Specific aims
Chapter 5: Yra1 and Chromosome Segregation
Background
Since the principal aim of this project was to understand for which function Yra1 is Ubiquitinated by Slx5-Slx8, we took in consideration a pathway where the E3 ubiquitin ligases play an important role to ensure genome stability: Chromosome Segregation. Loss of Slx5-Slx8 leads to a broad range of phenotypes linked to genome instability such as sensitivity to genotoxic stress, gross chromosomal rearrangements (GCR) and increased rate of DNA mutation and recombination (Burgess et al., 2007;
Nagai et al., 2008; Zhang et al., 2006). Their indirect effects on transcription were revealed by changes in gene expression as a result of general stress response (van de Pasch et al., 2013). Interestingly Slx5, but not Slx8, was shown to localize at Centromeres in a process dependent on the kinetochore protein Ndc10. Analysis of spindle morphologies in Δslx5 and Δslx8 shows spindle mis-positioning, fish hooks, and aberrant spindle kinetics (van de Pasch et al., 2013). Another study highlights the importance of Slx5-Slx8 in targeting the spindle positioning protein Kar9 to proteasomal degradation when located at kinetochore microtubules. Failure in this process causes defects in chromosome segregation and spindle positioning (Schweiggert et al., 2016).
A possible link of Yra1 with chromosome segregation is suggested by earlier studies showing that Yra1 interacts genetically and physically with Mlp2, an NPC-associated protein that co-purifies with the Spindle Pole Body component Spc42 and promotes efficient SPB assembly (Niepel et al., 2005; Vinciguerra et al., 2005).
Furthermore mass spectrometry analysis of Yra1 interactors (Oeffinger et al., 2007) identified components that show Chromosome Segregation defects when mutated.
These factors include Yhr127W, a protein of unknown function required for asymmetric localization of Kar9 during mitosis (Schoner et al., 2008); Shp1, important for mitotic progression; Hrr25, a kinase that regulates Chromosome Segregation, and Kap123 important for SPB duplication and microtubule stability (Kupke et al., 2011;
Ptak et al., 2009). Based on the described functional and physical links between Yra1, Slx5-Slx8 and Mlp2, we decided to analyze the effect of Yra1 overexpressing mutants on Chromosome Segregation.
Yra1 overexpression causes a Chromosome Segregation defect
We examined whether the HA-yra1 mutants induce Chromosome Segregation defects using the Chromosome loss assay (Figure 5.1 A). This system allows monitoring chromosome loss events using red/white colony sectoring. The assay is based on an ade2- yeast strain (red), which carries the SUP11 tRNATYR gene on a plasmid that fully suppresses the ade2- red phenotype and cells are white. Loss of the plasmid/minichromosome will lead to red-white sectoring or red colonies (Hieter et al., 1985). The HA-YRA1 WT, HA-yra1Δintron, HA-yra1(allKR) and HA-yra1(1-210) sequences were integrated into the relevant strain and examined for appearance of red colonies (Figure 5.1 B).
As indicated in Figure 5.1 C, only the HA-yra1Δintron causes a significant Chromosome Segregation defect compared to HA-YRA1 WT, as 9 out of 11 clones showed a significant number of red colonies (see accompanying Table). Although 4 out of 10 HA-yra1allKR clones and 3 out of 11 HA-yra1(1-210) clones present formation of red colonies, the chromosome segregation defect is not significant in these two mutants. The sen1-1 and Δthp2 were included as positive controls in this chromosome loss assay. Western blot analysis shows elevated levels of Yra1 protein in the three mutant strains compared to WT (Figure 5.1D). Thus, overexpression of wild-type Yra1 (HA-Yra1Δintron) causes a systematic chromosome segregation defect, while overexpression of the HA-yra1allKR and HA-yra1(1-210) mutant proteins promotes chromosome loss at a lower frequency.
Figure 5.1: The yra1Δintron mutant shows a ChromosomeSegregation defect
A) Scheme showing the principle of the Chromosome loss assay. The loss of the plasmid containing SUP11 reverts the white colonies to red color due to the ade2- mutation that is not anymore suppressed, resulting in the accumulation of a red pigment (Yuen et al., 2007).
B) Chromosome loss assay: HA-YRA1 WT, HA-yra1Δintron, HA-yra1allKR, HA-yra1(1-210) were integrated into the YPH1021 background ( from Philip Hieter lab). sen1-1 and Δthp2 mutants served as
positive controls for chromosome loss (from Hieter lab). 10 or 11 clones from each strain were grown for 5-10 generations in YPD and plated in SC with low concentrations of Adenine. Shown is the plating of one clone from each strain. Red colonies reflect loss of CFIII, SUP11.
C) Quantification of chromosome loss. The percentage of red colonies was determined for each of the 10 or 11 (N10, N11) clones analyzed from each strain (values shown in Table below). Standard deviation of the mean is shown and p values <0.01 relative to HA-YRA1 WT are indicated by **.
D) Western blot analysis of HA-Yra1 WT, HA-yra1Δintron, HA-yra1allKR, HA-yra1(1-210) integrated in YPH1021 background using αHA antibody. αPgk1 was used as control for loading. Protein levels are shown in the graph as ratio HA-Yra1/Pgk1. Experiment performed by Varinia-Garcia Molinero.
Yra1 mutant sensitivity to the anti-microtubule drug Benomyl
Chromosome Segregation is an essential process that is tightly regulated to ensure correct division of genetic content between two dividing cells. Two main surveillance mechanisms in S. cerevisiae ensure the faithful segregation of the DNA:
the Spindle Assembly Checkpoint (SAC), that delays the onset of the Anaphase when the spindles and chromatids are not correctly attached to the kinetochores (Musacchio and Salmon, 2007) and the Spindle Position Checkpoint (SPOC), that blocks the exit from mitosis when the spindle is not properly aligned with the cell polarity axis (Caydasi et al., 2010). Alterations of these surveillance mechanisms cause defects in Chromosome Segregation (Caydasi and Pereira, 2012).
We examined whether the HA-yra1 mutants are sensitive to the microtubule-destabilizing drug benomyl as an indication of defective SPOC or SAC. As shown in Figure 5.2, the HA-yra1(1-210) shows sensitivity to benomyl at 25°C, 30°C, and 34°C (Suppl. Figure 1). This sensitivity is enhanced in Δbub2 and Δmad3 background, which are SAC mutants sensitive to benomyl due to the inability of improperly assembled spindles to delay the exit from mitosis (Hoyt et al., 1991; Li and Murray, 1991). The benomyl sensitivity of HA-yra1(1-210) was not enhanced in the absence of Kar9, a factor important to correctly orient the spindle. Surprisingly, the HA-yra1Δintron and the HA-yra1allKR show no sensitivity to benomyl (Figure 5.2). The apparent inconsistency of mutant phenotypes in different assays could be due to the strain backgrounds used for the Chromosome Segregation assay (HA-yra1 mutants integrated in YPH1021) and for the benomyl sensitivity assay (shuffled HA-yra1 mutants).
Figure 5.2: The sensitivity of the HA-Yra1(1-210) mutant to the anti-microtubule drug benomylis enhanced in Δbub2 and Δmad3.
Spot test analysis of HA-Yra1 mutants in the presence of DMSO 2% (no drug control), benomyl 15µg/ml, benomyl 18µg/ml, benomyl 20µg/ml, benomyl 23µg/ml, at 25°C. Confluent cultures of HA-Yra1 mutants shuffled into WT or Δbub2, Δmad3, and Δkar9 strains were serially diluted and spotted on the indicated plates. Sensitivity and sickness is shown by *; additive effect by **. The corresponding spot tests at 30°C and 34°C.are shown in Supplementary Figure 1.
Table 4: Summary of HA-yra1 mutants sensitivity to Benomyl in combination with Δbub2, Δmad3 and Δkar9.
The Table summarizes the Benomyl sensitivity observed for the HA-yra1 shuffled mutants in WT or Δbub2, Δmad3 and Δkar9 strain backgrounds. (/)=not sensitive to the drug, (*)=sensitive to the drug, (**)=additive effects on drug sensitivity.
WT ∆bub2 ∆kar9 ∆mad3
Shuffled strains DMSO Benomil DMSO Benomil DMSO Benomil DMSO Benomil
HA-YRA1 WT / / / * / / / *
HA-yra1(1-210) / * / ** / * / **
HA-yra1allKR / / / / / / / *
HA-yra1∆intron / / / / / * / *
Analysis of spindle positioning and morphologies in HA-Yra1 mutants
To define whether the Chromosome segregation defects detected in HA-Yra1 mutants could be due to spindle positioning defects or aberrant spindle morphologies, we visualized the spindle by tagging Tub1 with GFP and the Spindle Pole Bodies (SPB) were detected by tagging Spc42 with mCherry. The cell cycle phase of each cell was established based on the criteria shown in Figure 5.3, which consider the size of the bud, the SPB, and the position of the spindle.
Figure 5.3: Spindle and spindle pole bodies over the cell cycle in S. cerevisiae
Scheme relating S. cerevisiae cell cycle phases with bud and mother (M) cell size, spindle pole body duplication and spindle positioning. Representative images of living cells were taken from the integrated HA-YRA1WT, Spc42-mCherry and Tub1-GFP strain. Shown is the merge of 1 z-stack for each channel of images taken with a spinning disc confocal 3i microscope.
Figure 5.4 shows the quantification of normal and aberrant spindles in each cell cycle of HA-yra1 shuffled mutants. The HA-YRA1 WT shuffled strain shows some spindle defects in Anaphase (Early and Late), albeit less than the HA-yra1 mutants; the
mutants also show defects in Late S/G2 phases. The most affected is the HA-yra1(1-210) shuffled strain, which shows defects even in early S phase.
Figure 5.4: Analysis of spindle position and morphology in the HA-Yra1 shuffled mutants.
A) Quantification of cells showing normal or aberrant spindles in Tub1-GFP, Spc42-mCherry HA-Yra1WT, HA-Yra1(1-210), HA-Yra1Δintron and HA-Yra1allKR shuffled strains. 25 Z-stack images were taken using a spinning disc confocal microscope. Cell cycle phases were established based on Figure 5.3. B) Percentage of cells counted for each cell cycle. C) Table summarizing the total number of cells counted for each condition.
Experiment performed by Audrey Zihlmann, supervised by Valentina Infantino.
To corroborate these results, we performed the same analysis in integrated HA-yra1 mutants strains. Figure 5.5 (A-C) shows that the HA-yra1(1-210) has mainly defects in late S/G2 phases with abnormal spindle orientations, while the HA-yra1Δintron and HA-yra1allKR have mainly defects in late Anaphase showing bent and short spindles (Figure 5.5 E, Table). The short spindle observed in late Anaphase in part reflects the spindle that is still not fully elongated (Winey and O'Toole, 2001).
The phenotypes observed in the integrated HA-yra1 mutants strains are not as severe as those observed in the shuffled strains, but defects in spindle positioning in late S/G2 and bent spindle in late anaphase are confirmed. Figure 5.5 D shows one representative image for each integrated HA-yra1 mutant pointing to the defect in spindle shape and positioning.
Figure 5.5: Analysis of spindle position and morphology in the HA-Yra1 integrated mutants.
A) Quantification of cells showing normal or aberrant spindle in Tub1-GFP, Spc42-mCherry HA-YRA1WT, HA-yra1(1-210), HA-yra1Δintron and HA-yra1allKR integrated strains. 25 Z-stack images were taken using a spinning disc 3i confocal microscope. Cell cycle phases were established based on Figure 5.3. B) Percentage of cells counted for each cell cycle.
C) Total number of cells counted for each condition.
D) One Z-stack for each channel is shown and merged images are presented for Spc42-mCherry and Tub1-GFP in the middle, and together with DIC in the last column, while the first column shows DIC alone. One example for each strain is shown. Small white arrows point to the observed phenotypes.
E) Table with the detailed phenotypes observed in each cell cycle for each strain analyzed.
F) Spot test analysis in YEPD 2% Glucose at 25°C, 30°C, 34°C of HA-YRA1WT, yra1(1-210), HA-yra1Δintron and HA-yra1allK, integrated into the Spc42-mCherry, Tub1-GFP strain.
Yra1 mutants are defective in the Kar9 pathway
Since Yra1 mutants exhibit spindle positioning defects in late S/G2, we tested whether the HA-yra1 mutants present genetic interactions with the two functionally redundant microtubule-associated pathways that rely on either KAR9 or DYN3 to ensure correct spindle positioning. We also tested functional interactions with ARP1, coding for a subunit of Dynactin necessary for Dynein function (Moore et al., 2008), and BUB2 encoding a component of the spindle positioning checkpoint (Caydasi et al., 2010). Figure 5.6 shows that HA-yra1(1-210) and HA-yra1Δintron are synthetic sick with Δarp1 and Δdyn3 but not with Δkar9, suggesting that Yra1 may function in the Kar9 pathway. The HA-yra1(1-210) mutant is synthetic sick with Δbub2 confirming the additive sensitivity of the HA-yra1(1-210), Δbub2 double mutant to Benomyl treatment (Figure 5.2).
Figure 5.6: Genetic interactions of yra1 mutants with spindle positioning components.
Spot test of HA-Yra1 shuffle strain in WT, Δkar9, Δarp1, Δdyn3, or Δbub2 background transformed with HA-Yra1 WT, HA-Yra1(1-210), HA-Yra1Δintron, HA-Yra1allKR CEN TRP1 plasmids on TRP- and 5-FOA plates. Synthetic sickness is shown by *. Experiment performed by Audrey Zihlmann, supervised by Valentina Infantino.
Strategy to address a potential physical link between Yra1 and the SPB.
To have more direct hints on a potential role of Yra1 in spindle positioning, we took advantage of the zoning assay (Figure 4.1 A) to establish whether the DNA locus bound by LexA-Yra1 and visualized by LacI-GFP is preferentially relocalizing towards the SPB. For this purpose, we tagged Spc42 with mCherry to visualize the SPBs and the position of LacI-GFP was defined relative to the SPBs. As control, the same analysis was performed with LexA-empty. Figure 5.7 A shows the cell cycle phases analyzed with examples of the three main categories considered for the LacI-GFP localization: i) close to SPB, when it is next to, partially overlapping or colocalizing with the SPB; ii) middle, when it is in between the two SPBs; iii) distant
when it is further away from the SPBs. No major differences were identified between LexA-Yra1 and LexA-empty (Figure 5.7 B-C-D-E), indicating that LacI-GFP localization relative to the SPBs is independent of LexA-Yra1 binding.
Figure 5.7: Localization of a LacI-GFP labeled locus with respect to the SPB in response to LexA-Yra1 or LexA-empty binding.
A) Examples of LacI-GFP localizations relative to Spc42-mCherry in each cell cycle phase illustrating the defined categories: close to, in between and distant from SPBs. DIC images and merge of one Z-stack of 25 images is shown for each channel. LacI-GFP: bright green dot; Nup49-GFP: nuclear periphery; Spc42-mCherry: SPBs in red.
B) Tetraspeck Fluorescent Microsphere 0.1-0.2 µm; images taken with the spinning disc confocal 3i microscope using the GFP and mCherry channels and corresponding merge to show that no correction was applied for dichroic or mechanical shift from different channels.
C) Quantification of LacI-GFP localization with respect to Spc42-mCherry in each cell cycle phase as shown in A in the presence of LexA-Yra1 or LexA-empty.
D) Quantification of LacI-GFP localization with respect to Spc42-mCherry (Yra1 or LexA-empty) in the overall population shown as percentage of cells.
E) Pie chart showing the percentage of cells counted for each cell cycle stage in LacI-GFP, LexA-Yra1 and LacI-GFP, LexA-empty. N=133 cells were counted for LexA-Yra1 and N=138 for LexA-empty from two independent experiments.