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Published 20 June 2005. doi:10.1083/jcb.200412058
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 169, Number 6, 965-976
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Article

 Quantitative electron microscopy and fluorescence spectroscopy of the membrane distribution of influenza hemagglutinin

Samuel T. Hess1, Mukesh Kumar1, Anil Verma1, Jane Farrington1, Anne Kenworthy2, and Joshua Zimmerberg1

1 Laboratory for Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892
2 Department of Molecular Physiology and Biophysics and Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232

Correspondence to Joshua Zimmerberg: joshz{at}helix.nih.gov

Although lipid-dependent protein clustering in biomembranes mediates numerous functions, there is little consensus among membrane models on cluster organization or size. Here, we use influenza viral envelope protein hemagglutinin (HA0) to test the hypothesis that clustering results from proteins partitioning into preexisting, fluid-ordered "raft" domains, wherein they have a random distribution. Japan HA0 expressed in fibroblasts was visualized by electron microscopy using immunogold labeling and probed by fluorescence resonance energy transfer (FRET). Labeled HA coincided with electron-dense, often noncircular membrane patches. Poisson and K-test (Ripley, B.D. 1977. J. R. Stat. Soc. Ser. B. 39:172–212) analyses reveal clustering on accessible length scales (20–900 nm). Membrane treatments with methyl-ß-cyclodextrin and glycosphingolipid synthesis inhibitors did not abolish clusters but did alter their pattern, especially at the shortest lengths, as was corroborated by changes in FRET efficiency. The magnitude and density dependence of the measured FRET efficiency also indicated a nonrandom distribution on molecular length scales (~6–7 nm). This work rules out the tested hypothesis for HA over the accessible length scales, yet shows clearly how the spatial distribution of HA depends on lipid composition.

S. Hess' present address is Department of Physics and Astronomy, University of Maine, Orono, ME 04469.

Abbreviations used in this paper: A({theta}), distribution of angles; CI99, Ripley's K-test 99% confidence interval; fclust, fraction of clustering; FRET, fluorescence resonance energy transfer; L(r) – r, Ripley's K-test metric; MßCD, methyl-ß-cyclodextrin; N(r), nearest neighbor distance distribution; PDMP, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol; PPMP, 1-phenyl-2-palmitoyl-amino-3-morpholino-1-propanol; {rho}nn, most common nearest neighbor distance; r0, correlation length.


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