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Published online 13 January 2003. doi:10.1083/jcb.200209091
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© The Rockefeller University Press, 0021-9525/2003/1/165 $5.00
The Journal of Cell Biology, Volume 160, Number 2, 165-170

Report

Direct visualization of Ras proteins in spatially distinct cell surface microdomains



Ian A. Prior1, Cornelia Muncke1, Robert G. Parton2 and John F. Hancock1

1 Department of Pathology and Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4006, Australia
2 Institute for Molecular Bioscience, Centre for Microscopy and Microanalysis and School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia

Address correspondence to John F. Hancock, Dept. of Pathology, University of Queensland Medical School, Herston Rd., Herston, Brisbane, Queensland 4006, Australia. Tel.: 61-7-3365-5288. Fax: 61-7-3365-5511. E-mail: j.hancock{at}mailbox.uq.edu.au; or Robert G. Parton, Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia. Tel.: 61-7-3365-6468. Fax: 61-7-3365-4422 E-mail: r.parton{at}imb.uq.edu.au

Localization of signaling complexes to specific microdomains coordinates signal transduction at the plasma membrane. Using immunogold electron microscopy of plasma membrane sheets coupled with spatial point pattern analysis, we have visualized morphologically featureless microdomains, including lipid rafts, in situ and at high resolution. We find that an inner-plasma membrane lipid raft marker displays cholesterol-dependent clustering in microdomains with a mean diameter of 44 nm that occupy 35% of the cell surface. Cross-linking an outer-leaflet raft protein results in the redistribution of inner leaflet rafts, but they retain their modular structure. Analysis of Ras microlocalization shows that inactive H-ras is distributed between lipid rafts and a cholesterol-independent microdomain. Conversely, activated H-ras and K-ras reside predominantly in nonoverlapping, cholesterol-independent microdomains. Galectin-1 stabilizes the association of activated H-ras with these nonraft microdomains, whereas K-ras clustering is supported by farnesylation, but not geranylgeranylation. These results illustrate that the inner plasma membrane comprises a complex mosaic of discrete microdomains. Differential spatial localization within this framework can likely account for the distinct signal outputs from the highly homologous Ras proteins.

Key Words: cholesterol; lipid rafts; immunogold; electron microscopy; statistical analysis


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