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

Abstract

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-β-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.

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

“…Alternatively, because some membrane regions appear to be inaccessible to HA, mobile HA molecules may undergo tethered diffusion. Such results are consistent with observations by confocal microscopy and EM that the clustered distribution of HA leaves many areas with significantly lower than average density (10).…”

“…Results (Fig. 4F) show clustering of HA on all length scales tested, consistent with previous results from EM (10) extended to longer length scales in both living and fixed cells (see SI Figs. 12 and 13 for additional images).…”

Progressive ordering with decreasing temperature of the phospholipids of influenza virus

“…Yet there must be a selective advantage for the virus to enrich itself in lipids that become progressively more immobile with decreasing temperature. We propose that the palmitoylation of hemagglutinin (maximum of three palmitates per trimer) provides a saturated lipid anchor that may itself induce the collection of other saturated-chain lipids and cholesterol when hemagglutinin is concentrated in the plasma membrane before budding (the viral pre-envelope 16 ) for the purpose of increasing viral stability during airborne transmission. Recent super-resolution microscopy studies 32 make clear that hemagglutinin remains mobile within Figure 6 Effect of temperature on lipid mixing assay of influenza virus fusion with RBC membranes.…”

“…These lipids are either selected at the time of budding or pre-selected as the 'pre-envelope' suggested by clusters of the viral envelope protein hemagglutinin seen in immunoelectron microscopy 16 .…”

Lipid rafts, cholesterol, and the brain

“…In some instances, clustering of raft proteins in microdomains can be detected by electron microscopy. The best studied examples include Ras, influenza hemagglutinin, and components of the high affinity IgE receptor signaling pathway (Wilson et al 2002;Parton and Hancock 2004;Wilson et al 2004;Hess et al 2005) (Takeda et al 2003). In addition, large-scale lipid phase separation can be observed in plasma membrane blebs (Baumgart et al 2007).…”