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.

Gag Induces the Coalescence of Clustered Lipid Rafts and Tetraspanin-Enriched Microdomains at HIV-1 Assembly Sites on the Plasma Membrane

“…If microdomain markers are not specifically clustered, FRET will occur by random molecular collisions dependent on YFP concentration. On the other hand, if markers are submicroscopically clustered, FRET will be less dependent on YFP concentration (33,122,142).…”

“…To determine whether FRET is highly dependent on YFP concentration, indicating no specific clustering of microdomain markers, or whether FRET is less dependent on YFP concentration, indicating clustering of markers, we used a previously described method of analysis (33,72,122,142). YFP fluorescence intensity was measured for each cell using ImageJ by taking the average pixel intensity within the ROIs determined as described above.…”

“…FRET efficiency (E) was plotted against YFP intensity (F). Data points were fitted according to the hyperbolic function E ϭ (E max ϫ F)/(F ϩ K d ), a simple saturable ligand binding (i.e., Michaelis-Menten) model, where K d is a variable analogous to a dissociation constant (33,122).…”

Membrane Organization and Lipid Rafts

“…Both TMD length and amino acid sequence will be involved in defining raftophilicity (Fig. 4A) (Scheiffele et al 1997;Barman and Nayak 2000;Engel et al 2010).…”

Simultaneous FRAP, FLIM and FAIM for measurements of protein mobility and interaction in living cells

“…Both fluorescence anisotropy and FRAP have previously been used independently for a study of aggregation states of alpha-synuclein, a protein which plays a role in Parkinson's disease [43], and an arrangement for dynamic FRAP and rotational diffusion measurements for colloids has been presented [44]. FRAP and FLIM have also recently been used independently in a study of keratinocyte migration [45], influenza virus association with lipid rafts [46], and cyanobacteria [47]. FLIM and anisotropy have recently been used for high content screening [48], and a system for widefield spectrallyresolved fluorescence lifetime and anisotropy imaging has been presented [49].…”