Complete removal of sphingolipids from the plasma membrane disrupts cell to substratum adhesion of mouse melanoma cells.

GM-95, a mutant cell line derived from mouse melanoma MEB-4 cells, is deficient in glycosphingolipids (GSLs) due to the lack of ceramide glucosyltransferase-1 activity (Ichikawa, S., Nakajo, N., Sakiyama, H., and Hirabayashi, Y. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2703-2707). In this study, we examined the involvement of the complex sphingolipids in cell to substratum adhesion. Immunofluorescent and chemical analyses revealed that the complex sphingolipids were significantly concentrated in the detergent-insoluble substrate attachment matrix of both GM-95 and MEB-4 cells. In spite of the absence of GSLs, GM-95 cells retained the ability to adhere to extracellular matrix (ECM) proteins such as fibronectin, collagen, and laminin. When both GM-95 and MEB-4 cells were treated with neutral sphingomyelinase, GM-95 cells were rounded up and detached from all ECM proteins examined. In contrast, neither the morphology nor the adherence of MEB-4 cells was altered. Under this treatment, sphingomyelin (SM) became undetectable in both cells. A similar inhibition was observed upon pretreatment of cells with fumonisin B1 or ISP-1, both of which block the synthesis of ceramide, a common precursor of both GSLs and SM. Stable transfectants expressing GSLs, which were established by transfection of glucosyltransferase-1 cDNA into GM-95 cells, became resistant to neutral sphingomyelinase-mediated rounding up and detachment from ECM proteins. In conclusion, the complex sphingolipids play critical roles in cell to substratum adhesion, and the presence of either GSLs or SM is sufficient for the adhesion.