Glycosphingolipids and Non-Raft Phospholipids Exhibit Very Similar Dynamics in Single-Molecule Observations

Abstract

The diffusion of typical raft-associated molecules, glycosphingolipids, GM1, GM3, GD1a, was observed using high-speed single-particle tracking (HS-SPT) at a temporal resolution of ∼6 μs. Each glycosphingolipid molecule was tagged with a 40-nm-gold particle conjugated by the respective Fab antibody or a Cy3-conjugated Fab antibody. At video rate, both probes exhibited the same diffusion behavior, undergoing apparent simple Brownian diffusion at the same effective diffusion coefficient (about 0.2 μm2/s). In HS-SPT observations, each glycosphingolipid molecule underwent actin-dependent hop diffusion over the membrane compartment of an average of 110 nm in diameter at an average frequency of once every 25 ms. Surprisingly, this behavior is the same as that of a typical non-raft phospholipid DOPE, qualitatively and quantitatively. These results are at variance with the previous FCS/FRAP studies. In addition, previous investigations concluded that GM1, labeled either with cholera toxin (CTX) in its headgroup or with Bodipy-FL on the alkyl chain, diffused much more slowly than non-raft phospholipid probes. Here, we found that CTX bound to GM1 in the plasma membrane diffused as fast as DOPE during the initial 0.5 s after its binding to the membrane, but slowed by a factor of 4 within 10 min after its binding. These results suggest that the slowed diffusion of GM1 found in previous observations would probably be due to the influences of CTX-induced GM1 crosslinking or of perturbation by Bodipy-FL on the alkyl chain. These results indicate that in the plasma membrane of non-stimulated cells, glycosphingolipids movements are not slowed by the possible presence of raft, further suggesting that the rafts in the steady-state cells are much smaller than the compartment size (110 nm) and/or short-lived (<<25 ms).