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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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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 Lbiv(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., Lbiv(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).
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fig3: 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 Lbiv(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., Lbiv(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).

Mentions: Full-length GFP–H-ras and GFP–H-rasG12V also have clustered distributions when expressed in BHK cells (Fig. 3). However, in contrast to GFP-tH, cyclodextrin treatment has relatively small effects on the clustering of GDP-bound H-ras (Fig. 3 a), GTP-bound H-rasG12V (Fig. 3 b) or GFP-targeted by the COOH-terminal 25 amino acids of H-ras (GFP-CTH; unpublished data). These data are consistent with a model where full-length H-ras has affinity for, and is resident in at least two plasma membrane microdomains; lipid rafts, and a noncholesterol-dependent nonraft microdomain (Jaumot et al., 2001; Prior et al., 2001). To formally test this hypothesis, we used bivariate K-function analysis to study the interaction of H-ras with lipid rafts. GFP-tH was coexpressed with untagged H-ras and plasma membrane sheets labeled with anti-GFP–2 nm and anti-Ras–4 nm gold. Bivariate analysis of the gold patterns (Fig. 3 d) reveals extensive colocalization of GDP-bound H-ras with GFP-tH in serum-starved cells, and shows that this decreases with serum stimulation. Constitutively activated H-rasG12V gives an even clearer picture of this phenomenon in that Lbiv(r) − r trends around zero, indicating no detectable colocalization of GFP-tH with H-rasG12V (Fig. 3 d). Together with the recent FRAP analysis of GFP-H-Ras in living cells, our results strongly suggest that wild-type H-ras is normally in a dynamic equilibrium between lipid rafts and other noncholesterol-dependent microdomains. Interaction with the nonraft site is favored experimentally by disrupting lipid rafts, i.e., removing one of the H-ras interaction sites, or physiologically by GTP loading. Thus, constitutively active H-ras is predominantly resident in the nonraft microdomain. The interaction of H-ras with the nonraft domain requires protein sequences in the hypervariable region because GFP-tH clustering is abolished by cyclodextrin treatment, whereas GFP-CTH is not.


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

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

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 Lbiv(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., Lbiv(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).
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Related In: Results  -  Collection

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fig3: 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 Lbiv(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., Lbiv(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).
Mentions: Full-length GFP–H-ras and GFP–H-rasG12V also have clustered distributions when expressed in BHK cells (Fig. 3). However, in contrast to GFP-tH, cyclodextrin treatment has relatively small effects on the clustering of GDP-bound H-ras (Fig. 3 a), GTP-bound H-rasG12V (Fig. 3 b) or GFP-targeted by the COOH-terminal 25 amino acids of H-ras (GFP-CTH; unpublished data). These data are consistent with a model where full-length H-ras has affinity for, and is resident in at least two plasma membrane microdomains; lipid rafts, and a noncholesterol-dependent nonraft microdomain (Jaumot et al., 2001; Prior et al., 2001). To formally test this hypothesis, we used bivariate K-function analysis to study the interaction of H-ras with lipid rafts. GFP-tH was coexpressed with untagged H-ras and plasma membrane sheets labeled with anti-GFP–2 nm and anti-Ras–4 nm gold. Bivariate analysis of the gold patterns (Fig. 3 d) reveals extensive colocalization of GDP-bound H-ras with GFP-tH in serum-starved cells, and shows that this decreases with serum stimulation. Constitutively activated H-rasG12V gives an even clearer picture of this phenomenon in that Lbiv(r) − r trends around zero, indicating no detectable colocalization of GFP-tH with H-rasG12V (Fig. 3 d). Together with the recent FRAP analysis of GFP-H-Ras in living cells, our results strongly suggest that wild-type H-ras is normally in a dynamic equilibrium between lipid rafts and other noncholesterol-dependent microdomains. Interaction with the nonraft site is favored experimentally by disrupting lipid rafts, i.e., removing one of the H-ras interaction sites, or physiologically by GTP loading. Thus, constitutively active H-ras is predominantly resident in the nonraft microdomain. The interaction of H-ras with the nonraft domain requires protein sequences in the hypervariable region because GFP-tH clustering is abolished by cyclodextrin treatment, whereas GFP-CTH is not.

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