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Vol. 15, Issue 6, 2580-2592, June 2004
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* Departments of Pathology and Cancer Research and Treatment Center, University of New Mexico, Albuquerque, New Mexico 87131;
Departments of Mathematics and Statistics, University of New Mexico, Albuquerque, New Mexico 87131;
Department of Computer Science, University of New Mexico, Albuquerque, New Mexico 87131;
|| Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892; and
¶ Materials Characterization Department, Sandia National Laboratories, Albuquerque, New Mexico 87185
Submitted August 8, 2003;
Revised December 29, 2003;
Accepted February 25, 2004
Monitoring Editor: Jennifer Lippincott-Schwartz
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, Fc
RI. 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 FcRI 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 FcRI 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 FcRI 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.
Abbreviations used: DNP-BSA, dinitrophenol-conjugated bovine serum albumin; FcRI, the high-affinity receptor for IgE; LAT, linker for activation of T cells; RBL-2H3, rat basophilic leukemia cell line 2H3.
Corresponding author. E-mail address: bwilson{at}salud.unm.edu.
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