Relationship of Lipid Rafts to Transient Confinement Zones Detected by Single Particle Tracking

We examined the physical and chemical characteristics of transient confinement zones (TCZs) that are detected in single particle trajectories of molecules moving within the membrane of C3H 10T1/2 murine fibroblasts and their relationship to "rafts." We studied the lateral movement of different membrane molecules thought to partition to varying degrees into or out of the putative lipid domains known as rafts. We found that lipid analogs spend significantly less time in TCZs compared with Thy-1, a glycosylphosphatidylinositol-anchored protein, and GM1, a glycosphingolipid. For Thy-1, we found that zone abundance was markedly reduced by cholesterol extraction, suggesting that a major source of the observed temporary confinement is related to the presence of raft domains. More detailed analysis of particle trajectories reveals that zones can be revisited even tens of seconds after the original escape and that diffusion within the zones is reduced by a factor of approximately 2, consistent with the zone being a cholesterol-rich liquid-ordered phase. Surprisingly, transient confinement was not strongly temperature dependent. Overall, our data demonstrate that there are raft-related domains present in certain regions of the plasma membrane of C3H cells, which can persist for tens of seconds.