The budding yeast nuclear envelope adjacent to the nucleolus serves as a membrane sink during mitotic delay

Abstract

The mechanisms that dictate nuclear shape are largely unknown. Here we screened the budding yeast deletion collection for mutants with abnormal nuclear shape. A common phenotype was the appearance of a nuclear extension, particularly in mutants in DNA repair and chromosome segregation genes. Our data suggest that these mutations led to the abnormal nuclear morphology indirectly, by causing a checkpoint-induced cell-cycle delay. Indeed, delaying cells in mitosis by other means also led to the appearance of nuclear extensions, whereas inactivating the DNA damage checkpoint pathway in a DNA repair mutant reduced the fraction of cells with nuclear extensions. Formation of a nuclear extension was specific to a mitotic delay, because cells arrested in S or G2 had round nuclei. Moreover, the nuclear extension always coincided with the nucleolus, while the morphology of the DNA mass remained largely unchanged. Finally, we found that phospholipid synthesis continued unperturbed when cells delayed in mitosis, and inhibiting phospholipid synthesis abolished the formation of nuclear extensions. Our data suggest a mechanism that promotes nuclear envelope expansion during mitosis. When mitotic progression is delayed, cells sequester the added membrane to the nuclear envelope associated with the nucleolus, possibly to avoid disruption of intranuclear organization.

Figure 1. Mitotic delay leads to the formation of a nuclear extension

(A) Images of strains from the automated microscopy screen. Cells are expressing a nuclear marker, Pus1p-GFP (green), and a cytoplasmic marker, TdTomato (red). Left: a field of wild type cells. The arrow points to a typical large budded cell at early stages of mitosis, with an elongated nucleus. Right: typical examples of cells of the indicated genetic backgrounds with nuclei exhibiting nuclear extensions. Scale bar =10μm. Additional images are in Supplemental Figure S1. (B and C) Wild type cells (KW913) expressing Pus1p-GFP (pane B) or Nup49p-GFP (pane C) were either untreated or treated with nocodazole (NZ) or zeocin for 3 hours. The nucleus in nocodazole treated cells does not traverse the bud neck due to the absence of microtubules. Scale bar =3μm. (D) Left panel: Wild type cells (KW926) were treated with hydroxyurea (HU) to induce an S phase arrest. Right panel: Cells over expressing SWE1 (MWY1152) and expressing Pus1p-GFP were arrested in G2 arrest by adding galactose. In both cases the arrest was achieved after three hours. Typical examples are shown. Scale bar =3μm. Quantification of the nuclear morphology of these strains and the control cells is presented in Table 1.

Figure 2. The mitotic nuclear extension coincides with the nucleolus

(A) The nucleolus in wild type cells. Wild type cells (KW926) were fixed, stained with DAPI and imaged for Pus1p-GFP (green in the overlays), Nsr1p-mCherry (Nsr1p-CR, red in the overlays) and DAPI (blue in the overlays). The green/red overlap causes Nsr1p-CR to appear orange. Scale bar =3μm. (B) Nuclear morphology in rad51Δ cells (opt two rows) and wild type cells treated with nocodazole (NZ, bottom two rows). rad51Δ and nocodazole treated wild type cells (KW926) were fixed and imaged as described in (A). Shown are typical examples of cells with nuclear extensions. Note that the DNA in these cells is similar to the controls (panel A), while the nuclear extension (i.e. the part of the nucleus containing Pus1p-GFP but no DNA) coincides with the nucleolar marker Nsr1p-CR. Scale bar =3μm. (C) Wild type cells (KW926), either without (control) or with nocodazole treatment were imaged at the indicated time points. Pus1p-GFP is in green and Nsr1p-CR is in red (appears orange due to the overlay). For nocodazole treated cells, panels with only Pus1p-GFP or Nsr1p-CR are shown in Supplemental Figure S2B. Scale bar = 3 μm.

Figure 3. The mitotic nuclear extension depends on continued phospholipid synthesis

(A) Wild type cells (KW913) were arrested in G1 with alpha factor mating pheromone and released from the arrest into regular growth media (left panels) or into media containing nocodazole (right panels). Samples for cell cycle distribution (upper panels) and phospholipid analysis (lower panels) were taken at the indicated time points. For cell cycle distribution, shown are unbudded cells (blue curves) or large budded cells with a single nucleus (LB1N, red curves). For phospholipid analysis, shown are the amounts of lipid phosphate relative to time 0, immediately before the release from the G1 arrest. (B) Examples of cells over-expressing SPO7 and NEM1 and treated with nocodazole. DNA (DAPI) is in blue and Nsr1p-CR is shown in red. (C) Analysis of lipid phosphate in the same cells shown in panel B. Cells were arrested in G1, induced for galactose-induced expression and released from the arrest into media containing galactose and nocodazole. Samples were taken at time 0 and 3 hours, and the graph shows the difference in the amount of lipid phosphate between these two time points per 10⁶ cells. Error bars indicate SEM. (D) Analysis of lipid phosphate for control and GAL-SWE1 cells, as described in panel C. The difference in lipid phosphate between the two strains is not statistically significant. The presence of nocodazole did not affect phospholipid accumulation (data not shown). (E) An example of a wild type cell (KW926), as it is released from a nocodazole induced mitotic arrest. Green: Pus1p-GFP, red: Nsr1p-CR. Images were taken at the indicated time points (minutes).