Origins of Enterovirus Replication Organelles Established by Whole-Cell Electron Microscopy

Figures

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  FIG 1. SBF-SEM imaging of CVB3-infected cells resolves ROs in their whole-cell context. Vero E6 cells were infected with CVB3, fixed at 6 h postinfection, and stained and processed for SBF-SEM imaging. (A) Left, a single slice from the SBF-SEM reconstruction of an infected cell. The cytosol is populated by clusters of ROs (example in boxed area), including single-membrane (white arrowhead), double-membrane (hatched arrowhead), and multilamellar (black arrowhead) ROs. Right, cellular organelles, including mitochondria (red), the ER (yellow), and the nucleus (blue), can be resolved alongside ROs (green). (B) Segmentations of two infected cells (A and B) highlight the positions of individual ROs clusters (multicolored) and the nucleus (blue) within the cell volumes (beige, semitransparent). (C) The number of RO clusters and their respective volumes were quantified in the cells presented in panel B, and the histogram of RO cluster sizes revealed a similar binary distribution in each cell, where the majority of RO volume in each cells falls within a single perinuclear cluster, while the majority of all other RO clusters are 2 orders of magnitude smaller. Cells A and B were each collected…

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  FIG 2. Correlative SBF-SEM and LM highlights the appearance of ROs connected to the ER early in infection. BGM(S1–10) cell transduced with mCherry-GM130, infected with CVB3 3A(S11), and monitored by live-cell imaging until the emergence of the first 3A-GFP puncta. (A) Volume rendering of the final confocal z-stacks prior to fixation and processing for SBF-SEM (mCherry, red; 3A-GFP, green; 4.5 hpi [top]), 3D rendering of the ROs (green) and Golgi cisternae (red) segmented from the corresponding SBF-SEM volume (middle), and a representative SBF-SEM slice (bottom). The intense GM130 signal in confocal data corresponds to recognizable Golgi apparatus cisternae in SBF-SEM data. A good overall correspondence can also be established between 3A-GFP puncta and RO foci (e.g., white arrowheads), despite small discrepancies in their relative position that suggest limited migration of ROs in the delay between LM imaging and fixation (e.g., asterisks). (B) Membrane continuities (hatched arrowheads) between all the RO clusters in the cell (white arrowheads) and ER membranes (black arrowheads) could be observed. (C) Continuities between the ER and ROs could also be found in high-resolution 2D TEM data.…

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  FIG 3. Correlative SBF-SEM and LM exposes RO clusters connected to the Golgi apparatus. BGM(S1–10) cell transduced with mCherry-GM130, infected with CVB3 3A(S11), and monitored by live-cell imaging until the emergence of signal in the Golgi region (∼5 h hpi). (A) Volume rendering of the confocal z-stacks acquired prior to fixation and processing for SBF-SEM (mCherry, red; 3A-GFP, green; 4.5 hpi [left]), 3D rendering of the ROs (green) and Golgi cisternae (red) segmented from the corresponding SBF-SEM volume (middle), and a representative SBF-SEM slice (right). The mCherry-GM130 and 3A-GFP signals correlated well with recognizable Golgi apparatus cisternae and RO clusters, respectively. (B) Assessment of a region (boxed area in panel A) containing Golgi-proximal RO foci reveals a membranous region (hatched arrowhead) bridging ROs (white arrowhead) and the Golgi apparatus (black arrowhead). (C) Overview of a similar region imaged by TEM (left) containing RO clusters (white arrowhead) and the Golgi apparatus (black arrowhead). High-magnification TEM imaging (right; boxed area on the left) reveals membrane continuities (hatched arrowhead) comprising tubules of the trans-Golgi network (black…

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  FIG 4. SBF-SEM and TEM data reveal extensive contact between ROs and LDs. Serial SBF-SEM sections from two regions of interest in the cell shown in Fig. 3 (boxed areas in the segmentation models shown; red, Golgi cisternae, green, ROs; yellow, lipid droplets) are presented, highlighting the association between RO tubules (white arrowheads) and lipid droplets (LD). Frame numbers (top left) indicate the relative depth from frame 0 through the SBF-SEM volume. Scale bars = 500 nm.