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. 2021 Jan 7;296:100236. doi: 10.1074/jbc.RA120.016571

Figure 3.

Figure 3

Remodeling of membrane compartments involved in glycoprotein trafficking and HSV-1 assembly imaged with SIM in fixed cells.A, HFF cells were infected with eYFP-ICP0/gC-mCherry HSV-1 and fixed at 3, 6, and 9 hpi. The Golgi apparatus was stained with an anti-58K Golgi protein antibody following standard immunofluorescence protocol and imaged using a custom-built SIM microscope. The 58K Golgi protein marker was used to measure the size of the Golgi compartment over the time course of the replication cycle. The area occupied by the Golgi apparatus was calculated for at least ten cells for each stage (stage 1: n = 11, stage 2: n = 14, stage 3: n = 11). The area continuously decreases from stage 1 to stage 3. During stage 4, the Golgi apparatus was fragmented in all imaged cells (n = 12) and no area was calculated. B, object-based colocalization of gC and TGN compartments from SIM data was analyzed by calculating their overlap. Overlap during stage 3 is very high but decreases during stage 4 since gC is trafficked to other cellular membrane compartments such as the plasma membrane. (Stage 3: n= 22 cells, stage 4: n = 26 cells). C, accumulation of early endosomes at the juxtanuclear region and spread correlated to fragmentation of juxtanuclear compartment. At stage 1, early endosomes are widely spread in the cytoplasm but start to accumulate at the juxtanuclear region during stage 2. At stage 3, early endosomes are further concentrated at the gC-enriched juxtanuclear region, and Rab4a is found within the assembly compartment. Fragmentation of the assembly compartment leads to respread of early endosomes in the cytoplasm during stage 4. Red arrows in the enlarged view highlight early endosomes clustered around the gC-enriched membrane compartments. The bottom panel illustrates Rab4 density in the cytoplasm, which is homogeneous at stage 1 but increases at the juxtanuclear region and gC-rich compartments during later stages. D, schematic illustration of early endosomes, gC, Golgi, and TGN compartments during HSV-1 replication. Scale bars 10 μm.