Figure 3. Imaging of reconstituted capsid-nuclei system confirms specific capsid binding to the NPCs at the nuclear membrane with and without PEG 8 kDa present.

(A) Representative super-resolution SIM image showing GFP-HSV-1 C-capsids (green) bound to isolated reconstituted rat liver nuclei (blue DAPI stain). A histogram of a capsid cross-section profile for a capsid GFP signal along the white line shows that individual C-capsids are resolved (HSV-1 C-capsid diameter ≈ 125 nm). (B) Confocal fluorescence microscopy images show that binding of GFP-HSV-1 C-capsids (green) to DAPI-stained isolated nuclei (blue), in the presence of cytosol (no ATP-regeneration system was added since it is not required for capsid binding [Ojala et al., 2000]), is not inhibited by the addition of 30% w/w PEG 8 kDa. The addition of wheat germ agglutinin (WGA) prevents most of the capsid binding to nuclei, which demonstrates that capsids bind specifically to NPCs as opposed to binding anywhere on the nuclear membrane [WGA associates with the specific glycoproteins within the NPC and competes with capsid binding (Ojala et al., 2000; Finlay et al., 1987)]. The images at the bottom of row two are a zoom-in of the individual nuclei.

Figure 3—figure supplement 1. Effect of osmotic pressure on morphology and permeability of nuclei.

(A) Purified rat liver nuclei incubated in CBB buffer containing 30% w/w PEG 8 kDa. Nuclei were stained with DAPI (blue) and visualized by confocal fluorescence microscopy (second row). A DIC image of each field was also obtained (first row). Scale bar in all images: 5 μm. Under hyperosmotic conditions (~18 atm), the nuclei slightly shrunk but the sub-nuclear structure of heterochromatin DNA was essentially unchanged. (B) Isolated rat liver nuclei were incubated at 37°C for 40 min in CBB buffer containing a fluorescently tagged 70 kDa dextran (FITC-Dextran, green) and 30% w/w PEG 8 kDa. The nuclei were stained with DAPI (blue). The integrity of the nuclei was not affected by the addition of PEG 8 kDa since fluorescently labeled 70 kDa dextran was excluded from the nuclei interior with nuclei remaining intact and structured. Representative images are shown.