Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories

doi:10.1073/pnas.0708066104

Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories

*Department of Physics and Astronomy and Institute for Molecular Biophysics, University of Maine, Orono, ME 04469;
^‡The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609; and
^§Laboratory for Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892-1855

Communicated by Thomas S. Reese, National Institutes of Health, Bethesda, MD, September 12, 2007 (received for review May 11, 2007)

Abstract

Organization in biological membranes spans many orders of magnitude in length scale, but limited resolution in far-field light microscopy has impeded distinction between numerous biomembrane models. One canonical example of a heterogeneously distributed membrane protein is hemagglutinin (HA) from influenza virus, which is associated with controversial cholesterol-rich lipid rafts. Using fluorescence photoactivation localization microscopy, we are able to image distributions of tens of thousands of HA molecules with subdiffraction resolution (≈40 nm) in live and fixed fibroblasts. HA molecules form irregular clusters on length scales from ≈40 nm up to many micrometers, consistent with results from electron microscopy. In live cells, the dynamics of HA molecules within clusters is observed and quantified to determine an effective diffusion coefficient. The results are interpreted in terms of several established models of biological membranes.

Footnotes

^†To whom correspondence should be addressed at:
Department of Physics and Astronomy, 313 Bennett Hall, University of Maine, Orono, ME 04469.
E-mail: sam.hess{at}umit.maine.edu
Author contributions: S.T.H. and J.Z. designed research; S.T.H., T.J.G., and M.V.G. performed research; S.T.H., S.A.M., and K.D.M. contributed new reagents/analytic tools; S.T.H., T.J.G., and M.V.G. analyzed data; and S.T.H. and J.Z. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0708066104/DC1.
Abbreviations:

FPALM,

fluorescence photoactivation localization microscopy;

FRAP,

fluorescence recovery after photobleaching;

PA-GFP,

photoactivatable green fluorescent protein.
Freely available online through the PNAS open access option.