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. 2003 Jan 20;160(2):165-70.
doi: 10.1083/jcb.200209091. Epub 2003 Jan 13.

Direct visualization of Ras proteins in spatially distinct cell surface microdomains

Ian A Prior  1 , Cornelia Muncke, Robert G Parton, John F Hancock
Affiliations

Direct visualization of Ras proteins in spatially distinct cell surface microdomains

Ian A Prior et al. J Cell Biol. .

Abstract

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.

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Figures

Figure 1.
Figure 1.
Visualizing lipid rafts using electron microscopy and spatial point pattern analysis. (a) Anti-GFP labeling is specific, ending at the edge of a typical GFP-tH sheet. (b) 60 min of 1% cyclodextrin treatment depletes cell surface cholesterol, detected by filipin labeling (blue). (c) Pooled K-function analyses of the spatial distributions of GFP-tH; L(r) − r values above the 99% confidence interval for CSR (99% CI; closed circles) indicate clustering at that value of r. Untreated GFP-tH (t = 0, red line) shows maximal deflection from CSR at r = 22 nm. Cyclodextrin-treated cells show a time-dependent loss of GFP-tH clustering such that at t = 60 min, GFP-tH is not clustered. K-functions are means (n ≥ 9 for each condition) standardized on the 99% CI. Bar, 100 nm.
Figure 2.
Figure 2.
Analysis of inner- and outer-leaflet lipid raft markers. (a) Inner-leaflet GFP-tH (5 nm gold) and outer-leaflet GFP-GPI (2 nm gold) were specifically labeled to visualize individual microdomains. Univariate K-function analysis of GFP-tH (b) and GFP-GPI (c) show that extensive GFP-GPI aggregation is induced by the patched protocol (closed diamonds), but that GFP-tH remains clustered in small microdomains. Bivariate K-function analysis shows that GFP-tH and GFP-GPI co-cluster when GFP-GPI is aggregated into large patches (d, closed diamonds). There is a tendency for GFP-tH and GFP-GPI to colocalize with the semi-patched technique (open diamonds), but this is not statistically significant. K-functions are means (n ≥ 9 for each condition) standardized on the 99% CI (closed circles). Bars, 50 nm.
Figure 3.
Figure 3.
H-ras also occupies nonraft microdomains. Clustering of GDP-bound H-ras (a; GFP-HG12) and activated H-ras (b; GFP-HG12V) changes little with cyclodextrin treatment. (c) Plasma membrane sheets expressing GFP-H-ras were labeled with anti-GFP–2 nm and anti-Ras–4 nm gold to derive expected values for L biv(r) − r when there is complete colocalization of antigens under these assay conditions. Plasma membrane sheets from cells coexpressing GFP-tH and H-ras were then labeled with anti-GFP–2 nm and anti-Ras–4 nm gold. Bivariate analysis shows extensive colocalization of wild-type, GDP-bound H-ras with GFP-tH (d, open squares); serum stimulated GTP-loading of H-ras decreases coclustering (d, closed squares). Constitutively active H-rasG12V shows no colocalization with GFP-tH, i.e., L biv(r) − r trends around zero (closed diamonds). (e) Transfection with antisense galectin-1 DNA results in loss of endogenous galectin-1 expression. Note the loss of galectin-1 labeling in the transfected cells (arrowheads) compared with control. (f) Activated H-rasG12V clustering is significantly reduced in the absence of galectin-1 expression (open squares) compared with control (closed squares). K-functions are means (n ≥ 8 for each condition) standardized on the 99% CI for univariates and 95% CI for bivariates (closed circles in all panels).
Figure 4.
Figure 4.
K-ras clusters in nonraft microdomains distinct from H-ras microdomains. Peak clustering of GFP-tK occurs at 16 nm; clustering is increased slightly with cyclodextrin treatment (a; t = 0 min, open squares; t = 15 min, open triangles; t = 60 min, open circles). Bivariate K-function analysis indicates no significant colocalization of activated K-ras with the lipid raft marker GFP-tH (b). (c) Replacement of the farnesyl group of GFP-tK (open squares) with a geranylgeranyl group results in a significant reduction in clustering (GFP-tKCCIL, open diamonds). Bivariate analysis of the association of activated Ras with microdomains marked by GFP-tK (d) shows significant colocalization of K-rasG12V with GFP-tK (open squares), but no significant colocalization of H-rasG12V with GFP-tK (open diamonds). Representative examples of electron microscopic images of GFP-tK (e) and GFP-tKCCIL (f) are shown. K-functions are means (n ≥ 9 for each condition) standardized on the 99% CI for univariates and 95% CI for bivariates (closed circles). Bars, 50 nm.
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