Spatio-temporal analysis of cell membrane molecules involved in the enhancement of malignant properties by glycosphingolipids

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We analyzed effects of GD3 expression on the cell signals triggered by hepatocyte growth factor (HGF)/Met interaction and by adhesion to collagen type I. The simultaneous treatment with both of them resulted in markedly increased activation of Akt and Erk in GD3+ cells. These results suggested that GD3 plays a crucial role in the convergence of PI3K-Akt and Ras-Erk signals, leading to the synergistic effects of those signals on malignant properties of melanomas. To search molecules existing in the vicinity of GD3 in living cells, enzyme-mediated activation of radical sources (EMARS) combined with mass spectrometry was performed. Neogenin and Integrin were identified to be interacting molecules with GD3 on lipid rafts of the cell membrane. We also tried spatio-temporal analysis of cell membrane molecules by single molecule observation, showing that glycosphingolipid GM1 specifically forms homodimer on the cell surface. Single molecule observation of GD3 on the membrane is now on-going.