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      Clusters of glycolipid and glycosylphosphatidylinositol-anchored proteins in lymphoid cells : accumulation of actin regulated by local tyrosine phosphorylation

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      European Journal of Immunology
      Wiley

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          Abstract

          Lateral cross-linking of glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipids can trigger a signaling cascade which leads to activation of lymphoid cells. A possible explanation how the signal is transduced through the plasma membrane has arisen from the concept of raft sphingolipid-cholesterol microdomains in cell membranes. Cross-linking of GPI-anchored proteins, glycolipids and other raft components leads to the formation of stabilized membrane patches in the plasma membrane which enrich members of the Src-tyrosine kinase family. We have studied cellular responses to raft patch formation in the Jurkat T cell line and in particular changes in the actin cytoskeleton. We found that raft patches formed by GPI-anchored CD59 protein and the ganglioside GM1 accumulate filamentous actin. Most interestingly, we observed a strong accumulation of tyrosine-phosphorylated proteins in raft patches, strongly supporting the view that they can function as centers of signal transduction. Using a Lck kinase-deficient variant of Jurkat cells and a specific Lck and Fyn tyrosine kinase inhibitor we found that enrichment of actin in raft patches is dependent on phosphotyrosine accumulation in the patches. These observations show a link between raft-mediated signaling and the interaction of actin cytoskeleton with raft membrane domains.

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          Functional rafts in cell membranes.

          A new aspect of cell membrane structure is presented, based on the dynamic clustering of sphingolipids and cholesterol to form rafts that move within the fluid bilayer. It is proposed that these rafts function as platforms for the attachment of proteins when membranes are moved around inside the cell and during signal transduction.
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            Lipid Domain Structure of the Plasma Membrane Revealed by Patching of Membrane Components

            Lateral assemblies of glycolipids and cholesterol, “rafts,” have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpressed plasma membrane markers were evenly distributed in the plasma membrane. We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers. For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin. The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T–lymphoma cells. Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components.
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              GPI-anchored proteins are organized in submicron domains at the cell surface.

              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 proteins. These rafts dictate the sorting of associated proteins and/or provide sites for assembling cytoplasmic signalling molecules. However, there is no direct evidence that rafts exist in living cells. 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 membranes. 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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                Author and article information

                Journal
                European Journal of Immunology
                Eur. J. Immunol.
                Wiley
                0014-2980
                1521-4141
                February 1999
                February 1999
                : 29
                : 2
                : 556-562
                Article
                10.1002/(SICI)1521-4141(199902)29:02<556::AID-IMMU556>3.0.CO;2-2
                10064071
                d0d8e20f-7f7d-472c-9fa9-266a2b99ee83
                © 1999

                http://doi.wiley.com/10.1002/tdm_license_1.1

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