Fig. 2: Immobilized IAVs recruit EGFR in a sialic acid-dependent way.
From: Single influenza A viruses induce nanoscale cellular reprogramming at the virus-cell interface
IAVs were immobilized on reactive glass surfaces, and A549 cells expressing EGFR-GFP (a) or EGFR-mEos3.2 (b) were cultivated on top. a TIRF microscopy reveals EGFR accumulation at the virus-binding sites. The bottom right line plot shows the normalized intensities along the white line. b SptPALM imaging was applied, and plots of EGFR-mEos3.2 tracks were superimposed on a still image of the immobilized viruses. Tracks are color-coded. Analysis was performed comparing tracks in viral proximity (red square) with virus-free membrane patches (blue square). Zoom of the green region shown on the right. Images shown in (a and b) are representative of 3 experiments with similar results. c MSD analysis of EGFR-mEos3.2 trajectories taken from cells growing on immobilized viruses (whole cell) shows a bimodal distribution with a mobile and an immobile/confined population. d Initial diffusion coefficients were calculated from the MSD curves of individual EGF receptors at different conditions (left panel). For each bar, the median initial diffusivities of EGFR from six experiments were used. The average median diffusivity of receptors in cells growing on reactive slides with and without viruses is not statistically different (whole cell (wc) ± virus). Looking exclusively in an 800 × 800 nm square around immobilized viruses (on the virus) shows a reduced diffusion coefficient (unpaired t test, two-tailed, p = 0.0040, n = 6). This effect is reversible by treating the cells with sialidase (on virus + sia and wc + virus + sia). Fractions of immobile receptors were analyzed (right panel). Receptors with an initial diffusion coefficient D1-3 < 0.01 µm2/s are considered immobile. In viral proximity, a statistically significant increase in immobile receptor fractions can be observed (unpaired t-test, two-tailed, p = 0.0398, n = 6 replicates from three experiments). Data are presented as mean values +/− SEM. e EGFR-mEos3.2 localizations were rendered using 30 sec time binning. The position of the labeled IAV particle is shown on the left and indicated as a dotted circle on the PALM reconstructions. Recurrent appearance of EGFR clusters can be observed. Scale bars: A, 10 µm; B, 5 µm; E, 1 µm. Source data are provided as a Source Data file.
