Figure 5.
Pol I reactivation is crucial for nucleolar reconstruction during mitosis. (A) Snapshots of live-cell imaging of 5.8S rRNA and nucleolar markers during mitosis. The time shown on the top of each panel indicates the time for the cell progressing through mitosis, and “0 min” indicates the time of nuclear membrane rupture. Arrows point to visible MS2-tagged rRNAs. (B) Schematic of the nucleolar architecture and the markers used to label nucleolar subcompartments in A. (C) The mean fluorescence intensities of nucleolar components RPA43, FBL, NPM1, and 5.8S rRNA were averaged, normalized, and plotted against time, where “0 min” is the time of nuclear membrane rupture (n = 15 cells). (D) The mean fluorescence intensities of nucleolar components RPA43, FBL, NPM1, and 5.8S rRNA within the reforming nuclei were averaged, normalized, and plotted against time, where time equals “0 min” when the chromosomes start to migrate toward respective poles (n = 15 cells). (E) Snapshots of live-cell imaging of MS2-tagged 5.8S rRNA labeled by stdMCP-tdTomato and BFP-NPM1 during mitosis in the absence or presence of ActD. The first image shown in each row is the first image taken intermediately after adding DMSO or ActD into the medium. “0 min” is the time of nuclear membrane rupture. See Videos 3 and 4 for dynamics. (F) Histograms illustrating the number of BFP-NPM1 condensates formed in the daughter cells upon mitosis exit in the absence or presence of ActD (n = 40). (G) Histograms demonstrating the size of the BFP-NPM1 condensates formed in the daughter cells upon mitosis exit in the absence or presence of ActD (n ≥ 98). All images in this figure are maximum-intensity projections from z stacks. The white circle highlights the area where the stdMCP-tdTomato is located, which represents the outline of the nucleus (before nuclear membrane rupture) or the cell (after nuclear membrane rupture). Scale bars, 5 µm.