Partitioning of Thy-1, GM1, and cross-linked phospholipid analogs into lipid rafts reconstituted in supported model membrane monolayers
- Departments of *Cell and Developmental Biology and †Chemistry, and §Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599; and ‡Department of Medicine, University of Texas Southwestern Medical Center and Veterans Administration Medical Center, Dallas, TX 75216
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Edited by Kai Simons, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany, and approved July 2, 2001 (received for review April 5, 2001)
Abstract
As shown earlier, raft-like domains resembling those thought to be present in natural cell membranes can be formed in supported planar lipid monolayers. These liquid-ordered domains coexist with a liquid-disordered phase and form in monolayers prepared both from synthetic lipid mixtures and lipid extracts of the brush border membrane of mouse kidney cells. The domains are detergent-resistant and are highly enriched in the glycosphingolipid GM1. In this work, the properties of these raft-like domains are further explored and compared with properties thought to be central to raft function in plasma membranes. First, it is shown that domain formation and disruption critically depends on the cholesterol density and can be controlled reversibly by treating the monolayers with the cholesterol-sequestering reagent methyl-β-cyclodextrin. Second, the glycosylphosphatidylinositol-anchored cell-surface protein Thy-1 significantly partitions into the raft-like domains. The extent of this partitioning is reduced when the monolayers contain GM1, indicating that different molecules can compete for domain occupation. Third, the partitioning of a saturated phospholipid analog into the raft phase is dramatically increased (15% to 65%) after cross-linking with antibodies, whereas the distribution of a doubly unsaturated phospholipid analog is not significantly affected by cross-linking (≈10%). This result demonstrates that cross-linking, a process known to be important for certain cell-signaling processes, can selectively translocate molecules to liquid-ordered domains.
Footnotes
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↵ ¶ To whom reprint requests should be addressed at: Department of Cell and Developmental Biology, University of North Carolina, CB# 7090, 108 Taylor Hall, Chapel Hill, NC 27599. E-mail: frap{at}med.unc.edu.
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This paper was submitted directly (Track II) to the PNAS office.
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See commentary on page 10517.
- Abbreviations:
- GPI,
- glycosylphosphatidylinositol;
- BBM,
- brush border membrane;
- DRM,
- detergent-resistant membrane fraction;
- DOPC,
- 1,2-dioleoyl-sn-glycero-3-phosphocholine;
- SM,
- sphingomyelin;
- FL-DOPE,
- 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-fluorescein;
- FL-DPPE,
- 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-fluorescein;
- TR-DPPE,
- 1,2- dipalmitoyl-sn-glycero-3-phospho-ethanol-amine-x-Texas red;
- CTB,
- cholera toxin B;
- FL-CTB,
- fluorescein-conjugated CTB;
- MβCD,
- methyl-β-cyclodextrin;
- F-Thy-1,
- Thy-1 labeled with fluorescein isothiocyanate;
- FRAP,
- fluorescence recovery after photobleaching
- Copyright © 2001, The National Academy of Sciences
