FIGURE 4.

Analysis of markers of early endosomes (EE), endosomal recycling compartment (ERC), and effector proteins that control scission at the EE-ERC-TGN interface at 48 hpi with MCMV. (A) 3D colocalization analysis of endomembrane markers with M55 protein based on M1/M2 coefficients of pixel overlap, determined on Costes-algorythm thresholded z-stacks of confocal images. Markers were visualized using Ab reagents (Supplementary Table S2), and the sites of intracellular accumulation of viral envelope glycoproteins were visualized using mAb reagents to M55. Antibody reagents used are listed in Supplementary Table S1. Data represent mean ± SEM per cell (n = 6–10). #, not available due to the low signal in infected cells. (B) Example of 3D reconstruction of the AC based on expression of Rab5 and M55 protein in 48-h infected MCMV cells. Upper panel presents confocal slices obtained through the focal plane and lower panel 3D reconstruction of the entire z-stack (14 slices) using Image J Volume Viewer plugin. Images shown on the right (* and **) present the view across the section of stack indicated by dashed arrows. (C–H)) Subcellular distribution of representative markers. Complete experiments are shown in the Supplementary Material (Supplementary Figures S11–S15). Examples of the subcellular distribution of markers of EEs (C), ERC (D), ERC-associated Arf6/Rab35 axis (E), ERC-associated effectors proteins (F), ARF system (G), and EE/ERC-associated scission machinery (H). Shown are images through the focal plane and colocalization analysis by plotting fluorescence intensity profiles along white dashed lines. Cell borders are indicated by fine dashed lines and nuclei by fine dotted lines. Bars, 10 μm. 2-column fitting image.