essential for the organization of juxtaparanodes. The molecular mechanisms underlying the segregation of these related glycoproteins within distinct complexes are poorly understood. Exit of paranodin from the endoplasmic reticulum (ER) is mediated by its interaction with F3/contactin. Using domain swapping with caspr2, we mapped a motif with Pro-Gly-Tyr repeats (PGY) in the ectodomain of paranodin responsible for its ER retention. Deletion of PGY allows cell surface delivery of paranodin bypassing the calnexin-calreticulin quality control. Conversely, insertion of PGY in caspr2 or NrCAM blocks these proteins in the ER. PGY is a novel type of processing signal that compels chaperoning of paranodin by contactin. Contactin associated with paranodin is expressed at the cell surface with high-mannose N-glycans. Using mutant CHO lines altered in the processing of N-linked carbohydrates, we show that the high-mannose glycoform of contactin strongly binds neurofascin-155, its glial partner at paranodes. Thus, the unconventional processing of paranodin and contactin may determine the selective association of axo-glial complexes at paranodes.