Partitioning of Thy-1, GM1, and cross-linked phospholipid analogs into lipid rafts reconstituted in supported model membrane monolayers.
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-beta-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 (approximately 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.
Dietrich
C
C
Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC 27599, USA.
Volovyk
Z N
ZN
Levi
M
M
Thompson
N L
NL
Jacobson
K
K
D000906:Antibodies; D002784:Cholesterol / Q000737:chemistry; D003432:Cross-Linking Reagents; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005677:G(M1) Ganglioside / Q000737:chemistry*; D020404:Glycerophospholipids / Q000737:chemistry*; D017261:Glycosylphosphatidylinositols / Q000737:chemistry; D021962:Membrane Microdomains / Q000737:chemistry*; D008567:Membranes, Artificial; D008958:Models, Molecular; D009930:Organic Chemicals; D010714:Phosphatidylethanolamines / Q000737:chemistry*; D018800:Thy-1 Antigens / Q000737:chemistry*
C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; C417664:Alexa594; D000906:Antibodies; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D020404:Glycerophospholipids; D017261:Glycosylphosphatidylinositols; D008567:Membranes, Artificial; D009930:Organic Chemicals; D010714:Phosphatidylethanolamines; D018800:Thy-1 Antigens; C007510:1,2-dipalmitoyl-3-phosphatidylethanolamine; D005677:G(M1) Ganglioside; D002784:Cholesterol; D019793:Fluorescein