Partitioning of Thy-1, GM1, and cross-linked phospholipid analogs into lipid rafts reconstituted in supported model membrane monolayers

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-␤-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 (Ϸ10%). 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.membrane domains ͉ receptor cross-linking ͉ cholesterol ͉ glycosylphosphatidylinositol-anchored proteins ͉ signal transduction T reatment of cell lysates with cold nonionic detergent and subsequent sucrose gradient centrifugation allows extraction of a detergent-resistant membrane fraction (DRM) (1, 2). The finding that this fraction is enriched in cholesterol and sphingolipids that form a liquid-ordered phase (3, 4) has led to the hypothesis that the DRM arises directly from discrete liquidordered domains in the plasma membrane termed lipid rafts (5, 6). Such domains could be important for membrane trafficking and sorting (1, 7). Moreover, cell signaling molecules are enriched in the DRM, and the partitioning of these molecules into the DRM is altered during signaling (8-12). Extraction of cholesterol from plasma membranes affects the abundance of molecules found in the DRM and impairs processes like membrane trafficking (13, 14) and cell activation (15, 16). These findings suggest that lipid rafts on plasma membranes are maintained by proper cholesterol levels as functional, preassembled signal transduction complexes (17)(18)(19).Although there is accumulating evidence suggesting that some form of lipid domains or clusters are present in cell membranes, there is still considerable controversy surrounding the basic physical properties of these domains (compositional diversity, size, structure, and dynamics) and their relation to the DRM (for review see refs. 20 and...

Lipid rafts: contentious only from simplistic standpoints

“…L o -L d phase separation in model membranes laterally segregates glycosylphosphatidylinositol (GPI)-anchored proteins, although the segregation is usually incomplete. For example, ~40% of the GPI-anchored protein THY1 partitions into L o domains of supported monolayers and unsupported bilayers 18,19 . Similarly, ~30% of GPI-PLAP (placental alkaline phosphatase) partitions into L o domains of giant unilamellar vesicles that are imaged using fluorescence correlation spectroscopy (FCS), although this fraction increases if the GPI-PLAP is crosslinked 20 .…”

Surface Patterning with Colloidal Monolayers

“…lipid rafts domains, immobilized protein domains on the cytoskeleton. [298][299] To explore effects of the barrier size and obstacle lattice on the diffusion within the membrane, the size of the colloids used in the substrate patterning process was varied. As the applied patterning procedure yields arrays of triangles corresponding to the voids in the colloidal monolayer, the dimensions of the triangular structures can be precisely adjusted by the used 206…”

Order of lipid phases in model and plasma membranes

“…Also in terms of protein segregation, GPI-anchored proteins, which are raft-associated in native membranes, partition into the Lo phase (19,20). However, one difference is striking: raft-associating transmembrane (TM) proteins have been shown to be excluded (21)(22)(23)(24), suggesting energetic constraints for TM ␣-helices partitioning into the tightly packed Lo phase as expected from the potential disordering effect of the amino acid side chains of the TM domain (18,24).…”