Figure 1.
Visualization of lipid droplets in dSTORM.
(A) Huh7 Lunet cells were incubated with LipidTox Red neutral lipid stain and analyzed by dSTORM. Time-dependent switch between “ON” and “OFF” states of LipidTox Red, shown is the signal of a dSTORM time series. (B) Huh7 Lunet cells were incubated with LipidTox Red neutral lipid stain and analyzed by widefield microscopy and dSTORM. Single channels are shown in black and white; the merged image is pseudocolored with widefield in white and dSTORM in blue (scale bar 5 µm, in magnified images 0.5 µm). (C) dSTORM of Huh7 Lunet cells after immunostaining with antibodies directed against endogenous ADRP/perilipin 2 and staining of lipid droplets with LipidTox red. Single channels are shown in black and white; the merged image is pseudocolored with ADRP in red and LipidTox in blue (scale bar 5 µm, in magnified images 0.5 µm).
Figure 2.
Characterization of Jc1Flag-E2 and JFH1Flag-E2 infectious HCVcc constructs.
(A) Scheme of the JFH1Flag-E2 and Jc1Flag-E2 constructs used in this study. (B) Western blot analysis of Huh7.5 cells transfected with in vitro transcribed JFH1Flag-E2 and Jc1Flag-E2 RNA with anti-Flag, anti-E2, anti-core, anti-actin antibodies. (C) TCID50 of Huh7.5 cells transfected with in vitro transcribed JFH1Flag-E2 and Jc1Flag-E2 RNA. (D) Confocal microscopy of Huh7 Lunet cells infected with JFH1Flag-E2 and Jc1Flag-E2 viral stocks after immunostaining with antibodies recognizing E2 and Flag. Single channels are shown in black and white; the merged image is pseudocolored with ADRP in red and LipidTox in blue (scale bars 5 µm).
Figure 3.
2D and 3D dSTORM images of core, Flag-E2, and lipid droplets in JFH1Flag-E2- and Jc1Flag-E2-infected cells.
Huh7 Lunet cells were infected with JFH1Flag-E2 and Jc1Flag-E2 viral stocks and processed for immunofluorescence staining. Cells were permeabilized with Triton X-100 followed by staining with anti-core and anti-Flag antibodies and LipidTox Red. 3D super-resolution datasets were acquired using astigmatism imaging. dSTORM images were reconstructed from a time series of 10,000–15,000 raw images per channel (A) Shown are the single channel 2D projections and the merge and 3D images with the axial position color-coded according to the scale on the right. Scale bars of the x–y image represent 5 µm. (B) dSTORM datasets were analyzed for the degree of colocalization using the JACoP (Just Another Colocalization Plugin) plugin for Image J [42]. We analyzed cytosolic regions of interest in 2D of 4 independent experiments and calculated the degree of colocalization using the Manders colocalization coefficient (mean ± sem, *p<0.05, unpaired two-tailed student's t-test).
Figure 4.
Analysis of the 3D distribution and colocalization of the viral proteins at lipid droplets.
dSTORM datasets were analyzed for the distribution and colocalization of core and Flag-E2. Cytosolic regions of interest were reconstructed in 3D and lipid droplets with core and Flag-E2 staining in close proximity were analyzed as follows: lipid droplet localization was scored A) core and Flag-E2 adjacent to lipid droplets, B) Flag-E2 adjacent to core, core adjacent to lipid droplets, and C) core adjacent to Flag-E2, Flag-E2 adjacent to lipid droplets. The degree of colocalization between the viral proteins was scored as I) complete, II) partial, or III) no colocalization. We analyzed reconstructed images from three independent experiments of Triton X-100–permeabilized cells and one experiment of saponin-permeabilized cells with a total amount of 40 lipid droplets in Jc1Flag-E2-infected cells and 36 lipid droplets in JFH1Flag-E2-infected cells. Colocalization and localization relative to lipid droplets was significantly different between cells infected with the two viral strains (*p<0.05, **p<0.01, chi-square test).
Figure 5.
3D reconstitution of core, Flag-E2, and lipid droplets in Jc1Flag-E2-infected cells.
Representative 3D reconstructions of core and Flag-E2 spatial localization and colocalization around lipid droplets. Shown are three examples of Jc1Flag-E2 -infected cells, first the image stacks used for the 3D reconstruction and the 3D reconstruction of all three channels, then core or Flag-E2 and the lipid droplet volume and finally volume of colocalization between core and Flag-E2 and the lipid droplet volume. Scale bars on the image stacks and ruler hatch marks in the 3D images are 100 nm.
Figure 6.
3D reconstitution of core, Flag-E2, and lipid droplets in JFH1Flag-E2-infected cells.
Representative 3D reconstructions of core and Flag-E2 spatial localization and colocalization around lipid droplets. Shown are three examples of JFH1Flag-E2-infected cells, first the image stacks used for the 3D reconstruction and the 3D reconstruction of all three channels, then core or Flag-E2 and the lipid droplet volume and finally volume of colocalization between core and Flag-E2 and the lipid droplet volume. Scale bars on the image stacks and ruler hatch marks in the 3D images are 100 nm.