Escape from mitotic catastrophe by actin-dependent nuclear displacement in fission yeast

Abstract

Eukaryotic cells position the nucleus within the proper intracellular space, thereby safeguarding a variety of cellular processes. In fission yeast, the interphase nucleus is placed in the cell middle in a microtubule-dependent manner. By contrast, how the mitotic nucleus is positioned remains elusive. Here we show that several cell-cycle mutants that arrest in mitosis all displace the nucleus toward one end of the cell. Intriguingly, the actin cytoskeleton is responsible for nuclear movement. Time-lapse live imaging indicates that mitosis-specific F-actin cables possibly push the nucleus through direct interaction with the nuclear envelope, and subsequently actomyosin ring constriction further shifts the nucleus away from the center. This nuclear movement is beneficial, because if the nuclei were retained in the center, unseparated chromosomes would be intersected by the contractile actin ring and the septum, imposing the lethal cut phenotype. Thus, fission yeast escapes from mitotic catastrophe by means of actin-dependent nuclear movement.

Keywords: Biological Sciences; Cell Biology; Chromosome Organization; Genetics; Molecular Biology; Molecular Genetics.

Graphical abstract

Figure 1

Mitotic arrest leads to nuclear displacement (A) Nuclear displacement in several mitotic mutant cells. Exponentially growing wild-type or indicated mitotic mutants grown at 27°C were shifted to 36°C and incubated for 3 h except for nda3-1828, which was incubated for 6 h. Cells were fixed with methanol and stained with DAPI (chromosomes) and Calcofluor (septa). Representative cells for wild-type (top row), mutants displaying displaced chromosomes (middle row), or cut (bottom row) are shown. Scale bar, 5 μm. (B) The percentage of cells showing displaced chromosomes or cut. The sample numbers (n) for individual strains are indicated on the bottom of columns. Data are presented as the means ± SD. See also Figure S1.

Figure 2

The nucleus becomes off-center as the medial actomyosin ring assembles (A) A schematic depiction of the nucleus and cytoskeletons during the mitotic cell cycle in fission yeast. (B) Time-lapse and kymograph images. Wild-type (top row) or cut7-22 cells (middle and bottom rows) grown at 27°C were shifted to 36°C and incubated for 2 h, when time-lapse imaging started. Cells contain mCherry-Atb2 (magenta, MTs), Cut11-mRFP (magenta, the NE), and LifeAct-GFP (green, actin). The first time points when LifeAct-GFP signals were observed in the middle of cells are indicated with white arrows, whereas those when the CAR initiated constriction are marked with yellow arrows. Corresponding kymographs are shown on the right (one-minute interval images were merged), in which the middle of the cell axis is shown with dotted lines and arrowheads. Scale bars, 10 min (horizontal) and 5 μm (vertical). See also Videos S1, S2, and S3. (C) Timing of nuclear displacement in relation to that of CAR assembly and constriction. cut7-22 cells containing mCherry-Atb2 (magenta, MTs), Cut11-mRFP (magenta, the NE), and Rlc1-GFP (green, the CAR) were grown at 27°C and shifted to 36°C for 2 h, when time-lapse imaging started (left, see Video S4). The duration of CAR assembly (CA) and constriction (CC) are marked with horizontal arrow bars. Scale bar, 5 μm. On the right graph, the position of the nucleus (the distance between the center of the cell axis and that of the nucleus) is plotted against initiation of CAR assembly (CA0), 5 min after CAR assembly (CA5), and initiation of CAR constriction (CC). Data are presented as the means ± SD. All p values were obtained from the two-tailed unpaired Student's t test. ∗∗∗∗p < 0.0001. The numbers on the bottom right corner of each image show times in minutes (B and C). See also Figure S2.

Figure 3

Polymerized actin structures other than endocytic patches drive nuclear movement (A) Time-lapse and kymograph images of the nuclear position in cut7-22 cells incubated at 36°C in the presence of LatA. cut7-22 mutant cells grown at 27°C were shifted to 36°C and incubated for 2 h (time 0). At this point, LatA (50 μM) was added and time-lapse imaging started. Cells contain mCherry-Atb2 (magenta, MTs), Cut11-mRFP (magenta, the NE), and LifeAct-GFP (green, actin). Note that actin signals already disappeared 4 min after LatA treatment. The position of the cell center is shown with dotted lines and arrowheads, and the numbers on the bottom right indicate times in minutes upon recording. Scale bars, 10 min (horizontal) and 5 μm (vertical). See also Video S5. (B) Profiles of the relative position of the nucleus in cut7-22 mutants in the absence (n = 18) or presence (n = 12) of LatA. The distance (x μm) between the center of the cell axis and that of the nucleus was measured at each time point and plotted against time. (C) The degree of nuclear movement in cut7-22 cells in the absence or presence of LatA. The percentage of cells that do or do not show nuclear displacement is shown. For each sample, the maximal distance (x μm) between the center of the cell axis and that of the nucleus was determined using the data shown in (B) and categorized into three classes: displaced (shown in magenta, in which x is ≥ 1 μm), mildly displaced (shown in pink, in which x is between 0.5 μm and 1 μm), and centered (shown in gray, in which x is ≤ 0.5 μm). See also Figure S3. (D) Lack of actin patches in cdc7-22 cells treated with CK-666. cut7-22 cells containing mCherry-Atb2 (MTs, magenta), Cut11-mRFP (the NE, magenta), and LifeAct-GFP (actin, green) were grown at 27°C and shifted to 36°C for 2 h. CK-666 (100 μM) was added, and images were taken 20 min after CK-666 addition. Scale bar, 5 μm. (E) The nuclear position in cut7-22 cells treated with CK-666. cut7-22 cells containing mCherry-Atb2 (MTs), Cut11-mRFP (the NE), and LifeAct-GFP (actin) were grown at 27°C and shifted to 36°C for 2 h. CK-666 (100 or 200 μM) or DMSO was added. Samples were observed 20 min after CK-666 addition. The distance (x μm) between the center of the cell axis and that of the nucleus was measured for each cell and plotted in the graph as dots: gray, x < 0.5 μm; pink, 0.5μm < x < 1.0 μm; magenta, x > 1.0 μm. Mitotic cells before CAR constriction were counted. Data are given as mean ± SD. p values were obtained from the two-tailed unpaired Student's t test. ∗∗∗∗p < 0.0001. (F) Patterns of nuclear positioning. Data shown in (E) are presented as columns. The sample numbers are shown on the bottom of each column. p values were obtained from the two-tailed χ² test. ∗∗∗∗p < 0.0001.

Figure 4

Cdc12/Formin, Myo2, and Myo51 are responsible for nuclear displacement (A) Lack of actin cables and the CAR in cut7-22cdc12-112 cells. cut7-22 or cut7-22cdc12-112 cells containing mCherry-Atb2 (MTs, magenta), Cut11-mRFP (the NE, magenta), and LifeAct-GFP (actin, green) were grown at 27°C and shifted to 36°C for 3 h. Actin cables (orange arrows) and the CAR (green arrowheads) are pointed. (B) Time-lapse images of the nuclear position. Indicated mutant cells grown at 27°C were incubated at 36°C for 2 h, when live imaging started. Individual mutants contain mCherry-Atb2 (magenta, MTs) and Cut11-GFP (green, NE). The numbers on the bottom right indicate times in minutes upon recording. The position of the cell center is shown with dotted lines and arrowheads. Scale bars, 5 μm (A and B). See also Videos S6, S7, and S8. (C) Profiles of the relative position of the nucleus in individual mutants. If x (the distance between the center of the cell axis and that of the nucleus) was <1 μm or a cell showed cut, it was classified as centered/cut. If x was >1 μm, it was classified as displaced. The sample numbers are shown on the bottom of each column. All p values were obtained from the two-tailed χ² test. ∗∗∗∗p < 0.0001. See also Figure S4.

Figure 5

Mitotic actin cables, but not actomyosin ring constriction, drive nuclear displacement (A) Selective disappearance of actin cables by treatment with a low concentration of LatA. cut7-22 cells containing mCherry-Atb2 (MTs, magenta), Cut11-mRFP (the NE, magenta), and LifeAct-GFP (actin, green) were grown at 27°C and shifted to 36°C for 2 h when LatA (0.15 μM) or DMSO was added. Still images were taken 10 min after LatA or DMSO addition. Actin cables (orange arrows) and the CAR (green arrowheads) are pointed. (B) Time-lapse images of the nuclear position in cut7-22 cells treated with a low concentration of LatA. cut7-22 cells containing mCherry-Atb2 (MTs, magenta), Cut11-mRFP (the NE, magenta), and Rlc1-GFP (the CAR, green) incubated as in (A) were imaged. Representative cells that successfully underwent CAR constriction (top) or failed to assemble/constrict the CAR (bottom) are shown. The numbers on the bottom right indicate times in minutes upon recording. Scale bars, 5 μm (A and B). (C) Suppression of nuclear displacement under a low concentration of LatA. If x (the distance between the center of the cell axis and that of the nucleus) was <1 μm or a cell showed cut, it was classified as centered/cut. If x was >1 μm, it was classified as displaced. The sample numbers are shown on the bottom of each column. (D) Visualization of interaction between F-actin cables and the nuclear envelope in cut7-22. A cut7-22 strain used in (A) was grown at 27°C and incubated at 36°C for 2 h, when time-lapse imaging started (see Video S9). The bottom row shows enlarged images in squares shown on the top row. The numbers on the bottom right indicate times in minutes upon recording. Arrows point the tips of F-actin cables (green) that interact with the NE (magenta). The position of the cell center is shown with an arrowhead. Scale bars, 5 μm (top) and 1 μm (bottom).

Figure 6

Inhibition of actomyosin ring constriction substantially but not completely abolishes nuclear displacement (A) Time-lapse and kymograph images of the nuclear position in cut7-22cdc7-24 cells incubated at 36°C in the absence or presence of LatA (see Videos S10 and S11). Mutant cells were grown at 27°C, shifted to 36°C, and incubated for 2 h, when imaging started. Two representative cells, in which the nucleus remains centered (top row) or is displaced (middle row), are shown. LatA (50 μM) was added at time 0. Cells contain mCherry-Atb2 (magenta, MTs), Cut11-mRFP (magenta, the NE), and LifeAct-GFP (green, actin). Corresponding kymograph images are shown on the right, in which the middle of the cell axis is shown with dotted lines and arrowheads. The numbers on the bottom right indicate times in minutes upon recording. Scale bars, 10 min (horizontal) and 5 μm (vertical). (B) Profiles of the relative position of the nucleus in cut7-22cdc7-24 mutants in the absence (n = 8) or presence (n = 7) of LatA. The distance (x) between the center of the cell axis and that of the nucleus was measured at each time point and plotted against time. (C) Classification of the patterns of nuclear movement in cut7-22cdc7-24 cells in the absence or presence of LatA. The percentage of cells that do or do not show nuclear displacement is shown. For each sample, the maximal distance (x μm) between the center of the cell axis and that of the nucleus was determined using the data shown in (B) and categorized into three classes: displaced (shown in magenta, in which x is ≥ 1 μm), mildly displaced (shown in pink, in which x is between 0.5 μm and 1 μm), and centered (shown in gray, in which x is ≤ 0.5 μm). The data for cut7-22 in (B) and (C) are the same as those in Figures 3B and 3C, respectively.

Figure 7

cut7 survivors become diploidized (A) Viability of wild-type and cut7-22 cells incubated at the restrictive temperature in the absence or presence of LatA. Wild-type and cut7-22 cells grown at 27°C were shifted to 36°C. After 2-h incubation at 36°C, DMSO or LatA (50 μM) was added, and cultures were incubated for additional 1 h. The cell number was measured, and after appropriate dilutions, cells were spread on rich YE5S plates containing Phloxine B. Plates were then incubated at 27°C to assess colony-forming ability (viability: the number of colonies formed divided by that of cells spread, and the value of wild-type cells were set as 100%). Data are presented as the means ± SEM (≥100 colonies). (B) Representative pictures of plates that contain survivor colonies (wild-type, left; cut7-22 cells, middle and right) are shown. Dark pink colonies (diploids) are pointed with arrowheads. Cell morphologies derived from light and dark pink colonies are shown on the bottom. Scale bar, 10 μm. (C) The percentage of dark pink colonies (diploid). At least 30 colonies were counted in three independent experiments. Data are presented as the means ± SEM. (D) A model depicting controlling mechanisms of mitotic nuclear positioning. During mitosis, actin cables nucleate in the cytoplasm and are recruited toward the cell center to be incorporated into the CAR. This process may generate a pushing force (white arrows) toward the nucleus. When mitosis is blocked, forces from either side of the nucleus become imbalanced, leading to nuclear displacement. Upon prolonged delay, cells are destined for two fates. In one type (i), the nucleus is further displaced from the cell center imposed by CAR constriction. Cells with displaced nuclei give rise to diploid and anucleate progenies. In another type (ii), the nucleus stays in the middle, leading to the lethal cut phenotype.