Nuclear size control in fission yeast

Abstract

A long-standing biological question is how a eukaryotic cell controls the size of its nucleus. We report here that in fission yeast, nuclear size is proportional to cell size over a 35-fold range, and use mutants to show that a 16-fold change in nuclear DNA content does not influence the relative size of the nucleus. Multi-nucleated cells with unevenly distributed nuclei reveal that nuclei surrounded by a greater volume of cytoplasm grow more rapidly. During interphase of the cell cycle nuclear growth is proportional to cell growth, and during mitosis there is a rapid expansion of the nuclear envelope. When the nuclear/cell (N/C) volume ratio is increased by centrifugation or genetic manipulation, nuclear growth is arrested while the cell continues to grow; in contrast, low N/C ratios are rapidly corrected by nuclear growth. We propose that there is a general cellular control linking nuclear growth to cell size.

Figure 1.

Nuclear and cell size correlate over a large range in cell size and shape. Quantitation of nuclear and cell size in cell size and cell shape mutants. (A) Overlay image of cell (differential interference contrast; DIC) and nuclear envelope (cut11-GFP). (B) Box-and-Whisker plots showing the N/C ratio. (C–E) Scatter plots of cell size and nuclear size. Wild type (WT): haploid (25°C, n = 382, r = 0.72), spores (25°C, n = 329. r = 0.63), nitrogen-starved cells (12 h EMM-N, 25°C, n = 275, r = 0.64), diploid (25°C, n = 308, r = 0.79). Cell size mutants: wee1-50, temperature-sensitive (ts) allele of the mitotic inhibitor wee1 resulting in a short G2 phase and mitosis at a smaller cell size (6 h 36°C, n = 179, r = 0.70), cdc10-V50, ts allele of a transcription factor controlling G1-S specific gene expression leading to a cell cycle arrest in G1 phase (3 h 36.5°C, n = 322, r = 0.82), cdc25-22, ts allele of the cdc25 phosphatase, an inducer of mitosis which arrests cells in G2 phase of the cell cycle (3 h 36.5°C, n = 188, r = 0.81), pnmt1-cdc13, a repressible allele of the b-type cyclin which leads to consecutive rounds of over-replication or arrest in G2 phase of the cell cycle (10 h release, n = 98, r = 0.62), orb2-34, a ts allele of PAK-related polarity kinase orb2 (5h 36.5°C, n = 122, r = 0.66), orb6-25, a ts allele of a kinase which coordinates cell morphogenesis with the cell cycle (5.5 h 36.5°C, n = 129, r = 0.69).

Figure 2.

DNA content does not directly determine nuclear size. Quantitation of nuclear and cell size in over-replicating cells. pnmt1-driven switch-off of the cyclin Cdc13 induces over-replication in a subset of cells (marked in red). Cells arrested with 2C DNA content are marked in blue. (A) For quantitative DAPI staining (right panel), cells were formaldehyde fixed 10 h after cdc13 switch-off. Left panel: overlay of DIC and Cut11-GFP. Bar, 10 μm. (B) FACS profile for DNA content shows the kinetics of cdc13 switch-off dependent over-replication (log scale FL2-H). Cell and nuclear size are represented in a scatter plot, N/C ratio in a Box-and-Whisker plot.

Figure 3.

N/C ratio is constant throughout cell cycle. Cell cycle analysis of nuclear and cell size using time-lapse microscopy (6 z-sections/min). (A) Time points of a selected field from Video 1. (B and C) The graphs represent the median of a 10 min moving averages from 5 independent cells, analyzed for an entire cell cycle as shown by the cartoons. Average cell volume (gray) and nuclear volume (black) cells and the respective N/C ratios (gray: nuclear surface area/cell volume, black: nuclear volume/cell volume). Video 1 is available at http://www.jcb.org/cgi/content/full/jcb.200708054/DC1.

Figure 4.

The proportional cytoplasmic volume directly influences nuclear size. (A) Multiple unevenly distributed nuclei within a cell are created using a cdc11-119 arrest for 4.5 to 6.5 h. During this interval, nuclear size of the outer nuclei (red) increases more rapidly than the size of the inner nuclei (blue). Proportional cytoplasmic volumes (defined as cytoplasmic volumes separated by the midpoints of two adjacent nuclear centers or the cell end) of the multinucleated cell are overlaid in color (bottom panel). (B) Scatter plot of nuclear size and proportional cytoplasmic volumes were measured at the end of interphase (blue: inner nuclei, red: outer nuclei). (C) cdc11-119 klp2Δ cells were grown at 36.5°C for 4.5 h. Then nuclei were displaced by centrifugation causing uneven distribution. Images taken 2 h after nuclear displacement show that nuclei which are surrounded by a larger proportional cytoplasmic volume are bigger. (D) An additional growth zone as formed in the cdc11-119 tea1Δ klp2Δ mutant increases nuclear size in its proximity. Left, overlay of DIC and cut11-GFP signal. Nuclei are numbered. (E) Average nuclear volume from four cells is plotted for every numbered nucleus. Error bars, SD. Bar, 5 μm. (F) cdc11-119 cells were arrested at 36.5°C for 9 h (4 cell cycles). Arrowheads on fluorescence image (inverted lookup table) point to representative large and small nuclei within the same cell. Bar, 10 μm.

Figure 5.

The cell can sense and correct changed N/C ratios. (A) N/C ratios are manipulated by block and release of cytokinesis using the cdc11-119 klp2Δ mutant. Before septa form, nuclei are displaced by centrifugation resulting in cells with reduced and elevated N/C ratios. (B) Time points from Video 2. A Gaussian blur (r = 2 pixels) was applied to the fluorescence channel. Kinetics of N/C ratio correction were monitored by 3D time-lapse microscopy (2-min intervals). Individual cells with a decreased N/C ratio are marked with colored lines; an asterisk marks the approximate frame when nuclear growth starts. (C and D) Quantitation of cell size and nuclear size and corresponding N/C ratio for a high N/C ratio. Time is indicated starting at cytokinesis. The graphs represent the median of 10-min moving averages from three independent cells. (E) Low N/C ratios get corrected more rapidly than high ratios. Video 2 is available at http://www.jcb.org/cgi/content/full/jcb.200708054/DC1.