Fluorescent gangliosides as probes for the retention and organization of fibronectin by ganglioside-deficient mouse cells

Abstract

Ganglioside-deficient transformed mouse fibroblasts (NCTC 2071A cells), which grow in serum-free medium, synthesize fibronectin but do not retain it on the cell surface. When fluorescent derivatives of gangliosides, containing either rhodamine or Lucifer yellow CH attached to the sialic acid residues, were added to the culture medium, the cells incorporated the derivatives and their surfaces became highly fluorescent. When the cells were stained with anti-fibronectin antibodies and a fluorescent second antibody, fibrillar strands of fibronectin were observed to be attached to the cell surface, with partial coincidence of the patterns of direct ganglioside fluorescence and indirect fibronectin immunofluorescence at the cell surface. When the cells were exposed to bacterial neuraminidase during the time of ganglioside insertion, similar patterns of fluorescence were observed. Because the fluorescent gangliosides are resistant to the enzyme, these results suggest that neuraminidase-sensitive endogenous glycoconjugates were not involved in the ganglioside-mediated retention and organization of endogenous fibronectin. After cells were exposed to exogenous chicken fibronectin, most of the fibronectin was attached to the substratum and only a few fibrils were attached to the cells. When exogenous gangliosides were included in the incubation, there was a striking increase in cell-associated exogenous fibronectin, which was highly organized into a fibrillar network. Conversely, cells incubated for 18 h with exogenous unmodified gangliosides exhibited a highly organized network of endogenously derived fibronectin. Upon further incubation of the cells for 2 h with fluorescent gangliosides, there was considerable co-distribution of the fluorescent gangliosides with the fibronectin network as revealed by immunofluorescence. Our results support the concept that gangliosides can mediate the attachment of fibronectin to the cell surface and its organization into a fibrillar network.