Influenza A viruses use multivalent sialic acid clusters for cell binding and receptor activation
Fig 4
EGFR is organized in nanodomains that overlap with SNA domains.
(A) A549 cells were labelled with antibodies against EGFR. STORM imaging revealed a clustered organization of EGFR. The clusters have an average diameter of 60 nm and contain about 5–10 molecules (B). Scale bar 1 μm. Inset: 100 nm. (C) Two-color STORM imaging of A549 cells labelled with SNA and anti-EGFR antibodies. The two panels on the right show larger magnification of the boxed areas in the left panel. Scale bars: 500 nm (left panel), 200 nm (right panel). The degree of colocalization was quantified using coordinate-based colocalization, where each localization is associated with a colocalization value CA. (D) Box plots of CA distribution of SNA localizations when colocalized with (1) SNA, (2) a random distribution of localizations at equal density as EGFR and (3) EGFR. After IAV-stimulation (MOI~100), we found that phosphorylated EGFR (Y1068) is also localized in nanodomains. Although a small population of clusters seems to be phosphorylated without stimulus, we observed an increase in the activated cluster population after stimulation with IAV (MOI~100) or EGF (100 ng/ml) (E, lower panel). To test for a potential redistribution of EGFR, we looked at the entire population after stimulation. While after EGF stimulation, we could observe a reduction of the clustered protein fraction as well as the cluster density per area, we could not detect such a protein redistribution after IAV stimulation (F).