Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172642&req=5

fig1: 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.

Mentions: Ras microlocalization was examined on intact 2-D sheets of apical plasma membrane, ripped off from adherent cells directly onto EM grids (Sanan and Anderson, 1991; Parton and Hancock, 2001). First, we investigated the distributions of GFP fused to the minimal plasma membrane targeting motifs of H-ras, GFP-tH (Fig. 1). GFP-tH, targeted by a combination of palmitoylation and a farnesylated CAAX motif, is an excellent lipid raft marker because even on high expression, it is localized exclusively to low density fractions on sucrose gradients (Prior et al., 2001). We visualized GFP-tH in plasma membrane sheets with anti-GFP–5 nm gold (Fig. 1 a). GFP-tH is extensively distributed in small, morphologically featureless patches. To analyze the complete gold patterns as rigorously and objectively as possible, we used statistical methods for point pattern analysis that are well established, but rarely used in cell biology. Ripley's K-function (Ripley, 1977, 1979; Philimonenko et al., 2000) evaluates an exhaustive map of all interparticle distances over the study area and compares the observed distribution with that expected from complete spatial randomness (CSR).* The analysis, which focuses on the second-order properties of the gold pattern, has the advantage that it analyzes spatial structure at multiple ranges simultaneously (Ripley, 1977, 1979; see Materials and methods and supplementary data).


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172642&req=5

fig1: 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.
Mentions: Ras microlocalization was examined on intact 2-D sheets of apical plasma membrane, ripped off from adherent cells directly onto EM grids (Sanan and Anderson, 1991; Parton and Hancock, 2001). First, we investigated the distributions of GFP fused to the minimal plasma membrane targeting motifs of H-ras, GFP-tH (Fig. 1). GFP-tH, targeted by a combination of palmitoylation and a farnesylated CAAX motif, is an excellent lipid raft marker because even on high expression, it is localized exclusively to low density fractions on sucrose gradients (Prior et al., 2001). We visualized GFP-tH in plasma membrane sheets with anti-GFP–5 nm gold (Fig. 1 a). GFP-tH is extensively distributed in small, morphologically featureless patches. To analyze the complete gold patterns as rigorously and objectively as possible, we used statistical methods for point pattern analysis that are well established, but rarely used in cell biology. Ripley's K-function (Ripley, 1977, 1979; Philimonenko et al., 2000) evaluates an exhaustive map of all interparticle distances over the study area and compares the observed distribution with that expected from complete spatial randomness (CSR).* The analysis, which focuses on the second-order properties of the gold pattern, has the advantage that it analyzes spatial structure at multiple ranges simultaneously (Ripley, 1977, 1979; see Materials and methods and supplementary data).

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