Cell surface beta-1,4-galactosyltransferase is associated with the detergent-insoluble cytoskeleton on migrating mesenchymal cells.
Cell surface beta-1,4-galactosyltransferase (GalTase) partially mediates a variety of cell interactions with laminin-containing matrices, including mesenchymal cell spreading and migration and neurite initiation, by binding to N-linked oligosaccharides within the E8 domain of laminin. Previous studies using indirect immunofluorescence have suggested that some surface GalTase colocalizes with actin-containing microfilaments in migrating cells. In this study, we present more direct biochemical evidence showing that surface GalTase is associated with the detergent-insoluble cytoskeleton and that this association is dependent upon the integrity of the cytoskeleton, valency of the anti-GalTase antibody, and migratory status of the cell. Two-thirds of the surface GalTase was associated with the detergent-insoluble cytoskeleton when assayed either by monovalent anti-GalTase Fab fragments or by extracting any detergent-soluble GalTase prior to labeling with intact anti-GalTase IgG. However, 80-100% of the surface GalTase could be induced to associate with the cytoskeleton when cross-linked with anti-GalTase IgG prior to detergent extraction. Destabilizing cytoskeleton-protein interactions with high levels of KCl, elevated pH, or cytochalasin B reduced the amount of surface GalTase retained in the detergent-insoluble cytoskeleton fraction. Finally, we have shown previously that laminin induces the expression of GalTase onto lamellipodia of migrating cells, and in this study, we show that the laminin-induced increase in surface GalTase is cytoskeletally associated. Collectively, these data suggest that cell surface GalTase participates in cell spreading and migration on laminin by virtue of its association with the cytoskeleton.
Eckstein
D J
DJ
Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030.
Shur
B D
BD
D016475:3T3 Cells / Q000201:enzymology; D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002465:Cell Movement; D003599:Cytoskeleton / Q000737:chemistry / Q000201:enzymology*; D003902:Detergents / Q000737:chemistry; D007797:Laminin; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009237:N-Acetyllactosamine Synthase / Q000378:metabolism*
D000199:Actins; D003902:Detergents; D007797:Laminin; D009237:N-Acetyllactosamine Synthase