Fibrillar organization of fibronectin is expressed coordinately with cell surface gangliosides in a variant murine fibroblast

Abstract

NCTC 2071A cells, a line of transformed murine fibroblasts, grow in serum-free medium, are deficient in gangliosides, synthesize fibronectin, but do not retain and organize it on the cell surface. When the cells are exposed to exogenous gangliosides, fibrillar strands of fibronectin become attached to the cell surface. A morphologically distinct variant of NCTC 2071A cells was observed to both retain cell surface fibronectin and organize it into a fibrillar network when the cells were stained with anti-fibronectin antibodies and a fluorescent second antibody. A revertant cell type appeared to resemble the parental NCTC 2071A cells in terms of morphology and fibronectin organization. All three cell types were subjected to mild NaIO4 oxidation and reduction with KB3H4 of very high specific radioactivity in order to label the sialic acid residues of surface gangliosides. The variant had much more surface gangliosides than the parental, particularly more complex gangliosides corresponding to GM1 and GD1a. The surface gangliosides of the revertant were intermediate between the parental and the variant. By using sialidase, which hydrolyzes GD1a to GM1, and 125I-labeled cholera toxin, which binds specifically to GM1, the identity and levels of these gangliosides were confirmed in the three cell types. When variant cells were exposed to sialidase for 2 d, there appeared to be little change in fibronectin organization. Concomitant treatment of the cells with the B subunit of cholera toxin, which bound to all the surface GM1 including that generated by the sialidase, however, eliminated the fibrillar network of fibronectin. In addition, exposure of the variant cells to a 70,000-mol-wt fragment of fibronectin, which lacks the cell attachment domain but contains a matrix assembly domain, inhibited the formation of fibers. Finally, all three cell types were assayed for their ability to attach to and spread on fibronectin-coated surfaces; no significant differences were found. Our results further establish that the ability of a cell to organize fibronectin into an extracellular matrix is dependent on certain gangliosides, but they also indicate that cell adhesion to fibronectin is independent of these gangliosides. We suggest that matrix organization and cell attachment and spreading are based on separate mechanisms and that these functions are associated with different cell surface "receptors."