Dissection of the Influenza A Virus Endocytic Routes Reveals Macropinocytosis as an Alternative Entry Pathway

de Vries, Erik; Tscherne, Donna M.; Wienholts, Marleen J.; Cobos-Jiménez, Viviana; Scholte, Florine; García-Sastre, Adolfo; Rottier, Peter J. M.; de Haan, Cornelis A. M.

One characteristic of macropinocytosis is the nonselective uptake of large amounts of extracellular solutes [33] . Therefore, the uptake of soluble FITC labeled dextran (Fdx) into relatively large vesicles (0.3 to 5 mM) has often been applied as a morphological marker for macropinosomes. Using this marker we found that the addition of 10% FCS to the culture medium slightly increased the uptake of Fdx into HeLa cells (Fig. 8A) . Notably, the distribution of Fdx changes in response to serum from a random distribution into a more granular pattern. At high magnification and at color settings adjusted to higher intensity it could be seen that these Fdx granules were free of actin staining (by phalloidin) indicating that they were in the lumen of vesicles (result not shown). Interestingly, in the presence of IAV (MOI of 10) the uptake of Fdx into vesicles was clearly enhanced. At a higher magnification viral particles could be found to co-localize in Fdx loaded vesicles as well as outside these vesicles (Fig. 8B) . Phalloidin staining of actin was used to demonstrate that many virus particles localized to actin-rich protrusions at the periphery of the cell. The uptake of Fdx was studied in a quantitative manner by flow cytometry (Fig. 8 C) . A moderate, but reproducible shift to higher Fdx fluorescence was observed at 37uC when virus was added in presence of 10% FCS whereas such a shift was absent when no serum or virus was added. This result confirms the observations by confocal microscopy (Fig. 8A) which showed that the combined presence of FCS and IAV increases the uptake of Fdx as compared to FCS alone. In a control experiment the uptake of Fdx in 10% FCS in presence of IAV was shown to be specifically inhibited by EIPA, but not by dynasore (Fig. S2, panel B) . In contrast, transferrin uptake, which serves as a specific marker for CME, was affected by dynasore, but not by EIPA (Fig. S2, panel A) .