FLIM-FRET and FRAP reveal association of influenza virus hemagglutinin with membrane rafts
Résumé
It has been supposed that the hemagglutinin (HA) of influenza virus must be recruited to membrane-rafts to perform its function in membrane fusion and virus budding. Here, we aimed at substantiating this association in living cells by biophysical methods. To this end, we fused the cyan-fluorescent protein Cerulean to the cytoplasmic tail of HA. Upon expression in CHO cells HA-Cer was glycosylated and transported to the plasma membrane similarly as authentic HA. We measured Förster's resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) and showed strong association of HA-Cer with Myr-Pal-YFP, 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. Fluorescence recovery after photobleaching (FRAP) 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 to 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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