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GPI-anchored proteins are organized in submicron domains at the cell surface

Nature volume 394, pages 798–801 (1998)Cite this article

Abstract

Lateral heterogeneities in the classical fluid-mosaic model of cell membranes are now envisaged as domains or ‘rafts’ that are enriched in (glyco)sphingolipids, cholesterol, specific membrane proteins and glycosylphosphatidylinositol (GPI)-anchored proteins1. These rafts dictate the sorting of associated proteins and/or provide sites for assembling cytoplasmic signalling molecules2. However, there is no direct evidence that rafts exist in living cells3,4. We have now measured the extent of energy transfer between isoforms of the folate receptor bound to a fluorescent analogue of folic acid, in terms of the dependence of fluorescence polarization on fluorophore densities in membranes5. We find that the extent of energy transfer for the GPI-anchored folate-receptor isoform is density-independent, which is characteristic of organization in sub-pixel-sized domains at the surface of living cells; however, the extent of energy transfer for the transmembrane-anchored folate-receptor isoform was density-dependent, which is consistent with a random distribution. These domains are likely to be less than 70 nm in diameter and are disrupted by removal of cellular cholesterol. These results indicate that lipid-linked proteins are organized in cholesterol-dependent submicron-sized domains. Our methodology offers a new way of monitoring nanometre-scale association between molecules in living cells.

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Figure 1: Possible outcomes of the fluorescence depolarization experiment for different arrangements of fluorescently labelled GPI-anchored proteinsin a microscope pixel.
Figure 2: Distribution of fluorescence intensity and anisotropy values at the surface of PLF-labelled FR-GPI- and FR-TM-expressing cells.
Figure 3: Anisotropy distributions at the surface of PLF-labelled cells obtained after photobleaching.
Figure 4: Effect of cholesterol depletion on the organization of GPI-anchored proteins at the surface of cells.

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Acknowledgements

We thank M. K. Mathew and G. Krishnamoorthy for their insight, F. Barrantes, A. K. Menon and J. Udgaonkar for critical reading of the text, S. Chitkala and N. S. Kiran for making the FR-TM construct, and R. Priya for help with cholesterol analysis. S.M. thanks the Human Frontier Science Program and the National Centre for Biological Sciences for support, and F. F. Bosphorus for inspiration.

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  1. National Centre for Biological Sciences, TIFR Centre, IISc Campus, Post Box 1234, Bangalore, 560012, India

    Rajat Varma & Satyajit Mayor

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Correspondence to Satyajit Mayor.

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Varma, R., Mayor, S. GPI-anchored proteins are organized in submicron domains at the cell surface. Nature 394, 798–801 (1998). https://doi.org/10.1038/29563

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