Localization of synthetic glycolipids in the cell and the dynamics of their insertion/loss

Modification of the cell surface with synthetic glycolipids opens up a wide range of possibilities for studying the function of glycolipids. Synthetic glycolipids called Function-Spacer-Lipids (FSL; where F is a glycan or label, S is a spacer, and L is dioleoylphosphatidyl ethanolamine) easily and controllably modify the membrane of a living cells. This current study investigates the dynamics and mechanism of the FSL insertion and release/loss. FSL insert into the cell membrane (~1 million molecules per cell) within tens of minutes, almost regardless of the nature of the cells (including the thickness of their glycocalyx) and the size of the FSL glycan. FSLs do not accumulate uniformly, but instead form patches >300 nm in size either entrapped in the glycocalyx, or integrated in the plane of the plasma membrane, but always outside the cell rafts. The natural release (loss) of FSL from the modified cell was two orders of magnitude slower than attachment/insertion and occurred mainly in the form of released microvesicles with a size of 140 ± 5 nm. The accumulation of FSL as patches in the cell membrane is similar to the coalescence of natural glycosphingolipids and supports (along with their long residence time in the membrane) the use of FSL as probes for the study of glycosphingolipid-protein interactions.