ARL1 and SYS1 affect nuclear morphology in spo7Δ cells. (A) The flare phenotype in spo7Δ cells. Images of fixed wild-type cells (WT) and spo7Δ cells are shown. The flare in spo7Δ cells is marked with an arrow. The NE is identified by the nucleoporin Nup49p fused to GFP (Nup49p-GFP); the nucleolus is identified by the nucleolar protein Nsr1p fused to mCherry red fluorescent protein (Nsr1p-CR); the DNA is stained with DAPI. (B) Diagram of the yeast nucleus showing the NE (green), the nucleolus (red), and the DNA (blue) in wild-type and spo7Δ cells, and hypothetical nuclear phenotypes that would result from a mutation leading to a multi-flare phenotype with (right) or without (left) loss of DNA tethering to the NE. (C) Nuclear phenotype of strain MWY254, carrying a mutation that is synthetically lethal with spo7Δ, observed after a 2-h temperature shift to 37°C. Nuclear morphology was assessed with the nucleoplasmic protein Pus1p fused to GFP (GFP-Pus1p). For comparison to nuclei of wild-type and spo7^ts cells, see D or F. (D) Nuclear phenotypes by GFP-Pus1p, associated with arl1Δ and sys1Δ mutations, alone or in combination with spo7^ts, after a 2-h temperature shift to 37°C. (E) Three-dimensional reconstruction of nuclei from arl1Δ (top left), spo7^ts (top right), and two arl1 spo7^ts (bottom) cells. Cells were shifted to 37°C for 2 h. (F) Spatial distribution of chromatin in wild-type, arl1Δ, spo7^ts, and arl1Δ spo7^ts strains. Chromatin is visualized by the histone H2B fused to mCherry (Htb2p-CR), and nuclear morphology is detected by GFP-Pus1p. Cells were shifted to 37°C for 2 h. Bars: (A, C, D, and F) 2 µm; (E) 1 µm.

Deleting ARL1 causes the coalescence of Nsr1p-CR to a single focus but does not affect overall nucleolar structure. (A) spo7^ts cells, with and without ARL1, were fixed either before (left) or after a 3-h temperature shift to 37°C (right), then examined for nuclear morphology (with GFP-Pus1p) and Nsr1p-CR distribution. Arrows show examples of Nsr1p-CR in single flares; the arrowhead shows an example of focal Nsr1p-CR. (B) Wild-type (WT) and arl1Δ strains were treated the same as in A. Arrows show examples of Nsr1p-CR in crescent form; the arrowhead shows an example of focal Nsr1p-CR. (C) Electron micrographs of wild-type and arl1Δ cells fixed after a 2-h temperature shift to 37°C. Nucleoli (labeled with asterisks) are the darker, more electron-dense regions within the nuclei (N). Bars, 2 µm.

arl1Δ spo7^ts cells with multi-flared nuclei can recover and divide. 1-h time points of live arl1Δ spo7^ts cells grown at 30°C after a 3-h temperature shift to 37°C. Three-dimensional reconstructions of nuclei (with GFP-Pus1) are overlaid on corresponding phase images of cells. (A) A cell with a multi-flared nucleus at t₀ divides over the 5-h time course. Mother and daughter cells are seen at the 5-h time point, each with its individual nucleus. (B) A cell with a multi-flared nucleus at t₀ does not divide, but undergoes dynamic shape changes. Bars, 2 µm.