title: Single-molecule imaging of the H-ras membrane-anchor reveals domains in the cytoplasmic leaflet of the cell membrane.
abstract: 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.
authors.affiliation: Department of Biophysics, Leiden University, Leiden, The Netherlands.
authors.forename: Piet H M
authors.initials: PH
authors.lastname: Lommerse
authors.forename: Gerhard A
authors.initials: GA
authors.lastname: Blab
authors.forename: Laurent
authors.initials: L
authors.lastname: Cognet
authors.forename: Gregory S
authors.initials: GS
authors.lastname: Harms
authors.forename: B Ewa
authors.initials: BE
authors.lastname: Snaar-Jagalska
authors.forename: Herman P
authors.initials: HP
authors.lastname: Spaink
authors.forename: Thomas
authors.initials: T
authors.lastname: Schmidt
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure*; D003593:Cytoplasm / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism* / Q000648:ultrastructure*; D051379:Mice; D021381:Protein Transport / Q000502:physiology; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism* / Q000648:ultrastructure*; D014018:Tissue Distribution
chemical_list: C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)