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