Figure 1.
Mia1p is exported from the nucleus via a Crm1p-dependent NES.
(A) Mia1p-GFP localized to mitotic spindles and interphase microtubules in crm1-809 Uch2p-mCherry expressing cells at 36°C but only to the nucleus at 18°C. (B) Positions of predicted NLS and NES on Mia1p. (C) Mia1p-ΔC17-GFP is enriched in the nucleus in interphase cells. (D) GFP-NES but not MutNES is excluded from the nucleus. Shown are single maximum intensity reconstructions of live cells. Scale bars = 5 µm.
Figure 2.
Nuclear retention of Mia1p leads to abnormal organization of interphase microtubule arrays.
(A) Localization of Mia1p-GFP in interphase cells, with Uch2-mCherry as an NE marker. (B) Relative intensities of nuclear Mia1p-GFP fluorescence in interphase cells of various mia1 mutant backgrounds (n = 100 cells). (C) Localization of Mia1p-GFP in interphase cells, with Pcp1p-mCherry as an SPB marker. (D) mia1-MutNES4 cells expressing α-tubulin-GFP exhibited fewer interphase microtubule bundles, similar to mia1Δ cells. (E) Quantification of the number of interphase microtubule bundles in wild type, mia1-MutNES4 and mia1Δcells (n = 100 cells). (F) Alp4p-GFP is detected as several distinct dots in wild type but not in mia1-MutNES4 cells treated with MBC. Shown are single maximum intensity reconstructions of live cells. Scale bars = 5 µm.
Figure 3.
Mutation in the Mia1p NES triggers mitotic spindle abnormalities.
(A) mia1-MutNES4 cells exhibit frequent spindle abnormalities such as monopolar and broken spindles. Shown are representative fields of wild type, mia1-MutNES4 and mia1Δcells expressing α-tubulin-GFP. Abnormal spindles are indicated by white arrowheads. (B) Percentages of aberrant mitoses in asynchronous cell populations (n = 300 cells). PAA, Post-anaphase microtubule arrays. (C) Percentages of cells exhibiting Mad2p-GFP at kinetochores (n = 300 cells). (D) mia1-MutNES4 mad2Δ cells are severely compromised for growth. Shown are three sets of segregants (from top to bottom: tetratype, parental ditype and non-parental ditype) from tetrads obtained from a cross between mia1-MutNES4 and mad2Δ cells, grown on YES agar (upper panel) and later replicated onto minimal medium lacking uracil (lower panel). Both the mia1+ and mad2+ genes were mutated using ura4+ gene. Note the smaller colony size in double mutants. (E) mia1-MutNES4 cells exhibit a decreased anaphase elongation rate. (F) Unlike Mia1p-GFP, Mia1p-MutNES4-GFP does not localize to kinetochores during mitosis. Pcp1p-mCherry was used as an SPB marker. Scale bars = 5 µm.
Figure 4.
Interaction between Mia1p and Alp14p is affected by mutating the Mia1p NES.
(A) Alp14p-GFP does not localize to microtubules in mia1-MutNES4 and mia1Δ cells. Shown are single maximum intensity reconstructions of live cells expressing Alp14p-GFP and Pcp1-mCherry. (B) Mia1p-GFP and Alp14p-TagRFP are spatially separated in mia1-MutNES4 cells. (C) MBP-Mia1p-MutNES4 showed weaker interaction with Alp14p-myc in pull-down assays, as compared to MBP-Mia1p. Alp14p-myc yeast lysates were extracted from mia1Δ cells and detected with anti-myc antibody. MBP-tagged proteins were detected by Coomassie staining. (D) When released from the nucleus using pim1-1 mutation at 36°C, Mia1p-MutNES4 is capable of loading Alp14p-GFP on cytoplasmic microtubules. Localization of Alp14p-GFP in wild type septated cells at 24°C and 36°C is included as a control. Shown are single maximum intensity reconstructions of live cells. (E) Overexpression of Mia1p-MutNES4-mCherry partially restores nuclear accumulation and spindle localization of Alp14p-GFP during mitosis. Shown are single maximum intensity reconstructions of live cells. Scale bars = 5 µm.
Figure 5.
Artificial accumulation of Alp14p in the nucleus partially rescues the mitotic defects observed in Mia1p NES mutant cells.
(A) Alp14p-NLS-TagRFP localizes to the nucleus during interphase, partially in mia1-GFP, and fully in mia1-MutNES4-GFP cells. Shown are single maximum intensity reconstructions of live cells. Scale bars = 5 µm. (B) Nuclear Alp14p-NLS-TagRFP partially restores localization of Mia1p-MutNES4-GFP to kinetochores during mitosis. Shown are single maximum intensity reconstructions of live cells. Scale bars = 5 µm. (C) Percentages of cells with various levels of Mia1p-MutNES4-GFP at kinetochores (n = 100 metaphase cells). Cells were binned into three classes based on intensity of GFP signal at kinetochores (strong, intermediate, none). Representative images are shown in the legend. (D) Percentages of aberrant mitoses and other cell cycle stages in asynchronous cell populations (n = 300 cells). Microtubules were immunostained with anti-α-tubulin antibody TAT-1. (E) Percentages of monopolar and broken short spindles (n = 100 spindles). Microtubules were immunostained with anti-α-tubulin antibody TAT-1.