Markers for detergent-resistant lipid rafts occupy distinct and dynamic domains in native membranes.
Lipid rafts isolated by detergent extraction and sucrose gradient fractionation from mast cells are enriched for the glycosylphosphatidylinositol-linked protein Thy-1, the ganglioside GM1, palmitoylated LAT, and cross-linked IgE receptors, FcepsilonRI. This study addresses the relationship of fractionation data to the organization of raft markers in native membranes. Immunogold labeling and electron microscopy shows there is little or no colocalization of the raft markers Thy-1, GM1, and LAT with each other or with FcepsilonRI on native membrane sheets prepared from unstimulated cells. External cross-linking of Thy-1 promotes coclustering of Thy-1 with LAT, but not with GM1. Thy-1 and LAT clusters occur on membrane regions without distinctive features. In contrast, external cross-linking of FcepsilonRI and GM1 causes their redistribution to electron-dense membrane patches independently of each other and of Thy-1. The distinctive patches that accumulate cross-linked FcepsilonRI and GM1 also accumulate osmium, a stain for unsaturated lipids, and are sites for coated vesicle budding. Electron microscopy reveals a more complex and dynamic topographical organization of membrane microdomains than is predicted by biochemical analysis of detergent-resistant membranes.
Wilson
Bridget S
BS
Department of Pathology and Cancer Research and Treatment Center, University of New Mexico, Albuquerque, New Mexico 87131, USA. bwilson@salud.unm.edu
Steinberg
Stanly L
SL
Liederman
Karin
K
Pfeiffer
Janet R
JR
Surviladze
Zurab
Z
Zhang
Jun
J
Samelson
Lawrence E
LE
Yang
Li-Hong
LH
Kotula
Paul G
PG
Oliver
Janet M
JM
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism*; D015704:CD4 Antigens / Q000378:metabolism; D002458:Cell Fractionation; D045744:Cell Line, Tumor; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D003902:Detergents / Q000737:chemistry / Q000494:pharmacology*; D004705:Endocytosis; D005677:G(M1) Ganglioside / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D009992:Osmium / Q000378:metabolism; D019308:Palmitic Acid / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D017455:Receptors, IgE / Q000378:metabolism; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D018800:Thy-1 Antigens / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D015415:Biomarkers; D015704:CD4 Antigens; D003902:Detergents; C491733:Lat protein, rat; D008565:Membrane Proteins; D010750:Phosphoproteins; D017455:Receptors, IgE; D018800:Thy-1 Antigens; D009992:Osmium; D019308:Palmitic Acid; D005677:G(M1) Ganglioside