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Direct visualization of Ras proteins in spatially distinct cell surface microdomains.

Prior IA, Muncke C, Parton RG, Hancock JF - J. Cell Biol. (2003)

Bottom Line: Cross-linking an outer-leaflet raft protein results in the redistribution of inner leaflet rafts, but they retain their modular structure.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4006, Australia.

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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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.
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fig2: 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.

Mentions: Next, we examined the relationship between inner- and outer-leaflet lipid rafts using a variation of the K-function analysis. When plasma membrane sheets are labeled for two different antigens with 2 nm and 4–5 nm gold, colocalization can be assessed using bivariate K-functions that determine whether one gold population is clustered with respect to the other (Diggle, 1986; see Materials and methods and supplementary data). We compared the distribution of GFP-tH with the outer-leaflet raft marker GFP-GPI; Fig. 2). Both proteins are GFP-tagged, but because only one membrane surface is exposed at any point in the labeling and rip-off procedure, no leakage of gold probes occurs (unpublished data). We used two protocols to induce different degrees of GFP-GPI aggregation, as revealed by univariate K-function analysis of the 2-nm gold patterns (Fig. 2 c). The semi-patched technique induces relatively little GFP-GPI aggregation (univariate K-function shows a mean cluster radius of 50 nm), whereas the patched protocol, routinely used to visualize lipid rafts by immunofluorescence, induces very large GFP-GPI aggregates (univariate K-function shows a mean radius of 180 nm). It is not possible to completely evaluate unpatched GFP-GPI because this necessitates ripping off apical membranes from prefixed cells, a technique that has to date proven unsuccessful. The bivariate K-function shows that there is significant colocalization of GFP-tH with GFP-GPI only when GFP-GPI is aggregated into very large patches (Fig. 2 d); this is indicated by significant positive deflections of the Lbiv(r) − r curve from zero. Interestingly, despite the major reorganization of GFP-GPI, GFP-tH remains in small clusters (Fig. 2 b; univariate K-function for GFP-tH). This result indicates that inner leaflet rafts, when aggregated by cross-linking GPI-anchored proteins, still retain their modular structure. Incomplete colocalization of inner- and outer-leaflet raft markers has been observed previously (Harder et al., 1998; Prior et al., 2001), but our new data show that inner and outer leaflet rafts are only loosely associated at steady state. This offers a potential new mechanism for regulating signaling by modulating the extent of coupling between inner and outer leaflet rafts.


Direct visualization of Ras proteins in spatially distinct cell surface microdomains.

Prior IA, Muncke C, Parton RG, Hancock JF - J. Cell Biol. (2003)

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.
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Related In: Results  -  Collection

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fig2: 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.
Mentions: Next, we examined the relationship between inner- and outer-leaflet lipid rafts using a variation of the K-function analysis. When plasma membrane sheets are labeled for two different antigens with 2 nm and 4–5 nm gold, colocalization can be assessed using bivariate K-functions that determine whether one gold population is clustered with respect to the other (Diggle, 1986; see Materials and methods and supplementary data). We compared the distribution of GFP-tH with the outer-leaflet raft marker GFP-GPI; Fig. 2). Both proteins are GFP-tagged, but because only one membrane surface is exposed at any point in the labeling and rip-off procedure, no leakage of gold probes occurs (unpublished data). We used two protocols to induce different degrees of GFP-GPI aggregation, as revealed by univariate K-function analysis of the 2-nm gold patterns (Fig. 2 c). The semi-patched technique induces relatively little GFP-GPI aggregation (univariate K-function shows a mean cluster radius of 50 nm), whereas the patched protocol, routinely used to visualize lipid rafts by immunofluorescence, induces very large GFP-GPI aggregates (univariate K-function shows a mean radius of 180 nm). It is not possible to completely evaluate unpatched GFP-GPI because this necessitates ripping off apical membranes from prefixed cells, a technique that has to date proven unsuccessful. The bivariate K-function shows that there is significant colocalization of GFP-tH with GFP-GPI only when GFP-GPI is aggregated into very large patches (Fig. 2 d); this is indicated by significant positive deflections of the Lbiv(r) − r curve from zero. Interestingly, despite the major reorganization of GFP-GPI, GFP-tH remains in small clusters (Fig. 2 b; univariate K-function for GFP-tH). This result indicates that inner leaflet rafts, when aggregated by cross-linking GPI-anchored proteins, still retain their modular structure. Incomplete colocalization of inner- and outer-leaflet raft markers has been observed previously (Harder et al., 1998; Prior et al., 2001), but our new data show that inner and outer leaflet rafts are only loosely associated at steady state. This offers a potential new mechanism for regulating signaling by modulating the extent of coupling between inner and outer leaflet rafts.

Bottom Line: Cross-linking an outer-leaflet raft protein results in the redistribution of inner leaflet rafts, but they retain their modular structure.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4006, Australia.

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.

Show MeSH
Related in: MedlinePlus