Characterization and intracellular distribution of microtubule-associated protein 2 in differentiating human neuroblastoma cells.
The use of a panel of monoclonal antibodies (mAbs) directed against different determinants of microtubule-associated protein 2 (MAP2) enabled us to identify two distinct high-molecular-mass MAP2 species (270 and 250 kDa) and a substantial amount of MAP2c (70 kDa) in human neuroblastoma cells. The 250-kDa MAP2 species appears to be confined to the human neuroblastoma cells and was not observed in microtubules (MTs) from bovine and rat brain, mouse neuroblastoma, or MTs from human cerebellum. A new overlay method was developed, which demonstrates binding of tubulin to human neuroblastoma high-molecular-mass MAP2 by exposing nitrocellulose-bound MT proteins under polymerization conditions to tubulin. Bound tubulin was detected with a mAb directed against beta-tubulin. The binding of tubulin to MAP2 could be abolished by a peptide homologous to positions 426-445 of the C-terminal region of beta-tubulin. Immunological cross-reactivity with several mAbs directed against bovine brain MAP2, taxol-promoted coassembly into MTs, and immunocytochemical visualization within cells were further criteria utilized to characterize these proteins as true MAPs. Indirect immunofluorescence with anti-MAP2 and anti-beta-tubulin mAbs demonstrated that there is a change in the spatial organization of MTs during induced cell differentiation, as indicated by the appearance of MT bundles and the redistribution of MAP2.
Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel.
J
J
Kirsch
A
A
Zutra
U Z
UZ
Littauer
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D001921:Brain / Q000648:ultrastructure; D002417:Cattle; D002454:Cell Differentiation; D000939:Epitopes / Q000276:immunology; D005455:Fluorescent Antibody Technique; D006801:Humans; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D008870:Microtubules / Q000032:analysis; D008970:Molecular Weight; D009447:Neuroblastoma / Q000032:analysis* / Q000648:ultrastructure; D010766:Phosphorylation; D051381:Rats; D014404:Tubulin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008869:Microtubule-Associated Proteins; D014404:Tubulin