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In the last decade evidence has accumulated that small domains of 50-700 nmin diameter are located in the exoplasmic leaflet of the plasma membrane. Mostof these domains supposedly consist of specific sets of lipids and proteins,and are believed to coordinate signal transduction cascades. Whether similardomains are also present in the cytoplasmic leaflet of the plasma membrane isunclear so far. To investigate the presence of cytoplasmic leaflet domains, theH-Ras membrane-targeting sequence was fused to the C-terminus of the enhancedyellow fluorescent protein. Using single-molecule fluorescence microscopy,trajectories of individual molecules diffusing in the cytoplasmic leaflet ofthe plasma membrane were recorded. From these trajectories, the diffusion ofindividual membrane-anchored enhanced yellow fluorescent protein molecules wasstudied in live cells on timescales from 5 to 200 ms. The results show that thediffusion of 30-40% of the molecules is constrained in domains with a typicalsize of 200 nm. Neither breakdown of actin nor cholesterol extraction changedthe domain characteristics significantly, indicating that the observed domainsmay not be related to the membrane domains identified so far.

Single-molecule imaging of the H-ras membrane-anchor reveals domains in the cytoplasmic leaflet of the cell membrane