Binding of cross-linked glycosylphosphatidylinositol-anchored proteins to discrete actin-associated sites and cholesterol-dependent domains.
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.
Suzuki
K
K
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Sheetz
M P
MP
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D021941:Caveolae / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002784:Cholesterol / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D010714:Phosphatidylethanolamines / Q000737:chemistry; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D013499:Surface Properties; D014407:Tumor Cells, Cultured / Q000378:metabolism
D000199:Actins; D003432:Cross-Linking Reagents; D017261:Glycosylphosphatidylinositols; D015395:Histocompatibility Antigens Class I; D010714:Phosphatidylethanolamines; C032907:Q surface antigens; D002784:Cholesterol; D019793:Fluorescein