PMC

Alm1 is required for proper accumulation of Cut8 and the proteasome to the NE. (A) WT cells expressing Cut8-GFP and Alm1-Tomato. Images are maximal projections of three z sections. (B) WT (red lectin stained) and alm1Δ cells expressing Cut8-GFP. Cut8-GFP intensity levels at the NE in WT and alm1Δ cells. Graphs represent mean and SD. n = 69. (C) Western blot analysis of total Cut8-GFP protein of WT and alm1Δ cells using anti-GFP mAb to detect Cut8-GFP and anti-PSTAIR mAb as a loading control. (D) Brightfield and immunofluorescence images of WT and alm1Δ cells expressing Mts2-8Myc, using anti-Myc antibodies against Mts2-8Myc (green) and DAPI to stain DNA (blue). (E) Western blot analysis of total Mts2-8Myc protein in WT and alm1Δ cells using anti-Myc antibodies to detect Mts2-8Myc and anti-PSTAIR antibodies as a loading control. (F) Brightfield and immunofluorescence images of WT and alm1Δ cells expressing Mts4-13Myc, using anti-Myc antibodies against Mts4-13Myc (green) and DAPI to stain DNA (blue). (G) Western blot analysis of total Mts4-13Myc protein in WT and alm1Δ cells using anti-Myc antibodies to detect Mts4-13Myc and anti-PSTAIR antibodies as a loading control. Positions of molecular mass markers are indicated in kilodaltons. Bars, 5 µm. A.U., arbitrary units. ***, P < 0.001.

Cnp3 overexpression phenocopies alm1Δ segregation defects. (A) Western blot analysis of total Cnp3-GFP using anti-GFP mAb or antitubulin (TAT1) mAb as a loading control. (B) Images of WT and alm1Δ cells expressing Cnp3-GFP from the endogenous locus and WT and alm1Δ cells expressing an additional copy of Cnp3-GFP from the medium-strength promoter nmt41 (pINT-Cnp3-GFP) in repressed conditions (YES media). (C) Centromeric Cnp3-GFP intensity levels in the indicated strains. n = 50. (D) Quantification of mitotic defects (lagging KTs during anaphase B). n = 50. (E) Time-lapse fluorescence images of the indicated strains. Time between frames is 2 min. Red asterisk denotes asynchronous KT segregation, and arrows denote lagging KTs. (F) Fluorescence images of alm1Δ cells expressing Cnp3-GFP from the endogenous promoter (marked with red lectins) and alm1Δ cells expressing Cnp3-GFP from the nmt81 promoter under repressed conditions (EMM + thiamine). (G) Western blot analysis of total Cnp3-GFP protein from cultures of the indicated genotypes grown in EMM + thiamine, using anti-GFP mAb or antitubulin (TAT1) mAb as a loading control. Molecular mass is given in kilodaltons. (H) Quantification of centromeric Cnp3-GFP intensity levels. n = 50 cells. (I) Quantification of mitotic defects (lagging KTs during anaphase B) of the indicated strains. n = 50. Graphs represent mean and SD. Bars, 5 µm. A.U., arbitrary units. *, P < 0.05; ***, P < 0.001.

Alm1 localizes to the NE and NPC, and its absence results in chromosome segregation defects. (A) Alm1-Tomato colocalizes with Nup211-GFP. Maximal projection of three z sections is shown. (Inset) Magnification of the indicated nucleus. Bars, 2.5 µm. Fluorescence intensity levels along the nuclear rim of the selected nucleus from an arbitrary starting point. (B) Images of WT and alm1Δ cells expressing Nup211-GFP. Mean fluorescence intensity of Nup211-GFP at the NE and in the nucleoplasm. n = 30. (C) WT and alm1Δ cells expressing the NE marker Cut11-GFP. Arrow indicates smaller daughter nuclei. Time is indicated in minutes. Ratio of the perimeter of sister nuclei. n = 25. (D) Minichromosome loss assay. Three independent experiments. n > 1,500. (E) Images of WT and alm1Δ mitotic cells expressing Sid2-Tomato and Mis6-GFP. Time between frames is 1 min. Magnifications of the regions indicated by dashed boxes are shown below. Bar, 2.5 µm. Asterisk marks asynchronous KT segregation. Arrow indicates lagging KT. Bars, 5 µm. A.U., arbitrary units. Error bars represent SD. ***, P < 0.001; ****, P < 0.0001.

alm1Δ mutant shows altered accumulation of centromere and KT proteins. (A) WT (green lectin stained) and alm1Δ cells expressing Cnp1-mCherry (top) or Cnp3-Tomato (bottom). Bars, 5 µm. (B and C) Mean fluorescence intensity in alm1Δ cells relative to WT cells of the indicated proteins (Cnp1-mCherry, Mhf1-GFP, Mhf2-GFP, Cnp20-GFP, Pcs1-GFP, Mde4-GFP, Fta1-GFP, Sim4-Tomato, and Cnp3-Tomato) at KTs during interphase. n = 50. (D) Western blot analysis of total Cnp3-GFP protein from WT and alm1Δ cells using anti-GFP mAb to detect Cnp3-GFP (top) and TAT1 as a loading control (bottom). Positions of molecular mass markers are indicated in kilodaltons. (E) RT-qPCR analysis of act1, cnp1, and cnp3 mRNA levels in WT and alm1Δ cells. Three biological repeats were performed. (B, C, and E) Error bars represent SD. (F) RT-qPCR analysis of centromere I (dh, dg, imr, and cnt) transcript levels in WT and alm1Δ mutant (normalized to act1 transcript levels). n = 5. Error bars represent SEM. (G) ChIP-qPCR analysis of Cnp3-GFP and H3K9me levels. ChIP data have been normalized to act1 and are shown relative to the maximal enrichment in WT cells at the cen1 region. *, P < 0.05; **, P < 0.001; ***, P < 0.001.

Proteasome function and localization are required for stoichiometric accumulation of Cnp3 at KTs. (A) Centromeric Cnp3-GFP levels in the indicated backgrounds and temperatures. n = 50. (B) Western blot analysis of total Cnp3-GFP, using anti-GFP mAb to detect Cnp3-GFP and TAT1 as a loading control. (C) Quantification of mitotic defects (lagging KTs during anaphase) of WT and mts4 cells at 25ºC by in vivo fluorescence microscopy, using Cnp3-GFP as a KT marker. n > 140. (D) Quantification of centromeric Cnp3-Tomato levels in the indicated backgrounds and temperatures. n = 50. (E) Western blot analysis of total Cnp3-GFP protein in the indicated strains, using anti-GFP mAb to detect Cnp3-GFP and antitubulin (TAT1) mAb as a loading control. (F) Cnp3 protein stability in WT and alm1Δ cells in the presence of CHX. Cnp3-GFP was detected by immunoblotting with anti-GFP mAb, and anti-PSTAIR mAb was used as a loading control. Quantification of Cnp3-GFP protein stability. Cnp3-GFP band intensities were quantified using ImageJ and normalized to PSTAIR signals. Relative intensity at time 0 was set up as 100% in each case. Error bars represent SD from three independent experiments. (G) mts4 expressing Cnp3-GFP and untagged mts4 cells were grown to midlog phase at 25ºC and then shifted to 36ºC for 3 h. Samples were collected and subjected to anti-GFP immunoprecipitation (IP). Whole lysate (left) was immunoblotted with anti-GFP mAb to detect Cnp3-GFP and antitubulin (TAT1) mAb as a loading control. Immunoprecipitated proteins were immunoblotted with anti-GFP mAb to detect Cnp3-GFP and antiubiquitin pAb to detect ubiquitinated proteins. (H) mts4 cells overexpressing Cnp3-GFP or Cnp3-GFP and His₆-ubiquitin were grown in EMM to midlog phase at 25ºC and then shifted to 36ºC for 3 h. Samples were collected, and polyubiquitinated proteins were purified with Ni²⁺–nitrilotriacetic acid beads in denaturing conditions. Ubiquitinated proteins were detected by immunoblotting with antiubiquitin pAb (right), and ubiquitinated forms of Cnp3 were detected by immunoblotting using anti-GFP mAb (left). A fraction of the whole cell extract (left) with an equal amount of total protein was immunoblotted with anti-GFP mAb to detect Cnp3-GFP and antitubulin (TAT1) mAb as a loading control. Positions of molecular mass makers are indicated in kilodaltons. Graphs represent mean and SD. A.U., arbitrary units. *, P < 0.05; **, P < 0.001; ***, P < 0.001.

SGA assay based on TBZ sensitivity identifies genetic factors that contribute to maintain KT structure and functionality. (A) TBZ sensitivity assay of alm1Δ mutant. clr4Δ mutant was used as a positive control (). (B) Flow-through of the SGA to screen for genetic interactors of alm1Δ mutant in TBZ. WT and alm1Δ query strains were crossed with the Bioneer haploid deletion mutant library (v. 3) and spotted on YES- and TBZ-containing plates. Examples of colony growth on YES and YES + TBZ plates. The growth of the single and double mutants was quantified in both media, and we compared the ratio with the median ratio. Blue indicates small colony size, and yellow indicates large colony size (see Materials and methods). (C) Clustering analysis showing one of the clusters of deletion mutants that have a synthetic effect on TBZ sensitivity with alm1Δ. For each experiment, four replicates were performed. Clustering analysis showing one group of genes with no sensitivity to TBZ, neither in the single nor the double mutant with alm1Δ. Blue indicates synthetic interaction, yellow indicates suppressive interaction, black indicates no interaction, and gray indicates the absence of data. (D) GO enrichment analysis for biological processes of all of the genes identified in the three synthetic clusters. The table contains the percentage between the total number of genes of the indicated GO group and the total number of genes of S. pombe (upper value), and the percentage between the number of gene deletion mutants of the synthetic clusters belonging to the mentioned GO group and the total number of gene deletion mutants of the synthetic clusters (lower value). (E) Tetrad dissection analysis of crosses between alm1Δ and strains with the indicated genotypes. Double mutants between alm1Δ and the indicated mutant backgrounds are encircled.