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. 2001 Oct;81(4):2181–2189. doi: 10.1016/S0006-3495(01)75866-3

Binding of cross-linked glycosylphosphatidylinositol-anchored proteins to discrete actin-associated sites and cholesterol-dependent domains.

K Suzuki 1, M P Sheetz 1
PMCID: PMC1301690  PMID: 11566789

Abstract

The mechanism by which cross-linked glycosylphosphatidylinositol (GPI)-anchored proteins are immobilized has been a mystery because both the binding to a transmembrane protein and attachment to a rigid cytoskeleton are needed. Using laser tweezers surface scanning resistance (SSR) technology, we obtained physical evidence for cross-linked GPI-anchored protein, Qa-2, binding to a transmembrane protein and for diffusion to discrete cytoskeleton attachment sites. At low levels of cross-linking of Qa-2 molecules, the resistance to lateral movement was that expected of monomeric lipid-anchored proteins, and no specific binding to cytoskeleton-attached structures was observed. When aggregates of the GPI-anchored protein, Qa-2, were scanned across plasma membranes, the background resistance was much higher than expected for a GPI-anchored protein alone and submicron domains of even higher resistance were observed (designated as elastic or non-elastic barriers) at a density of 82 (61 elastic and 21 small non-elastic barriers) per 100 microm(2). Elastic barriers involved weak but specific bonds to the actin cytoskeleton (broken by forces of 2 or 4 pN and were removed by cytochalasin D). Small non-elastic barriers (50-100 nm) depended upon membrane cholesterol and were closely correlated with caveolae density. Thus, cross-linked GPI-anchored proteins can diffuse through the membrane in complex with a transmembrane protein and bind weakly to discrete cytoskeleton attachment sites either associated with flexible actin networks or sphingolipid-cholesterol rich microdomains in live cell membranes. Our SSR measurements provide the first description of the physical characteristics of the interactions between rafts and stable membrane structures.

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Selected References

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  1. Bagatolli L. A., Gratton E. A correlation between lipid domain shape and binary phospholipid mixture composition in free standing bilayers: A two-photon fluorescence microscopy study. Biophys J. 2000 Jul;79(1):434–447. doi: 10.1016/S0006-3495(00)76305-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brown D. A., London E. Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol. 1998;14:111–136. doi: 10.1146/annurev.cellbio.14.1.111. [DOI] [PubMed] [Google Scholar]
  3. Bussell S. J., Koch D. L., Hammer D. A. Effect of hydrodynamic interactions on the diffusion of integral membrane proteins: diffusion in plasma membranes. Biophys J. 1995 May;68(5):1836–1849. doi: 10.1016/S0006-3495(95)80360-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carrion-Vazquez M., Oberhauser A. F., Fisher T. E., Marszalek P. E., Li H., Fernandez J. M. Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering. Prog Biophys Mol Biol. 2000;74(1-2):63–91. doi: 10.1016/s0079-6107(00)00017-1. [DOI] [PubMed] [Google Scholar]
  5. Choquet D., Felsenfeld D. P., Sheetz M. P. Extracellular matrix rigidity causes strengthening of integrin-cytoskeleton linkages. Cell. 1997 Jan 10;88(1):39–48. doi: 10.1016/s0092-8674(00)81856-5. [DOI] [PubMed] [Google Scholar]
  6. Edidin M. Lipid microdomains in cell surface membranes. Curr Opin Struct Biol. 1997 Aug;7(4):528–532. doi: 10.1016/s0959-440x(97)80117-0. [DOI] [PubMed] [Google Scholar]
  7. Felsenfeld D. P., Choquet D., Sheetz M. P. Ligand binding regulates the directed movement of beta1 integrins on fibroblasts. Nature. 1996 Oct 3;383(6599):438–440. doi: 10.1038/383438a0. [DOI] [PubMed] [Google Scholar]
  8. Ferraretto A., Pitto M., Palestini P., Masserini M. Lipid domains in the membrane: thermotropic properties of sphingomyelin vesicles containing GM1 ganglioside and cholesterol. Biochemistry. 1997 Jul 29;36(30):9232–9236. doi: 10.1021/bi970428j. [DOI] [PubMed] [Google Scholar]
  9. Friedrichson T., Kurzchalia T. V. Microdomains of GPI-anchored proteins in living cells revealed by crosslinking. Nature. 1998 Aug 20;394(6695):802–805. doi: 10.1038/29570. [DOI] [PubMed] [Google Scholar]
  10. Ge M., Field K. A., Aneja R., Holowka D., Baird B., Freed J. H. Electron spin resonance characterization of liquid ordered phase of detergent-resistant membranes from RBL-2H3 cells. Biophys J. 1999 Aug;77(2):925–933. doi: 10.1016/S0006-3495(99)76943-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hahn A. B., Soloski M. J. Anti-Qa-2-induced T cell activation. The parameters of activation, the definition of mitogenic and nonmitogenic antibodies, and the differential effects on CD4+ vs CD8+ T cells. J Immunol. 1989 Jul 15;143(2):407–413. [PubMed] [Google Scholar]
  12. Harder T., Scheiffele P., Verkade P., Simons K. Lipid domain structure of the plasma membrane revealed by patching of membrane components. J Cell Biol. 1998 May 18;141(4):929–942. doi: 10.1083/jcb.141.4.929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harder T., Simons K. Clusters of glycolipid and glycosylphosphatidylinositol-anchored proteins in lymphoid cells: accumulation of actin regulated by local tyrosine phosphorylation. Eur J Immunol. 1999 Feb;29(2):556–562. doi: 10.1002/(SICI)1521-4141(199902)29:02<556::AID-IMMU556>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  14. Jacobson K., Dietrich C. Looking at lipid rafts? Trends Cell Biol. 1999 Mar;9(3):87–91. doi: 10.1016/s0962-8924(98)01495-0. [DOI] [PubMed] [Google Scholar]
  15. Kenworthy A. K., Petranova N., Edidin M. High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes. Mol Biol Cell. 2000 May;11(5):1645–1655. doi: 10.1091/mbc.11.5.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Khan S., Sheetz M. P. Force effects on biochemical kinetics. Annu Rev Biochem. 1997;66:785–805. doi: 10.1146/annurev.biochem.66.1.785. [DOI] [PubMed] [Google Scholar]
  17. Korlach J., Schwille P., Webb W. W., Feigenson G. W. Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy. Proc Natl Acad Sci U S A. 1999 Jul 20;96(15):8461–8466. doi: 10.1073/pnas.96.15.8461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kucik D. F., Elson E. L., Sheetz M. P. Weak dependence of mobility of membrane protein aggregates on aggregate size supports a viscous model of retardation of diffusion. Biophys J. 1999 Jan;76(1 Pt 1):314–322. doi: 10.1016/S0006-3495(99)77198-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kusumi A., Suzuki K., Koyasako K. Mobility and cytoskeletal interactions of cell adhesion receptors. Curr Opin Cell Biol. 1999 Oct;11(5):582–590. doi: 10.1016/s0955-0674(99)00020-4. [DOI] [PubMed] [Google Scholar]
  20. Lisanti M. P., Sargiacomo M., Scherer P. E. Purification of caveolae-derived membrane microdomains containing lipid-anchored signaling molecules, such as GPI-anchored proteins, H-Ras, Src-family tyrosine kinases, eNOS, and G-protein alpha-, beta-, and gamma-subunits. Methods Mol Biol. 1999;116:51–60. doi: 10.1385/1-59259-264-3:51. [DOI] [PubMed] [Google Scholar]
  21. Murray E. W., Robbins S. M. Antibody cross-linking of the glycosylphosphatidylinositol-linked protein CD59 on hematopoietic cells induces signaling pathways resembling activation by complement. J Biol Chem. 1998 Sep 25;273(39):25279–25284. doi: 10.1074/jbc.273.39.25279. [DOI] [PubMed] [Google Scholar]
  22. Parton R. G., Joggerst B., Simons K. Regulated internalization of caveolae. J Cell Biol. 1994 Dec;127(5):1199–1215. doi: 10.1083/jcb.127.5.1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pralle A., Keller P., Florin E. L., Simons K., Hörber J. K. Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells. J Cell Biol. 2000 Mar 6;148(5):997–1008. doi: 10.1083/jcb.148.5.997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Saffman P. G., Delbrück M. Brownian motion in biological membranes. Proc Natl Acad Sci U S A. 1975 Aug;72(8):3111–3113. doi: 10.1073/pnas.72.8.3111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sako Y., Kusumi A. Barriers for lateral diffusion of transferrin receptor in the plasma membrane as characterized by receptor dragging by laser tweezers: fence versus tether. J Cell Biol. 1995 Jun;129(6):1559–1574. doi: 10.1083/jcb.129.6.1559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sako Y., Nagafuchi A., Tsukita S., Takeichi M., Kusumi A. Cytoplasmic regulation of the movement of E-cadherin on the free cell surface as studied by optical tweezers and single particle tracking: corralling and tethering by the membrane skeleton. J Cell Biol. 1998 Mar 9;140(5):1227–1240. doi: 10.1083/jcb.140.5.1227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Saxton M. J. Lateral diffusion in a mixture of mobile and immobile particles. A Monte Carlo study. Biophys J. 1990 Nov;58(5):1303–1306. doi: 10.1016/S0006-3495(90)82470-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schneider W. J., Goldstein J. L., Brown M. S. Purification of the LDL receptor. Methods Enzymol. 1985;109:405–417. doi: 10.1016/0076-6879(85)09106-6. [DOI] [PubMed] [Google Scholar]
  29. Schütz G. J., Kada G., Pastushenko V. P., Schindler H. Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy. EMBO J. 2000 Mar 1;19(5):892–901. doi: 10.1093/emboj/19.5.892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sheets E. D., Lee G. M., Simson R., Jacobson K. Transient confinement of a glycosylphosphatidylinositol-anchored protein in the plasma membrane. Biochemistry. 1997 Oct 14;36(41):12449–12458. doi: 10.1021/bi9710939. [DOI] [PubMed] [Google Scholar]
  31. Sheetz M. P. Glycoprotein motility and dynamic domains in fluid plasma membranes. Annu Rev Biophys Biomol Struct. 1993;22:417–431. doi: 10.1146/annurev.bb.22.060193.002221. [DOI] [PubMed] [Google Scholar]
  32. Simons K., Ikonen E. Functional rafts in cell membranes. Nature. 1997 Jun 5;387(6633):569–572. doi: 10.1038/42408. [DOI] [PubMed] [Google Scholar]
  33. Simson R., Yang B., Moore S. E., Doherty P., Walsh F. S., Jacobson K. A. Structural mosaicism on the submicron scale in the plasma membrane. Biophys J. 1998 Jan;74(1):297–308. doi: 10.1016/S0006-3495(98)77787-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Solomon K. R., Rudd C. E., Finberg R. W. The association between glycosylphosphatidylinositol-anchored proteins and heterotrimeric G protein alpha subunits in lymphocytes. Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):6053–6058. doi: 10.1073/pnas.93.12.6053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stahlhut M., van Deurs B. Identification of filamin as a novel ligand for caveolin-1: evidence for the organization of caveolin-1-associated membrane domains by the actin cytoskeleton. Mol Biol Cell. 2000 Jan;11(1):325–337. doi: 10.1091/mbc.11.1.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stauffer T. P., Meyer T. Compartmentalized IgE receptor-mediated signal transduction in living cells. J Cell Biol. 1997 Dec 15;139(6):1447–1454. doi: 10.1083/jcb.139.6.1447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stefanová I., Horejsí V., Ansotegui I. J., Knapp W., Stockinger H. GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Science. 1991 Nov 15;254(5034):1016–1019. doi: 10.1126/science.1719635. [DOI] [PubMed] [Google Scholar]
  38. Suzuki K., Sterba R. E., Sheetz M. P. Outer membrane monolayer domains from two-dimensional surface scanning resistance measurements. Biophys J. 2000 Jul;79(1):448–459. doi: 10.1016/S0006-3495(00)76306-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Varma R., Mayor S. GPI-anchored proteins are organized in submicron domains at the cell surface. Nature. 1998 Aug 20;394(6695):798–801. doi: 10.1038/29563. [DOI] [PubMed] [Google Scholar]
  40. Verkade P., Harder T., Lafont F., Simons K. Induction of caveolae in the apical plasma membrane of Madin-Darby canine kidney cells. J Cell Biol. 2000 Feb 21;148(4):727–739. doi: 10.1083/jcb.148.4.727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Xue W., Kindzelskii A. L., Todd R. F., 3rd, Petty H. R. Physical association of complement receptor type 3 and urokinase-type plasminogen activator receptor in neutrophil membranes. J Immunol. 1994 May 1;152(9):4630–4640. [PubMed] [Google Scholar]

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