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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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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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fig4: 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.

Mentions: Little is known about the microlocalization of K-ras targeted by a polylysine domain and a farnesylated CAAX motif. Therefore, we investigated the distribution of GFP fused to the minimal plasma membrane targeting motifs of K-ras, GFP-tK. Analysis of the GFP-tK–labeled gold patterns reveals that they are clustered, but with different characteristics from GFP-tH (Fig. 4 a and Fig. S1 a). Modeling establishes that the GFP-tK domains have a mean radius of 16 ± 3 nm and occupy 20% of the plasma membrane (Fig. S1 c). In contrast to GFP-tH, cholesterol depletion causes a small rise in GFP-tK clustering after 60 min of cyclodextrin treatment. The subtle effect of cyclodextrin on GFP-tK microdomains may reflect a general role of cholesterol in maintaining overall plasma membrane integrity. Wild-type and constitutively active K-ras show identical clustering to GFP-tK, both in the presence or absence of cyclodextrin (unpublished data), and bivariate analysis of plasma membranes coexpressing GFP-tH and activated K-rasG12V showed no significant colocalization of the lipid raft marker with K-ras (Fig. 4 b). Clustering of GFP-tK and K-ras was unexpected, although biophysical studies have shown that myristoylated polybasic peptides can sequester negatively charged lipids to generate novel membrane domains (Murray et al., 1999). Intriguingly, clustering was strikingly reduced when the wild-type K-ras CVIM motif was replaced with CCIL, to direct geranylgeranylation rather than farnesylation (Fig. 4 c, GFP-tKCCIL). Therefore, our data show that nature of the prenoid group profoundly affects the ability of polybasic K-ras to organize into specific microdomains, an observation that may have functional implications. Overall, our results clearly demonstrate for the first time the existence of K-ras microdomains within disordered plasma membranes that are distinct from classical lipid rafts.


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

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

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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fig4: 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.
Mentions: Little is known about the microlocalization of K-ras targeted by a polylysine domain and a farnesylated CAAX motif. Therefore, we investigated the distribution of GFP fused to the minimal plasma membrane targeting motifs of K-ras, GFP-tK. Analysis of the GFP-tK–labeled gold patterns reveals that they are clustered, but with different characteristics from GFP-tH (Fig. 4 a and Fig. S1 a). Modeling establishes that the GFP-tK domains have a mean radius of 16 ± 3 nm and occupy 20% of the plasma membrane (Fig. S1 c). In contrast to GFP-tH, cholesterol depletion causes a small rise in GFP-tK clustering after 60 min of cyclodextrin treatment. The subtle effect of cyclodextrin on GFP-tK microdomains may reflect a general role of cholesterol in maintaining overall plasma membrane integrity. Wild-type and constitutively active K-ras show identical clustering to GFP-tK, both in the presence or absence of cyclodextrin (unpublished data), and bivariate analysis of plasma membranes coexpressing GFP-tH and activated K-rasG12V showed no significant colocalization of the lipid raft marker with K-ras (Fig. 4 b). Clustering of GFP-tK and K-ras was unexpected, although biophysical studies have shown that myristoylated polybasic peptides can sequester negatively charged lipids to generate novel membrane domains (Murray et al., 1999). Intriguingly, clustering was strikingly reduced when the wild-type K-ras CVIM motif was replaced with CCIL, to direct geranylgeranylation rather than farnesylation (Fig. 4 c, GFP-tKCCIL). Therefore, our data show that nature of the prenoid group profoundly affects the ability of polybasic K-ras to organize into specific microdomains, an observation that may have functional implications. Overall, our results clearly demonstrate for the first time the existence of K-ras microdomains within disordered plasma membranes that are distinct from classical lipid 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