Figure 6.
Nuclear volume scales with cell volume in A. pullulans. (A) Schematic showing the integration strategy to replace ura3::HYGR with URA3:3xmCherry:NLS-GFP. (B) Representative maximum intensity projection of a cell expressing 3xmCherry (magenta, cytosol) and NLS-GFP (nucleus, cyan), with 3D segmentation of cell and nuclei from the same image. Scale bar, 5 μm. (C) Relationship between cell volume and nuclear volume. Each point is a measurement from a single cell. The data are fit with a linear model and the line of best fit with 95% confidence intervals is shown along with the R2 value, number of cells, and slope. (D) Volumes of cells with the indicated numbers of nuclei. Each point is an individual cell (n = 566, 232, 23, 53, 18, 12, 15, 32, 16, 8, 7, 5, 2, 3, 2, 3, and 3, cells for cells with 1–17 nuclei). (E) Relationship between cell volume and the average volume of an individual nucleus for cells with the indicated number of nuclei (n = 566, 232, 48, and 25 cells for cells with 1, 2, 4, or 8 nuclei). The slopes of each line are 5.08 ± 0.28, 15.1 ± 1.2, 14.8 ± 4.0, and 30.2 ± 8.1 (slope ±standard error), with R2 values of 0.38, 0.40, 0.23, and 0.39 for fits to data from cells with 1, 2, 4, and 8 nuclei, respectively. (F) Relationship between the average volumes of individual nuclei and the number of nuclei for cells that are close in size (625–660 µm3). Error bars indicate standard deviation. (G) Maximum intensity projected confocal images of the cells quantified in F. The cell volume measured is outlined with a yellow dashed line and volumes are indicated in each image. The nuclei are numbered. Scale bar, 5 μm. (H) Example maximum intensity projected confocal image of a cell expressing NLS-3xGFP (cyan) and spH2B-mCherry (magenta) with three daughter cells, two with one nucleus and one with two nuclei (yellow arrowhead). Top images show each channel individually and the bottom image shows the merged image. The NLS-GFP signal in the nuclei of the two-nucleus daughter cell is dimmer than in the one-nucleus daughter cells. Scale bar, 5 µm. (I) Volumes of nuclei in sister cells from the same mother. Nuclear volumes in sister cells were normalized to the average nuclear volume in the one-nucleus sister cells (n = 130 one-nucleus sisters and 62 two-nucleus sisters). (J) Total H2B-mCherry signal per nucleus in sister cells from the same mother. Nuclear H2B-mCherry signals in sister cells were normalized to the average signal in the one-nucleus sister cells (n = 87 one-nucleus sisters and 38 two-nucleus sisters). (K) NLS-GFP signal per nucleus in the same sister cells shown in J. Nuclear NLS-GFP signals in sister cells were normalized to the average signal in the one-nucleus sister cells (n = 87 one-nucleus sisters and 38 two-nucleus sisters). In all graphs, the mean and standard deviation are shown. Statistical significance is calculated by two-tailed Student’s t test, and the P value is shown (****, P ≤ 0.0001).

Nuclear volume scales with cell volume in A. pullulans. (A) Schematic showing the integration strategy to replace ura3::HYGR with URA3:3xmCherry:NLS-GFP. (B) Representative maximum intensity projection of a cell expressing 3xmCherry (magenta, cytosol) and NLS-GFP (nucleus, cyan), with 3D segmentation of cell and nuclei from the same image. Scale bar, 5 μm. (C) Relationship between cell volume and nuclear volume. Each point is a measurement from a single cell. The data are fit with a linear model and the line of best fit with 95% confidence intervals is shown along with the R2 value, number of cells, and slope. (D) Volumes of cells with the indicated numbers of nuclei. Each point is an individual cell (n = 566, 232, 23, 53, 18, 12, 15, 32, 16, 8, 7, 5, 2, 3, 2, 3, and 3, cells for cells with 1–17 nuclei). (E) Relationship between cell volume and the average volume of an individual nucleus for cells with the indicated number of nuclei (n = 566, 232, 48, and 25 cells for cells with 1, 2, 4, or 8 nuclei). The slopes of each line are 5.08 ± 0.28, 15.1 ± 1.2, 14.8 ± 4.0, and 30.2 ± 8.1 (slope ±standard error), with R2 values of 0.38, 0.40, 0.23, and 0.39 for fits to data from cells with 1, 2, 4, and 8 nuclei, respectively. (F) Relationship between the average volumes of individual nuclei and the number of nuclei for cells that are close in size (625–660 µm3). Error bars indicate standard deviation. (G) Maximum intensity projected confocal images of the cells quantified in F. The cell volume measured is outlined with a yellow dashed line and volumes are indicated in each image. The nuclei are numbered. Scale bar, 5 μm. (H) Example maximum intensity projected confocal image of a cell expressing NLS-3xGFP (cyan) and spH2B-mCherry (magenta) with three daughter cells, two with one nucleus and one with two nuclei (yellow arrowhead). Top images show each channel individually and the bottom image shows the merged image. The NLS-GFP signal in the nuclei of the two-nucleus daughter cell is dimmer than in the one-nucleus daughter cells. Scale bar, 5 µm. (I) Volumes of nuclei in sister cells from the same mother. Nuclear volumes in sister cells were normalized to the average nuclear volume in the one-nucleus sister cells (n = 130 one-nucleus sisters and 62 two-nucleus sisters). (J) Total H2B-mCherry signal per nucleus in sister cells from the same mother. Nuclear H2B-mCherry signals in sister cells were normalized to the average signal in the one-nucleus sister cells (n = 87 one-nucleus sisters and 38 two-nucleus sisters). (K) NLS-GFP signal per nucleus in the same sister cells shown in J. Nuclear NLS-GFP signals in sister cells were normalized to the average signal in the one-nucleus sister cells (n = 87 one-nucleus sisters and 38 two-nucleus sisters). In all graphs, the mean and standard deviation are shown. Statistical significance is calculated by two-tailed Student’s t test, and the P value is shown (****, P ≤ 0.0001).

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