It has been supposed that the HA (haemagglutinin) of influenza virus must be recruited to membrane rafts to perform its function in membrane fusion and virus budding. In the present study, we aimed at substantiating this association in living cells by biophysical methods. To this end, we fused the cyan fluorescent protein Cer (Cerulean) to the cytoplasmic tail of HA. Upon expression in CHO (Chinese-hamster ovary) cells HA–Cer was glycosylated and transported to the plasma membrane in a similar manner to authentic HA. We measured FLIM-FRET (Förster resonance energy transfer by fluorescence lifetime imaging microscopy) and showed strong association of HA–Cer with Myr-Pal–YFP (myristoylated and palmitoylated peptide fused to yellow fluorescent protein), an established marker for rafts of the inner leaflet of the plasma membrane. Clustering was significantly reduced when rafts were disintegrated by cholesterol extraction and when the known raft-targeting signals of HA, the palmitoylation sites and amino acids in its transmembrane region, were removed. FRAP (fluorescence recovery after photobleaching) showed that removal of raft-targeting signals moderately increased the mobility of HA in the plasma membrane, indicating that the signals influence access of HA to slowly diffusing rafts. However, Myr-Pal–YFP exhibited a much faster mobility compared with HA–Cer, demonstrating that HA and the raft marker do not diffuse together in a stable raft complex for long periods of time.
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Research Article|
January 15 2010
FLIM-FRET and FRAP reveal association of influenza virus haemagglutinin with membrane rafts
Stephanie Engel;
Stephanie Engel
*Immunology and Molecular Biology, Faculty of Veterinary Medicine, Free University Berlin, Philippstr. 13, 10115 Berlin, Germany
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Silvia Scolari;
Silvia Scolari
†Department of Biology and Molecular Biophysics, Humboldt University Berlin, Invalidenstr. 42, 10115 Berlin, Germany
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Bastian Thaa;
Bastian Thaa
*Immunology and Molecular Biology, Faculty of Veterinary Medicine, Free University Berlin, Philippstr. 13, 10115 Berlin, Germany
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Nils Krebs;
Nils Krebs
†Department of Biology and Molecular Biophysics, Humboldt University Berlin, Invalidenstr. 42, 10115 Berlin, Germany
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Thomas Korte;
Thomas Korte
†Department of Biology and Molecular Biophysics, Humboldt University Berlin, Invalidenstr. 42, 10115 Berlin, Germany
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Andreas Herrmann;
Andreas Herrmann
†Department of Biology and Molecular Biophysics, Humboldt University Berlin, Invalidenstr. 42, 10115 Berlin, Germany
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Michael Veit
Michael Veit
1
*Immunology and Molecular Biology, Faculty of Veterinary Medicine, Free University Berlin, Philippstr. 13, 10115 Berlin, Germany
1To whom correspondence should be addressed (email mveit@zedat.fu-berlin.de).
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Publisher: Portland Press Ltd
Received:
September 21 2009
Revision Received:
October 27 2009
Accepted:
November 04 2009
Accepted Manuscript online:
November 04 2009
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2010 Biochemical Society
2010
Biochem J (2010) 425 (3): 567–573.
Article history
Received:
September 21 2009
Revision Received:
October 27 2009
Accepted:
November 04 2009
Accepted Manuscript online:
November 04 2009
Citation
Stephanie Engel, Silvia Scolari, Bastian Thaa, Nils Krebs, Thomas Korte, Andreas Herrmann, Michael Veit; FLIM-FRET and FRAP reveal association of influenza virus haemagglutinin with membrane rafts. Biochem J 1 February 2010; 425 (3): 567–573. doi: https://doi.org/10.1042/BJ20091388
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