Article Figures & Data
Figures
- Fig. 1.
Subcellular localization of GFP-22 expressed by transient transfection. COS-1 cells grown in a two-well coverslip chamber were transfected with either plasmid pEGFPC1 (A) or plasmid pGE155 (B to F), expressing GFP or GFP-22, respectively. The cells were examined live by confocal microscopy at 28 (A and B), 16 (C and D), or 44 (E and F) h after transfection.
- Fig. 2.
The fate of VP22-induced microtubule bundles in transiently transfected cells. COS-1 cells grown on 42-mm-diameter coverslips were transfected with the expression vector for GFP-22 and transferred to a heated chamber 20 h later. A single cell containing bundled microtubules was selected (0 hrs), and images were collected every 10 min for a period of 24 h. Two-hourly images are shown in the gallery, and the corresponding animation can be found elsewhere (http://www.mcri.ac.uk/VirusAssembly/timelapse.html ).
- Fig. 3.
Translocation of GFP-22 from cytoplasm to nucleus during cell division. COS-1 cells grown on 42-mm-diameter coverslips were transfected with the expression vector for GFP-22 and transferred to a heated chamber 24 h later. A single cell containing diffuse cytoplasmic GFP-22, which had the appearance of a cell entering mitosis, was selected (0′), and images were collected every 2 min for a total time of 80 min. Representative images are shown in the gallery, and the corresponding animation can be found elsewhere (http://www.mcri.ac.uk/VirusAssembly/timelapse.html ).
- Fig. 4.
Chromatin-associated GFP-22 is retained in the nucleus after cell division. COS-1 cells were treated as described in the legend to Fig. 3, and a cell in the early stages of mitosis was selected for further analysis (0′). Images of this cell were collected every minute for 150 min, with 10-min intervals presented in the gallery. The corresponding animation can be found elsewhere (http://www.mcri.ac.uk/VirusAssembly/timelapse.html ).
- Fig. 5.
Subcellular localization of GFP-22 in HSV-1 plaques. Vero cells grown in two-coverslip chambers were infected with HSV-1 expressing GFP-22, 166v, at a multiplicity of 0.001. Thirty-six hours later, the cells were examined live by confocal microscopy and fluorescent foci of infection were analyzed. Four representative foci are shown (A to D). Arrows indicate infected cells either in mitosis or containing nuclear GFP-22.
- Fig. 6.
Heterogeneity in GFP-22 subcellular localization at early times in a low-multiplicity HSV-1 infection. Vero cells grown in two-coverslip chambers were infected with HSV-1 expressing GFP-22 at a multiplicity of 0.01. Sixteen hours later, the cells were examined live by confocal microscopy and fluorescent cells were analyzed for variations in GFP-22 localization. GFP-22 was found localized exclusively in the cytoplasm (A), bound to mitotic chromatin (B), and in pairs of nuclei (C) or in single nuclei (D).
- Fig. 7.
Schematic diagram illustrating the four pathways of mitosis identified in cells infected with the GFP-22-expressing virus. Vero cells grown on 42-mm-diameter coverslips were infected with the GFP-22-expressing virus at a multiplicity of 0.01. Sixteen hours later, the cells were transferred to the heated stage, and cells in the early stages of mitosis were identified by their content of GFP-22-decorated condensed chromatin. Time-lapse analysis was carried out on a range of these mitotic cells for periods up to 16 h, and four different outcomes were identified: (1) mitosis proceeds as normal; (2) mitosis proceeds as normal with the exception that cytokinesis fails; (3) condensed chromatin decondenses at metaphase without separation of chromosomes, resulting in a single nucleus; and (4) cells remain in mitosis for prolonged periods and then continue with any of the above three pathways.
- Fig. 8.
Model for the compartmentalization of VP22 through the cell cycle. In both transfected and infected cells, we propose that VP22 localizes primarily to the cytoplasm of interphase-expressing cells. If such a cell enters mitosis, VP22 translocates from the cytoplasm to the nucleus and remains there through chromatin decondensation and nuclear envelope reformation (2). However, in both transfected and infected cells mitosis may be inhibited either by VP22-induced microtubule bundling (1) or by later stages in virus replication (3).
