Fig 1.
Sialylated IAV attachment factors are organized in nanodomains on A549 cells.
(A) Influenza virus is an enveloped particle that encapsulates its segmented (-)vRNA genome built of 8 viral ribonucleoprotein complexes (vRNPs). The viral membrane harbors the two glycoproteins hemagglutinin (HA) and neuraminidase (NA). HA is responsible for binding sialic acid (SA) containing attachment factors on the host cell plasma membrane. Upon cell-binding, the virus needs to activate functional receptors to trigger endocytosis. (B) Confocal imaging of live A549 cells labelled with SNA (conjugated to JF549). The cells feature a non-uniform SNA distribution across the plasma membrane. Large finger-like protrusions can be observed on the dorsal side of the cell. (C) Confocal and STED live-cell imaging of A549 cells labelled with SNA (conjugated to StarRed) confirms the existence of finger-like protrusions as well as a population of smaller nanodomains with diameter of ~100 nm (C, right, inset). (D) To increase the resolution and focus on the small nanodomain we performed STORM imaging of A549 cells labelled with SNA (conjugated to Alexa647). Reconstructed STORM images confirm two major structural features (1) finger-like protrusions as well as (2) small nanodomains. Cell treatment with neuraminidase (NA, 0.01 U/ml for 2h) led to a strong reduction of the localization density due to the cleavage and hence decrease local concentration of SA (D, right, inset).
Fig 2.
Density-based localization analysis reveals small SA clusters between microvilli.
(A) Spatial distribution of STORM localizations from SNA-A647 on A549 cells showing the coexistence of two structural features, (1) large microvilli as well as (2) small nanodomains. The inset in A shows a pixel image reconstruction (10 nm/pxl) of the localization map in A. (B) Density distribution of localizations shown in A within a search radius of 50 nm. Color scale according to number of neighbor localizations. (C) Final clustering result with identified clusters allows quantification of the cluster area. After all identified clusters were filtered according to their size to selectively analyze non-microvilli structures, we found clusters with an area between 0.005–0.04 μm2 (D). The large cluster labelled by the red dashed line was filtered out (see also S4 Fig). Distribution of the number of molecules per cluster as estimated according to the number of localizations (D, inset). The inner structure of non-microvilli clusters was analyzed according to their local localization density (B). (E) Representative example of two identified clusters showing their inner density gradient. The color code represents the number of nearest neighbor localizations within a radius of 30 nm (i.e. the local localization density). The localization density is plotted on the vertical axis. Distribution of the density difference between background and the cluster center over all identified cluster (F).
Fig 3.
IAV performs a receptor concentration-driven random walk on the plasma membrane.
Based on our quantitative analysis of the AF distribution on A549 cells, we hypothesize a motion behavior that is driven by the local AF concentration. We simulate this behavior initially as a 2D random walk with free diffusion coefficient Dfree (A). (B) Next, we simulate AF clusters (red circles, r = 100 nm), which would, due to the increased SA concentration, lead to a temporal confinement (Dconf < Dfree). To identify confined regions within the simulated virus trajectories, we establish a confinement probability Iconf. Accordingly, a free diffusing particle shows only fluctuation in of Iconf (D), while the addition of temporal confinement leads to a clear increase that overlaps with stationary phases of the particle as visible in the XY displacement plot (E). We used the confinement probability to analyze experimental virus trajectories in particular the mixed type of trajectories (C) (see also S8 Fig) (C). Iconf shows a clear signature of temporal confinement (F) similar to the model prediction (E). As a further challenge for our model, we performed a subtrajectory analysis, thereby extracting the dwell time, Dconf as well as the area of the respective temporal confinement in our virus trajectories. (G) shows an overlay of the perimeters of the extracted confined regions as well as the average radius (R). From our simulated data, correlation of Dconf with the dwell time shows that a local increase in AF concentration (i.e. decreased diffusion) due to the encounter of an SA nanodomain leads to an increased local dwell time (H, red markers). We observed a similar behavior, when we tested the confinement dwell time in experimental virus trajectories (H, black markers).
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).
Fig 5.
Live-cell super-resolution imaging reveals long-lived EGFR clusters in A549 cells.
EGFR coupled to the photo-convertible protein mEos3 was expressed in A549 cells. Subsequent PALM imaging allows to study EGFR distribution in live cells at the single protein level. In the absence of any stimulus, we could detect nanodomains of EGFR within the dorsal and also the ventral plasma membrane (A). Scale bar: left panel, 1 μm. The image in A shows a maximum projected map of single molecule localizations recorded over a period of 10 min. B shows two cluster examples as a cumulative density distribution (upper panel) as well as XY scatter with the colorscale according to time at which the localization was detected (lower panel). While the projection of all localization allows to identify protein clusters, we can use the time information to further estimate the cluster lifetime. As shown in C, cumulative counting of individual localizations within a clustered region gives direct information of the minimum cluster lifetime. D shows the corresponding lifetime distribution of EGFR clusters recorded at the dorsal as well as the ventral side of the cell in the absence of any stimulus.
Fig 6.
Model for IAV-mediated cell binding, receptor search and activation.
Using quantitative STORM imaging, we could show that SA-conjugated IAV AF as well as one functional receptor, EGFR, form nanodomains in the plasma membrane of A549 cells. While dense AF nanodomains constitute an attractive multivalent binding platform, their diversity in local AF concentration suggests a variety of different residence times for which IAV would stay bound within these domains. Using single-virus tracking, we observed a mixed diffusive—confined motion, that could be simulated using our quantitative SA cluster information. These data suggest a receptor concentration-driven lateral search mechanism between SA enriched nanodomains. Eventually, since AF domains partly overlap with EGFR, IAV encounters a functional receptor that can be activated to signal cell entry. Our data further suggest that a stable preformed EGFR cluster population is activated during IAV stimulation, thereby possibly facilitating efficient signal transduction. EGFR clusters are stabilized by lipid rafts as well as cortical actin.