Recently developed glycosphingolipid probes and their dynamic behavior in cell plasma membranes as revealed by single-molecule imaging.

Glycosphingolipids, including gangliosides, are representative lipid raft markers that perform a variety of physiological roles in cell membranes. However, studies aimed at revealing their dynamic behavior in living cells are rare, mostly due to a lack of suitable fluorescent probes. Recently, the ganglio-series, lacto-series, and globo-series glycosphingolipid probes, which mimic the behavior of the parental molecules in terms of partitioning to the raft fraction, were developed by conjugating hydrophilic dyes to the terminal glycans of glycosphingolipids using state-of-art entirely chemical-based synthetic techniques. High-speed, single-molecule observation of these fluorescent probes revealed that gangliosides were scarcely trapped in small domains (100 nm in diameter) for more than 5 ms in steady-state cells, suggesting that rafts including gangliosides were always moving and very small. Furthermore, dual-color, single-molecule observations clearly showed that homodimers and clusters of GPI-anchored proteins were stabilized by transiently recruiting sphingolipids, including gangliosides, to form homodimer rafts and the cluster rafts, respectively. In this review, we briefly summarize recent studies, the development of a variety of glycosphingolipid probes as well as the identification of the raft structures including gangliosides in living cells by single-molecule imaging.