title: FLIM-FRET and FRAP reveal association of influenza virus haemagglutinin with membrane rafts.
abstract: 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.
authors.affiliation: Immunology and Molecular Biology, Faculty of Veterinary Medicine, Free University Berlin, Philippstr. 13, 10115 Berlin, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Engel
authors.forename: Silvia
authors.initials: S
authors.lastname: Scolari
authors.forename: Bastian
authors.initials: B
authors.lastname: Thaa
authors.forename: Nils
authors.initials: N
authors.lastname: Krebs
authors.forename: Thomas
authors.initials: T
authors.lastname: Korte
authors.forename: Andreas
authors.initials: A
authors.lastname: Herrmann
authors.forename: Michael
authors.initials: M
authors.lastname: Veit
mesh_terms: D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006388:Hemagglutinins / Q000737:chemistry*; D021962:Membrane Microdomains / Q000737:chemistry*; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D009975:Orthomyxoviridae / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: C485182:Cyan Fluorescent Protein; D006388:Hemagglutinins; D049452:Green Fluorescent Proteins