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doi: 10.1242/10.1242/jcs.00135

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An evolutionarily conserved fission yeast protein, Ned1, implicated in normal nuclear morphology and chromosome stability, interacts with Dis3, Pim1/RCC1 and an essential nucleoporin

¹ Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan
² Department of Integrated Bioscience, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba 277-8562, Japan

View larger version (58K): [in a new window]   Fig. 1. Chromosome missegregation in the ned1-1 mutant. (A) Diploid cells were incubated in a rich medium YPD at 36°C for indicated periods and plated on MR-plates at 26°C. Haploid (white in color) and aneuploid colonies (deep red, sectored) were scored. *For wild-type (WT), deep red colonies are categorized as aneuploid in the figure, however those with sectors were very rare, with undetermined genetic constituents. (B) FISH analysis of segregating chromosomes. Diploid cells were incubated in YPD medium at 36°C for 4 hours. Red, chromosome III (rDNA); green, chromosome II (cos713); blue, chromatin regions (DAPI). In the second panel, non-disjoined chromosome II is left in the mid-zone. In the bottom panel, one of the two chromosome III shows nondisjunction. Bar, 2 µm.

View larger version (79K): [in a new window]   Fig. 6. Ned1-Dis3 interactions. (A) Strains indicated are incubated at 36°C or at 26°C for 3 days and at 22°C for 4 days. (B) Two-hybrid interaction between Ned1 (1-121) and Dis3 (927-970). The activity of Ned1 protein segment containing a substitution mutation G80A or G80R as well as blank plasmid is shown on the —Ade, His plate. (C) The rate of minichromosome loss per cell division was determined by the half-sectored colony assay (Allshire et al., 1995). In each experiment, approximately 3x104 divisions were scored. The values are the average of two experiments.

View larger version (106K): [in a new window]   Fig. 2. Deformed nuclei and abnormal ER-like membranes in the ned1-1 mutant. (A) Haploid cells were incubated in YPD medium at 30°C. Merged images: blue, DAPI; red, ethidium bromide; green, MAb414. (B) Haploid cells are incubated in EMM2 medium at 30°C. The anti-Nup189 antibody was used instead of MAb414 (green), chromosomes were in red. (C) S. cerevisiae diploid nuclei stained with anti-Nup189. (D) Electron micrographs of diploid cells cultured at 36°C for 3 hours. N, nucleus; arrowheads, ER in wild-type; arrows, mesh-like (left bottom) and multi-layered structure (right) probably formed from enormously overdeveloped ER-like membranes in ned1-1 cells. (E) Haploid fission yeast cells transformed with pSec61-GFP. Cells are incubated in thiamine-free EMM at 30°C for 20 hours. Green, GFP; Red, DAPI. Bars, 2 µm.

View larger version (45K): [in a new window]   Fig. 3. Identification of Ned1 protein. (A) Extracts prepared from a wild-type (a) or a ned1 strain (b) were examined by western blot analysis. Numbers on the left indicate the molecular weight (kDa) of protein standards. (B) Wild-type fission yeast cell extracts prepared from a logarithmic (a) or from a stationary culture (b) in EMM2 at 30°C. Each of the protein samples was digested with alkaline phosphatase in the absence (-) or presence (+) of phosphatase inhibitors. Numbers on the left indicate the molecular weight (kDa) of each band. (C) Protein samples prepared from a synchronized culture at indicated times are analyzed by western blotting (top). The arrow indicates the position of a 90 kDa band. The synchrony was shown by the profile of the septation index (bottom).

View larger version (65K): [in a new window]   Fig. 4. Analysis of Nup189 protein and the defective phenotype of nup189-disrupted cells. (A) The two-hybrid interaction activity of indicated protein segments is shown (see text for details). (B) Specificity of the anti-Nup189 antibody. Protein samples prepared from a wild-type strain carrying the vector pKD 10 (a) or pKD 10-nup189+ (b) were electrophoresed in a gel and immunoblotted. The upper band in b is more intense than in a, while the lower bands are of the same intensity. (C) Colocalization of Nup189 and MAb414 antigen. Merged: Nup189, red; MAb414, green; DAPI, blue. (D) Wild-type (a) and mutant spores (b-d) containing pD817 germinated in an appropriate selective medium at 30°C. pD817 carries a segment of cytochrome P450 reductase gene that is fused with the GFP sequence and is used to visualize the nuclear envelope (Tange et al., 1998). (a) 25 hours, (b) 19 hours, and (c,d) 38.5 hours after transfer into the medium. Red, DAPI; green, GFP. Bars, 2 µm.

View larger version (70K): [in a new window]   Fig. 5. Genetic interactions of ned1-1 mutant with crm1, pim1 and spi1. (A) In the upper panels, indicated strains were incubated at 33°C for 3 days on YEA containing the indicated amounts of staurosporin (sta) or caffeine (caf). In the lower panel, the same strains were incubated on YEA at 24°C for 4 days. (B) Cell extracts prepared from the indicated strains were run on a 10% SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue. The p25 band was only visible in the lane for crm1 single mutant. (C) Indicated strains were incubated at 33°C for 4 days. (D) The ned1-1 mutant cells were transformed with multicopy plasmids that carried the indicated gene. Transformants were plated on YEA containing 15 µg/ml TBZ, incubated at 30°C for 3 days.

View larger version (110K): [in a new window]   Fig. 7. The effect of Ned1-overproduction on the nuclear structure. (A) A 3D image of a cell with overproduced Ned1. Electron micrographs from serial thin sections were used to construct the image (see Materials and Methods). The cell was incubated at 30°C for 24 hours to induce the expression of Ned1 protein. Blue, microtubule bundle in the nucleus; red, the spindle pole body (SPB). (B) Representing parts of electron micrographs used for construction of the 3D image shown in A. N, nucleus; MT, microtubules. (a) Middle part of the cell; arrowhead, the SPB situated in the cytoplasm. (b,c) Left and right parts of the cell, respectively; arrows indicate the ends of extended nuclear envelope. (C) Wild-type cells carrying both pREP82tubGFP and pREP1ned1+cDNA are incubated in the absence of thiamine at 30°C for 24 hours. (a,c) Localization of Nup189. (b,d) Microtubules (green), Nup189 (red) and DAPI (blue). (D) Suppression of the nuclear elongation by the pim1-46 mutation. pim1+ or pim1-46 cells were induced for full expression of Ned1 (26°C for 28.5 hours) and the nuclei were stained with DAPI. (E,F) Cells carrying either Cut12-GFP (E) or Mis6-GFP (F) were incubated as in A for the Ned1-overproduction (bottom panels; in upper panels cells without Ned1-overproduction) and stained with DAPI and anti-Sad1 antibody. Merged: cyan, DAPI; green, GFP; red, Sad1. Bars in B: 0.2 µm (a), 0.4 µm (b, c); other bars, 2 µm.