Direct visualization of Ras proteins in spatially distinct cell surface microdomains

By I. Prior, C. Muncke, Robert G. Parton, John Hancock. Journal of Cell Biology, 2003

777 citations recorded by the source corpus.

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.

Subfields: Cell Biology, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/83b3ceebde0244212f1296eae560d0d10f5d8720

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Citations in the corpus

A comprehensive survey of Ras mutations in cancer (2012, neutral): “Each isoform is present in these locations in different concentrations due to differences in total abundance, specific targetting and relative affinity (43-49).”
Ras proteins: different signals from different locations (2003, methodology): “Data obtained using a totally different experimental approac”
Galectin-1: a small protein with major functions. (2006, background): “P HeLa, HEK293, Rat-1, 293T cells ↑ ras activation with selective activation of Raf-1/ERK pathway Paz et al., 2001; Elad-Sfadia et al., 2002; Prior et al., 2003; Rotblat et al., 2004 Laminin NS P–”
Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis (2011, background): “Thus, in the special case where the proteins are randomly distributed, the protein correlation function g(r)protein (r > 0) ~ 1, and the second term of equation (6) becomes unity.”
Nonequilibrium physics in biology (2019, neutral): “From (Prior et al., 2003).”
PTRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function. (2008, methodology): “To confirm that PTRF is localizing to caveolae, we performed immunoelectron microscopy (immuno-EM) on plasma membrane lawns (Prior et al., 2003) generated from baby hamster kidney (BHK) cells cotransfected with Cav1-mGFP and PTRF-RFP.”
Lipid rafts: contentious only from simplistic standpoints (2006, background): “Clustering of GFP-tH is abrogated by cholesterol depletion or disassembly of the actin cytoskeleton and, as with GFP–GPI, the ratio of GFP-tH clusters to monomers does not change as a function of GFP-tH expression 24 , 25 .”