No abstract

The alpha‐2,8‐linked sialic acid polymer (PSA) on the neural cell adhesion molecule (NCAM) is an important regulator of cell surface interactions. We have examined the translocation of PSA‐NCAM to the surface of cultured cortical neurons and insulin secreting beta cells under different conditions of cell activity. Endoneuraminidase N, an enzyme that specifically cleaves PSA chains, was used to remove pre‐existing PSA from the plasma membrane and the re‐expression of the molecule was monitored by immunocytochemistry. Punctate PSA immunostaining was restored on the surface of 68% of neurons within 1 h. This recovery was almost completely prevented by tetrodotoxin, suggesting that spontaneous electrical activity is required. K+ depolarization (50 mM) allowed recovery of PSA surface staining in the presence of tetrodotoxin and this effect required the presence of extracellular Ca2+. Rapid redistribution of PSA‐NCAM to the surface of beta cells was observed under conditions that stimulate insulin secretion. Ca2+ channel inhibition decreased both PSA‐NCAM expression and insulin secretion to control, non‐stimulated levels. Finally, subcellular fractionation of an insulin‐secreting cell line showed that the secretory vesicle fraction is highly enriched in PSA‐NCAM. These results suggest that PSA‐NCAM can be translocated to the cell surface via regulated exocytosis. Taken together, our results provide unprecedented evidence linking cell activity and PSA‐NCAM expression, and suggest a mechanism for rapid modulation of cell surface interactions.

The F3 molecule is a member of the immunoglobulin superfamily anchored to plasma membranes by a glycosylphosphatidylinositol group. In adult mouse cerebellum, F3 is predominantly expressed on a subset of axons, the parallel fibers, and at their synapses. In vitro studies established that it is a plurifunctional molecule that, depending on the cellular context and the ligand with which it interacts, either mediates repulsive interactions or promotes neurite outgrowth. In the present study, we report the isolation of two fractions of F3‐containing microdomains from adult cerebellum on the basis of their resistance to solubilization by Triton X‐100 at 4°C. Both fractions were composed of vesicles, ranging from 100 to 200 nm in diameter. Lipid composition analysis indicated that the lighter fraction was enriched in cerebrosides and sulfatides. F3 sensitivity to phosphatidylinositol phospholipase C differed between the two fractions, possibly reflecting structural differences in the lipid anchor of the F3 molecule. Both fractions were highly enriched in other glycosylphosphatidylinositol‐anchored proteins such as NCAM 120 and Thy‐1. It is interesting that these vesicles were devoid of the transmembrane forms (NCAM 180 and NCAM 140), which were recovered in Triton X‐100‐soluble fractions, but contained the L1 transmembrane adhesion molecule that is coexpressed with F3 on parallel fibers and the fyn tyrosine kinase. Immunoprecipitation experiments indicated that F3, but not NCAM 120 or Thy‐1, was physically associated in a complex with both L1 and fyn tyrosine kinase. This strongly suggests that the interaction between L1 and F3, already described to occur with isolated molecules, is present in neural tissue. More important is that our study provides information on the molecular machinery likely to be involved in F3 signaling.

No abstract