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Published online 1 June 2004. doi:10.1083/jcb.200312170
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 165, Number 5, 735-746
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Article

 Dynamics of putative raft-associated proteins at the cell surface

Anne K. Kenworthy1,2,3, Benjamin J. Nichols1,4, Catha L. Remmert2, Glenn M. Hendrix2, Mukesh Kumar5, Joshua Zimmerberg5, and Jennifer Lippincott-Schwartz1

1 Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20895
2 Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232
3 Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232
4 Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, England, UK
5 Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20817

Address correspondence to J. Lippincott-Schwartz, Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bldg. 18T, Rm. 101, 18 Library Dr., Bethesda, MD 20895. Tel.: (301) 402-1010. Fax: (301) 402-0078. email: jlippin{at}helix.nih.gov

Lipid rafts are conceptualized as membrane microdomains enriched in cholesterol and glycosphingolipid that serve as platforms for protein segregation and signaling. The properties of these domains in vivo are unclear. Here, we use fluorescence recovery after photobleaching to test if raft association affects a protein's ability to laterally diffuse large distances across the cell surface. The diffusion coefficients (D) of several types of putative raft and nonraft proteins were systematically measured under steady-state conditions and in response to raft perturbations. Raft proteins diffused freely over large distances (>4 µm), exhibiting Ds that varied 10-fold. This finding indicates that raft proteins do not undergo long-range diffusion as part of discrete, stable raft domains. Perturbations reported to affect lipid rafts in model membrane systems or by biochemical fractionation (cholesterol depletion, decreased temperature, and cholesterol loading) had similar effects on the diffusional mobility of raft and nonraft proteins. Thus, raft association is not the dominant factor in determining long-range protein mobility at the cell surface.

Key Words: lipid rafts; membrane microdomains; lateral diffusion; fluorescence recovery after photobleaching; cholesterol

Abbreviations used in this paper: CTXB, cholera toxin B subunit; D, diffusion coefficient; DRM, detergent-resistant membrane; GPI, glycosylphosphatidylinositol; MßCD, methyl ß-cyclodextrin; Mf, mobile fraction; NPC, Niemann-Pick type C; NRK, normal rat kidney; TX-100, Triton X-100.


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