IFITM3 Clusters on Virus Containing Endosomes and Lysosomes Early in the Influenza A Infection of Human Airway Epithelial Cells

Kummer, Susann; Avinoam, Ori; Kräusslich, Hans-Georg

To unravel the distribution pattern of IFITM3 in A549 cells at early stages of infection, we made use of super-resolution microscopy. The blurring effect of diffraction limited imaging can obfuscate the potential clustering of IFITM3. Stimulated emission depletion (STED) super-resolution microscopy was therefore applied to resolve IFITM3 clusters in early IAV-infected A549 cells. Cells infected with IAV HK/1/68 were fixed at 1-6 h post infection (h p.i.), stained with anti-IFITM3 and subjected to confocal and STED microscopy (resolution < 50 nm) of the same region ( Figure 3A ). An increased IFITM3 signal intensity was again observed in the early phase of the IAV infection, and individual IFITM3 clusters could be resolved by STED microscopy. We computationally analysed the IFITM3 subcellular localization based on raw STED images using a 2D cluster analysis (objects counter). The clusters were defined as extranuclear IFITM3 signal accumulations with a size of > 10 5 nm 2 . The threshold was chosen by an iterative approach searching for the proportion of clusters, with the critical size being altered upon the infection progression.