(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



Meiotic cells escape prolonged spindle checkpoint activity through kinetochore silencing and slippage [1]

['Anne Mackenzie', 'Department Of Biology', 'Indiana University', 'Bloomington', 'Indiana', 'United States Of America', 'Victoria Vicory', 'Soni Lacefield', 'Department Of Biochemistry', 'Cell Biology']

Date: 2023-05

To prevent chromosome mis-segregation, a surveillance mechanism known as the spindle checkpoint delays the cell cycle if kinetochores are not attached to spindle microtubules, allowing the cell additional time to correct improper attachments. During spindle checkpoint activation, checkpoint proteins bind the unattached kinetochore and send a diffusible signal to inhibit the anaphase promoting complex/cyclosome (APC/C). Previous work has shown that mitotic cells with depolymerized microtubules can escape prolonged spindle checkpoint activation in a process called mitotic slippage. During slippage, spindle checkpoint proteins bind unattached kinetochores, but the cells cannot maintain the checkpoint arrest. We asked if meiotic cells had as robust of a spindle checkpoint response as mitotic cells and whether they also undergo slippage after prolonged spindle checkpoint activity. We performed a direct comparison between mitotic and meiotic budding yeast cells that signal the spindle checkpoint through two different assays. We find that the spindle checkpoint delay is shorter in meiosis I or meiosis II compared to mitosis, overcoming a checkpoint arrest approximately 150 minutes earlier in meiosis than in mitosis. In addition, cells in meiosis I escape spindle checkpoint signaling using two mechanisms, silencing the checkpoint at the kinetochore and through slippage. We propose that meiotic cells undertake developmentally-regulated mechanisms to prevent persistent spindle checkpoint activity to ensure the production of gametes.

Mitosis and meiosis are the two major types of cell divisions. Mitosis gives rise to genetically identical daughter cells, while meiosis is a reductional division that gives rise to gametes. Cell cycle checkpoints are highly regulated surveillance mechanisms that prevent cell cycle progression when circumstances are unfavorable. The spindle checkpoint promotes faithful chromosome segregation to safeguard against aneuploidy, in which cells have too many or too few chromosomes. The spindle checkpoint is activated at the kinetochore and then diffuses to inhibit cell cycle progression. Although the checkpoint is active in both mitosis and meiosis, most studies involving checkpoint regulation have been performed in mitosis. By activating the spindle checkpoint in both mitosis and meiosis in budding yeast, we show that cells in meiosis elicit a less persistent checkpoint signal compared to cells in mitosis. Further, we show that cells use distinct mechanisms to escape the checkpoint in mitosis and meiosis I. While cells in mitosis and meiosis II undergo anaphase onset while retaining checkpoint proteins at the kinetochore, cells in meiosis I prematurely lose checkpoint protein localization at the kinetochore. If the mechanism to remove the checkpoint components from the kinetochore is disrupted, meiosis I cells can still escape checkpoint activity. Together, these results highlight that cell cycle checkpoints are differentially regulated during meiosis and mitosis.

We performed a direct comparison of the duration of spindle checkpoint activity between mitosis and meiosis in budding yeast. We monitored the length of the checkpoint delay signaled through two different ways, either through addition of nocodazole to disrupt kinetochore-microtubule attachments or through a lack of tension-bearing kinetochore-microtubule attachments. The lack of tension-bearing attachments signals the spindle checkpoint through the activity of Aurora B kinase, which phosphorylates kinetochore proteins to release microtubules, creating unattached kinetochores [ 33 ]. Whether there is a difference in checkpoint strength between a lack of attachment versus lack of tension is an important question because Aurora B kinase localizes to both the kinetochore and microtubules and is involved in maintenance of spindle checkpoint activity [ 41 – 46 ]. Whether the pool of Aurora B kinase on the microtubules affects checkpoint activity and whether there are differences between mitosis and meiosis in the signaling is currently unknown. To this end, we found that cells escaped spindle checkpoint signaling significantly faster in both meiosis I and meiosis II when compared to mitosis, signaled through either addition of a microtubule-depolymerizing drug or a genetic background that caused a lack of intra-kinetochore tension. In contrast to mitosis and meiosis II, in which cells undergo mitotic slippage, cells in meiosis I can escape the spindle checkpoint through two mechanisms, slippage and silencing. Our data in budding yeast support the model that meiosis has a developmentally controlled regulation to turn off spindle checkpoint activity to ensure completion of meiosis for gamete formation.

Most experiments on spindle checkpoint activity and slippage have been performed in mitosis. However, meiosis poses additional challenges to chromosome segregation that may require differences in spindle checkpoint regulation. Meiosis consists of two divisions in which homologous chromosomes segregate during meiosis I and sister chromatids separate during meiosis II. Setting up the specialized segregation pattern in meiosis I requires that homologous chromosomes pair and undergo crossover recombination to form linkages between the chromosomes [ 33 ]. The two sister chromatid kinetochores are held together so that they co-orient [ 34 – 36 ]. This allows a connection of homologous chromosome kinetochores to microtubules emanating from opposite spindle poles for biorientation. The poleward spindle forces are resisted by the crossover and the cohesins along the chromosome arms until the cohesins are cleaved in anaphase I for chromosome segregation [ 37 – 40 ]. The meiosis II chromosome segregation pattern is similar to that of mitosis, in that sister chromatid kinetochores attach to microtubules emanating from opposite spindle poles [ 33 ]. The spindle forces are resisted by cohesins in metaphase II until they are cleaved and sister chromatids separate. Whether these differences in chromosome segregation and cell cycle regulation affect spindle checkpoint activity is not known.

Cells can escape the spindle checkpoint after a prolonged delay [ 24 ]. For example, experimental conditions that completely disrupt kinetochore-microtubule attachments, such as with the addition of the microtubule depolymerizing drug nocodazole, cause an arrest for several hours. Interestingly, cells prevent a permanent arrest by escaping the spindle checkpoint in a process known as mitotic slippage or adaptation [ 25 – 28 ]. Mitotic slippage occurs in budding yeast and animal cells despite the presence of spindle checkpoint components bound to the kinetochore [ 25 , 28 ]. During mitotic slippage, securin and cyclin B are degraded for anaphase onset due to activation of the APC/C, MCC disassembly, cyclin B turnover, or Cdk1 inhibition [ 25 , 29 , 30 ]. Previous studies in animal cells have shown that even with checkpoint signaling, cyclin B and securin are slowly degraded, allowing slippage [ 25 , 31 , 32 ]. One mechanism that allows mitotic slippage in budding yeast is through PP1. Instead of PP1 targeting the kinetochore, as occurs during spindle checkpoint silencing, PP1 targets the MCC component Mad3 during mitotic slippage [ 30 ]. The dephosphorylation of Mad3 destabilizes the MCC, allowing APC/C activation for anaphase onset.

The spindle checkpoint produces a diffusible signal at an unattached kinetochore that ultimately inhibits the anaphase promoting complex/ cyclosome (APC/C), a ubiquitin ligase that targets substrates for ubiquitination and proteasomal degradation for anaphase onset [ 1 ]. At an unattached kinetochore, the spindle checkpoint kinase Mps1 phosphorylates the kinetochore protein Knl1 Spc105 for the Bub3-Bub1 complex to bind [ 3 – 7 ]. Mps1 also phosphorylates Bub1 and Mad1 so that Bub1 can recruit the Mad1-Mad2 complex and Mad2 can be converted into a form that forms a scaffold, ultimately leading to assembly of the mitotic checkpoint complex (MCC) composed of Bub3, Mad2, Mad3/BubR1, and Cdc20 [ 8 – 16 ]. The Mps1 phosphorylation of Mad1 is also important for Cdc20 binding and incorporation into the MCC [ 10 , 11 , 17 ]. The MCC serves as the diffusible signal that inhibits the APC/C [ 18 – 20 ]. The spindle checkpoint is turned off once proper kinetochore-microtubule attachments are made in a process called spindle checkpoint silencing. Protein phosphatase I (PP1) binds and dephosphorylates Spc105 Knl1 to remove Bub3, preventing production of the MCC [ 3 , 21 – 23 ]. Furthermore, the MCC is disassembled allowing the APC/C to become active for anaphase onset by targeting securin and cyclin B for ubiquitination and subsequent degradation[ 1 ].

A conserved surveillance mechanism known as the spindle checkpoint delays chromosome segregation in both mitosis and meiosis if kinetochores are not properly attached to spindle microtubules [ 1 ]. The delay provides cells additional time to make proper attachments prior to the onset of chromosome segregation. Although the spindle checkpoint reduces chromosome mis-segregation, segregation errors can still occur and cause aneuploidy, in which cells have missing or extra chromosomes. Aneuploidy can be detrimental to normal cellular function and can contribute to human disease [ 2 ]. For example, aneuploidy is a hallmark of cancer and leads to genetic instability and cancer progression. Aneuploidy during meiosis contributes to trisomy conditions, miscarriage, and infertility. Therefore, understanding how the spindle checkpoint normally functions and how it can sometimes fail to prevent chromosome mis-segregation is important for understanding the etiology of these human diseases.

Results

The spindle checkpoint is less persistent in meiosis compared to mitosis To investigate differences in spindle checkpoint strength between mitosis and meiosis, we used live-cell fluorescence microscopy to monitor the duration of the spindle checkpoint delay. The cells expressed the spindle pole body (SPB) component Spc42 tagged with mCherry and a biosensor for the enzyme separase, which cleaves cohesin at the metaphase-to-anaphase transition [47]. The biosensor consists of a chromosomal locus tagged with a fluorescent focus that disappears upon separase activation; a LacO array was integrated into a chromosome and the cells expressed a GFP-tagged Lac repressor engineered with an Scc1 cleavage site (GFP-Scc1-LacI) for mitosis or a Rec8 cleavage site for meiosis (GFP-Rec8-LacI). With separase activation, the cohesin subunit Scc1 or Rec8 will be cleaved and a GFP focus will no longer be visible, although the nucleus will still have a GFP haze (Fig 1A and 1B). The advantage of using the separase biosensor is that we can then monitor anaphase onset in conditions or mutant backgrounds that may have depolymerized microtubules or spindle elongation prior to cohesin cleavage (Fig 1C and 1D). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. The spindle checkpoint is less persistent in meiosis compared to mitosis. (A-B) Representative time lapse images of a cell harboring Spc42-mCherry and the separase biosensor in mitosis (A) and meiosis I (B). Time from SPB separation to separase activation. Time 0 indicates SPB separation. Scale bar = 5 μm. (C-D) Cartoons depicting activation of the spindle checkpoint through loss of kinetochore (KT) tension and loss of kinetochore-microtubule (KT-MT) attachments in mitosis (C) and meiosis I (D) Wildtype = WT. E) Graph depicting mean time from start of movie to anaphase onset in mitosis following treatment with DMSO or nocodazole. Asterisk indicates statistically significant difference between DMSO-treated and nocodazole-treated cells (p <0.0001, Mann-Whitney test) and error bars show standard deviation (SD) n≥ 50 cells per genotype. F) Graph depicting mean time from start of movie to anaphase onset in meiosis following treatment with DMSO or nocodazole. (G-H) Graph showing the mean time from SPB separation to anaphase onset in mitosis (G) and meiosis I (H). Asterisks indicate statistically significant difference between wildtype and mutants (p < 0.0001, Mann-Whitney test). Error bars show SD. n≥100 cells per genotype. https://doi.org/10.1371/journal.pgen.1010707.g001 We first measured the duration of a spindle checkpoint delay in a condition in which kinetochore-microtubule attachments are disrupted due to treatment with the microtubule-depolymerizing drug nocodazole (Fig 1C and 1D). In mitosis, we released haploid cells from an alpha-factor-induced G1 arrest and treated cells with 15 μg/mL of nocodazole after we observed separation of SPBs in most of the cells. We then started the movie and measured the duration from the start of the movie until loss of the GFP focus, which represents the time of separase cleavage of cohesin. We observed an average duration of 348 ± 93 minutes when cells were treated with nocodazole, which is on average a delay of 323 minutes compared to cells treated with the solvent control (DMSO) (average ± SD; Fig 1E). As previously shown for mitotic cells, we observed cell-to-cell variability in the duration of the delay [28]. This delay is dependent on the spindle checkpoint because mad3Δ cells treated with nocodazole progressed into anaphase with a similar duration as the DMSO control cells. To assess the duration of a checkpoint delay in meiosis I, we treated cells with 30 μg/mL of nocodazole after release from a prophase I arrest using the GAL-NDT80 arrest and release system [48,49]. We treated cells with nocodazole 80-minutes after release from prophase I, which is when we observed SPB separation. We specifically waited to add the nocodazole until after prophase I because microtubule perturbation earlier causes a G2 arrest [50]. In the presence of nocodazole, cells had a metaphase I duration of 103 ± 56 minutes, which is 69 minutes longer than the control cells treated with DMSO alone (average ± SD; Fig 1F). The mad3Δ cells treated with nocodazole had a similar time of anaphase onset as the DMSO control, suggesting that the delay was spindle checkpoint dependent. We conclude that the spindle checkpoint is more persistent in mitosis than in meiosis when most kinetochore-microtubule attachments are disrupted. Chromosome segregation errors that cause single chromosome aneuploidies often arise from the misattachment of kinetochores and microtubules, such that the two sister chromatids in mitosis or the two homologous chromosomes in meiosis I are attached to the same pole. The monopolar attachment creates a lack of intra-kinetochore stretch, or tension [51]. The kinetochore-microtubule attachments that are not under tension are preferentially released due to the activation of the Ipl1Aurora B kinase, ultimately leading to spindle checkpoint activation [52–55]. We therefore wanted to compare the persistence of the spindle checkpoint delay between mitosis and meiosis I upon disruption of kinetochore tension. We engineered strains to create a condition in which all kinetochores can attach to spindle microtubules but cannot generate tension (Fig 1C and 1D). To prevent kinetochore tension in mitosis, we depleted the essential protein Cdc6, which is required for initiation of DNA replication (Piatti, 1995). Because only one microtubule binds per kinetochore in budding yeast, without DNA replication, the single sister chromatid will be unable to biorient, creating a lack of kinetochore tension. To deplete Cdc6, CDC6 was placed under control of the GAL1 galactose-inducible promoter and fused to a Ubi degron (Cdc6 depletion or Cdc6-dp) [56]. When cells are grown in the presence of glucose, CDC6 expression is repressed, and any remaining Cdc6 is degraded. We assessed the duration of the spindle checkpoint delay in diploid Cdc6-dp cells by measuring the time from SPB separation to separase activation, as assessed by the loss of the GFP focus. Cdc6-dp cells were in metaphase for a duration of 352 ± 148 minutes, which is an average delay of 295 minutes when compared to wildtype cells (average ± SD; Fig 1G). There was also substantial cell-to-cell variability in the duration of the checkpoint delay in Cdc6-dp cells, similar to nocodazole-treated cells. The Cdc6-dp haploid cells had a similar delay as the diploid cells, with metaphase duration of 339 ± 157 minutes, suggesting that ploidy did not cause major differences in the delay time (S1A Fig). The observed delay in Cdc6-dp cells was due to spindle checkpoint activation because Cdc6-dp mad3Δ cells progressed through metaphase with similar timings as the wildtype control cells. We note that the timings from the lack of tension experiments cannot be directly compared to the timings from the nocodazole experiments. In the nocodazole experiments, we did not measure the full duration of metaphase because time-lapse imaging was started after the addition of nocodazole, to prevent the drug from interfering with SPB separation (see Materials and Methods). In contrast, we measured the time from SPB separation to anaphase onset in the Cdc6-dp experiments. However, the results show that both nocodazole and Cdc6-dp cause an extended spindle checkpoint delay that cells can eventually escape. To prevent intra-kinetochore tension during meiosis I, we disrupted proteins needed for crossover formation. Normally, crossovers, held in place by arm cohesin, link homologs together and resist the spindle forces when homologous chromosomes biorient, which creates intra-kinetochore tension [33]. We prevented crossover formation by using a catalytically inactive allele of SPO11, spo11-Y135F, which is a mutant form of the enzyme required to make double-stranded breaks for crossover formation [57]. Without crossovers, homologs are not physically linked together, causing a lack of tension across the kinetochores. In these cells, chromosomes move to the spindle pole prematurely in an anaphase-like prometaphase I because they cannot resist spindle forces [58]. The spo11-Y135F cells exhibited a metaphase I duration of 194 ± 81 minutes, which is an average delay of 140 minutes compared to the wildtype control cells, although there is substantial cell-to-cell variability (average ± SD; Fig 1H). The spo11-Y135F mad3Δ cells showed similar timings to the wildtype control cells, suggesting that the delay was spindle checkpoint dependent. Like the mitotic studies, we cannot directly compare the nocodazole-treated cells with the spo11-Y135F cells. In the nocodazole experiment, we started the time-lapse imaging after addition of the nocodazole to cells in early metaphase I, just after SPB separation. In contrast, the spo11-Y135F cells were imaged throughout meiosis, providing the full duration of the metaphase I delay. To determine if the duration of the spindle checkpoint delay is similar in other mutants with reduced crossovers, we deleted MEK1, which biases repair of double-strand breaks (DSBs) to the homolog, allowing for crossover formation. In cells harboring MEK1 mutants, crossover formation is reduced by ~85%, so some homologs will retain crossovers [59–61]. The mek1Δ cells displayed an average metaphase I delay of approximately 130 minutes with cell-to-cell variability, which was similar to spo11-Y135F cells (Fig 1H). Overall, our results demonstrate that the spindle checkpoint is not as persistent in meiosis as it is in mitosis, signaled through either addition of nocodazole or through genetic backgrounds that cause a lack of kinetochore tension. We wondered whether the shorter duration of the spindle checkpoint in meiosis was due to the size differences between budding yeast meiotic and mitotic cells. Mouse oocytes have a weaker spindle checkpoint than mitotic cells, which is thought to be due to their larger size [62–69]. Similarly, checkpoint strength is thought to be at least partially dependent on cell size in the oocyte and developing C. elegans embryo [70–72]. To determine if a size difference could be causing a weaker spindle checkpoint in meiotic budding yeast cells, we measured the volume of budding yeast cells at the time point just before anaphase onset and found that mek1Δ cells in meiosis had a volume of 254 ± 46 fL, while Cdc6-dp cells had an average volume of 343 ± 89 fL (S1B Fig). From this we conclude that cells arrested in mitosis have a larger average volume compared to cells arrested in meiosis, so it is not likely that the inherent weakness of the meiotic checkpoint is due to a larger cell volume.

The meiotic spindle checkpoint prevents some chromosome mis-segregation when crossover formation is drastically reduced Previous work revealed that the spindle checkpoint proteins Mad1, Mad2, and Mad3 have different functions in meiosis. Mad3 provides a checkpoint delay, but this delay is not needed for faithful chromosome segregation in normal growth conditions [58,73,74]. The checkpoint components Mad1 and Mad2 are also needed for a delay but have additional roles in cell cycle regulation and chromosome biorientation [73–76]. Interestingly, the spindle checkpoint delay strongly reduces the mis-segregation of a pair of non-exchange homeologous chromosomes [74,77]. We asked whether the spindle checkpoint could decrease chromosome mis-segregation in mek1Δ cells, which have a much higher fraction of non-exchange chromosomes than the strains previously tested. To address this question, we compared chromosome segregation fidelity between mek1Δ cells and mek1Δ mad3Δ cells, which lacked spindle checkpoint activity. We monitored the segregation of a single pair of homologs with LacO repeats near the centromere of chromosome IV in cells that expressed LacI-GFP under the control of a copper-inducible CUP1 promoter [78]. Upon the addition of copper sulfate, LacI-GFP was expressed and bound the LacO repeats, which allowed visualization of each homolog as a single focus (Fig 2A). In wildtype and mad3Δ cells, the percent of chromosome IV mis-segregation was less than 2%. In contrast, 24% of mek1Δ and 38% of mek1Δ mad3Δ cells mis-segregated chromosome IV in anaphase I (Fig 2B). Thus, the meiotic spindle checkpoint prevented some chromosome mis-segregation in anaphase I, despite not having as long of a delay as the mitotic checkpoint. We note that this is experiment is only monitoring one of the 16 pairs of homologous chromosomes, suggesting that each mek1Δ cell likely contains many chromosomes that are not bioriented when they escape the spindle checkpoint arrest. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. Three nonexchange chromosomes signal a similar average duration of the spindle checkpoint delay as 32 non-exchange chromosomes. A) Representative images of a cell in which both homologs of chromosome IV are tagged with a GFP focus at anaphase I. Scale bar = 5 μm. B) Graph of percent mis-segregation of chromosome IV during meiosis I. Asterisk indicates a statistically significant difference from wildtype (Fisher’s Exact test; p = 0.0327). n≥ 100 cells per genotype. C) Graph of mean time from SPB separation to separase biosensor activation. Numbers above bars indicate the number of non-exchange chromosomes in each genotype. Asterisk represents statistically significant difference between wildtype and the indicated genotype (p <0.0001, Mann-Whitney test), and error bars show SD. n≥100 cells per genotype. KT = kinetochore. SLC = short linear chromosome. https://doi.org/10.1371/journal.pgen.1010707.g002

Three non-crossover chromosomes are needed to signal the same duration of a meiotic spindle checkpoint delay as 32 non-crossover chromosomes Previous work in animal cells and fission yeast mitosis have shown that the spindle checkpoint strength depends on the number of unattached kinetochores and the amount of spindle checkpoint components recruited to kinetochores [31,32,79]. Given the similarity in checkpoint duration between spo11-Y135F and mek1Δ cells in meiosis I, we questioned whether a threshold number of non-exchange chromosomes was needed for the full duration of the spindle checkpoint delay in meiosis or if a single chromosome without a partner could cause a full delay. To this end, we engineered budding yeast strains with increasing numbers of non-exchange chromosomes and measured the time from SPB separation to separase activation. First, we introduced one short linear chromosome (SLC) into an otherwise wildtype yeast strain (WT + SLC). This SLC has telomere sequences at each end and is approximately 27kb, containing a marker as well as 256 repeats of the LacO array. SLCs are thought not to form crossovers due to their short size and inability to hold the arm cohesins in place to stabilize the crossover [80–82]. But, the SLC has a centromere on which a kinetochore can build. The WT + SLC strain displayed a metaphase I duration of 75 ± 20 minutes, which is 24 minutes longer than wildtype cells (average ± SD; Fig 2C). We next constructed a strain in which we introduced a single pair of homeologous chromosomes, which are chromosomes from two different yeast species that cannot form crossovers due to their sequence divergence [74,83,84]. The spindle checkpoint helps homeologous chromosomes segregate accurately, but the mechanism that ensures their disjunction is unknown [74,77]. The strain harboring the homeologs displayed a checkpoint delay of 62 minutes compared to wildtype cells (Fig 2C). Next, we created a strain with three non-exchange chromosomes, by adding a SLC to the strain harboring the homeologs (homeologs +1SLC). These cells underwent metaphase I after 176 ± 41 minutes, which is a checkpoint delay of 125 minutes, similar to that of spo11-Y135F cells (Fig 2C). We conclude that there is a graded checkpoint response, and 3 non-exchange chromosomes are sufficient to signal a similar average duration of the spindle checkpoint delay as cells with 32 non-exchange chromosomes.

Mps1 and Ipl1 maintain the spindle checkpoint delay In meiosis, Ipl1Aurora B releases improper attachments and Mps1 helps form force-generating attachments [85]. Previous work has shown that inhibition of Mps1 and Ipl1Aurora B shortened metaphase I in spo11Δ cells, suggesting that both kinases are needed for spindle checkpoint activity [86]. We asked if the kinases are also needed to maintain the meiotic spindle checkpoint delay after it has been initiated in mek1Δ cells that have a reduced number of crossovers. We used the anchor away technique to deplete Ipl1Aurora B or Mps1 from the nucleus [87] (S2A Fig). We tagged endogenous IPL1 or MPS1 at its C-terminus with FRB in a strain with the ribosome protein RPL13A tagged with FKBP12. Because Rpl13A shuttles from the nucleus into the cytosol during ribosome biogenesis, the addition of rapamycin, which allows the stable interaction between FRB and FKBP12, depleted Ipl1-FRB or Mps1-FRB from the nucleus. We added rapamycin at 8 hours after cells were switched to sporulation media, a timepoint when many of the cells were in metaphase I, and then began live-cell imaging. We measured the time of anaphase I onset only in the cells that were in metaphase I at the start of imaging. We found that anchoring away of either Ipl1 or Mps1 in mek1Δ cells shortened metaphase by 42 minutes and 53 minutes, respectively (S2B Fig). Therefore, both Ipl1Aurora B and Mps1 are needed to maintain the spindle checkpoint delay in mek1Δ cells.

Chromosomes undergo error correction attempts throughout the delay We hypothesized that perhaps anaphase onset occurred prior to having all the chromosomes bioriented in mek1Δ cells because either Ipl1Aurora B localization or error correction activity decreased over time in mek1Δ cells. We first measured the level of Ipl1-3GFP that co-localized with the kinetochore protein Mtw1-mRuby2 at anaphase I onset. We found no significant difference in Ipl1-3GFP kinetochore levels in wildtype compared to mek1Δ cells (S3A and S3B Fig). Because Ipl1Aurora B kinetochore-localization did not decline, we tested our next hypothesis that Ipl1Aurora B error correction activity declined during the delay. We monitored the number of error correction attempts of one pair of homologs throughout a mek1Δ delay. To visualize the chromosomes, we added a LacO array approximately 12kb from the centromere of both copies of chromosome IV. In the absence of Ipl1Aurora B in meiosis, improper kinetochore-microtubule attachments are not corrected and both homologs stay attached to the same SPB [85,88]. Therefore, to ask whether Ipl1 activity was attenuated during the meiotic spindle checkpoint delay, we counted the number of error correction attempts during a mek1Δ spindle checkpoint delay by following the detachment and reattachment of a single pair of homologs between SPBs, taking images every 5 minutes. We scored chromosomes as bioriented and non-bioriented and noted when a chromosome traversed between two SPBs. We considered any traversing from one pole to another or switching from non-bioriented to bioriented as an error correction attempt (Fig 3A). In wildtype cells, biorientation occurs very quickly when the spindle is short in prometaphase I, making it difficult to monitor re-orientation [85]. But, biorientation is prolonged in mek1Δ cells with non-exchange chromosomes. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. Chromosomes undergo error correction attempts throughout the spindle checkpoint delay in mek1Δ cells. A) Cartoon showing an example of a mek1Δ cell undergoing four error correction attempts. B) Representative time-lapse images of a mek1Δ cell undergoing two error correction events in the first 50 minutes of the delay. Time 0 is the time at which SPBs separate. Arrowheads show movement of chromosome between SPBs. Scale bar = 5 μm. C) Time-lapse images of a mek1Δ cell undergoing error correction after 50 minutes of the metaphase I delay. Time 0 indicates SPB separation. Scale bar is 5 μm. Arrowheads show error correction event. D) Percent of cells that underwent error correction in the first 50 minutes of metaphase I and after 50 minutes of metaphase I. n≥ 100 cells. E) Graph of individual error correction attempts per cell within the first 50 minutes and after the first 50 minutes of metaphase I. n≥ 100 cells. Error bars show SD. F) Graph showing spindle length, measured 10 minutes prior to anaphase onset. n≥ 50 cells per genotype. Error bars show SD. Asterisk indicates a statistically significant difference between wildtype and mek1Δ (p < 0.0001, Mann-Whitney test). G) Time-lapse images of a cell that does not undergo error correction after 50 minutes of metaphase I. Time 0 is the time at which SPBs separate. Arrowheads show chromosome movement between SPBs. Asterisk indicates chromosomes straying from SPB. Scale bar = 5 μm. https://doi.org/10.1371/journal.pgen.1010707.g003 Because the time of anaphase onset was approximately 50 minutes after SPB separation in wildtype cells, we reasoned that the additional spindle checkpoint delay in mek1Δ cells was beyond 50 minutes. We hypothesized that we may see a dampening of error correction events during the delay beyond 50 minutes. Therefore, we split our analysis into two categories, the number of error correction events in the first 50 minutes of metaphase I and the number of events after cells were delayed for 50 minutes (Fig 3B and 3C). We found that 78% of mek1Δ cells underwent error correction events in the first 50 minutes of the delay, with an average of 2.3 ± 1.7 error correction attempts per cell (average ± standard deviation) (Fig 3D and 3E). After 50 minutes, 47% of mek1Δ cells still underwent error correction attempts, with a total average of 2.1 ± 3 error correction attempts. Unfortunately, the 47% may be an underestimation because there may have been more cells that attempted error correction, but the event did not hold up to our criteria. For example, the mek1Δ cells had a longer spindle length than wildtype cells prior to anaphase I onset, likely due to fewer crossovers that resist the forces to keep the appropriate spindle size (3.3um ± 0.9 μm in wildtype compared to 4.5um ± 1.2 μm in mek1Δ cells; average ± SD; Fig 3F). The increased spindle length may have made it harder for kinetochores to attach to microtubules emanating from the opposite SPB, and instead, the released kinetochore reattached to the same SPB. We noticed that the LacI-GFP focus often drifted from the SPB, suggesting that the chromosome was released from the SPB, but may not have been able to traverse or biorient (Fig 3G). Therefore, we conclude that because we observed error correction throughout the spindle checkpoint delay in mek1Δ cells, premature attenuation of Ipl1 activity is likely not responsible for the short meiotic spindle checkpoint delay.

The spindle checkpoint is primarily silenced in meiosis I despite non-bioriented chromosomes We wondered why the spindle checkpoint delay was shorter in meiosis compared to mitosis. To address this question, we asked how cells escaped the spindle checkpoint arrest. Progression past the spindle checkpoint occurs through various mechanisms, which can be classified into two major categories: checkpoint silencing and mitotic slippage. Checkpoint silencing is how the spindle checkpoint is turned off under normal conditions with the establishment of correct kinetochore-microtubule attachments. Checkpoint proteins are removed from the kinetochore, which ultimately leads to APC/C activation [89]. For example, the normal mechanism of turning off the checkpoint after chromosomes have bioriented is through PP1’s dephosphorylation of the kinetochore protein Spc105KNL1 during mitosis in yeast, worms, and animal cells [3,21,22,90–95]. Dephosphorylation of Spc105KNL1 reduces the binding affinity of Bub3, such that the kinetochore can no longer serve as a scaffold to build the MCC. During checkpoint slippage, cells can escape the spindle checkpoint arrest through various mechanisms despite the persistence of checkpoint proteins at the kinetochore [25,28,30]. We asked if differences in how cells escaped the spindle checkpoint could provide an explanation for why spindle checkpoint delays are shorter in meiosis than in mitosis. To distinguish between accelerated kinetochore silencing or slippage, we monitored spindle checkpoint proteins localized at the kinetochore at anaphase onset. We reasoned that with silencing, cells would disperse checkpoint proteins from the kinetochore prior to anaphase, while with slippage, cells would retain checkpoint proteins at the kinetochore during anaphase. A previous study using a similar assay found that mitotic budding yeast cells retained spindle checkpoint proteins at the kinetochore upon escaping the spindle checkpoint after the addition of microtubule depolymerizing drugs [28]. However, whether the same mechanism is used in cells that signal the checkpoint through another mechanism or during meiosis I is not known. We tagged the spindle checkpoint protein Bub3 with three copies of mCherry (Bub3-3mCherry) in cells containing the separase biosensor (Fig 4A). Using live-cell imaging, we monitored Bub3-3mCherry at the kinetochore and categorized its localization at anaphase onset. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. The spindle checkpoint is inappropriately silenced in meiosis I but not in mitosis. A) Representative time-lapse images show cells entering anaphase, as indicated by the dispersal of separase biosensor focus. Time 0 indicates anaphase onset. Scale bars = 5 μm. Arrowhead shows Bub3-3mcherry focus after anaphase onset. B) Quantification of Bub3-3mCherry localization at anaphase onset in mitosis. n≥50 cells per genotype. C) Representative time-lapse images of a nocodazole-treated cell in meiosis I. Arrowhead shows the absence of Bub3 focus at the kinetochore. Scale bars = 5 μm. D) Quantification of Bub3-3mCherry localization at anaphase onset in meiosis I. n≥50 cells per genotype. E) Representative time-lapse images of a mek1Δ cell entering anaphase I. Time 0 indicates anaphase onset. Arrowhead shows the absence of Bub3 focus at the kinetochore. F-G) Quantification of fluorescence intensity at the kinetochore. At each time point indicated, the fluorescence intensity of the separase biosensor and Bub3-3mcherry were measured and compared to the measurements taken at -15 minutes. Time 0 indicates anaphase onset. Graphs display the percent of cells that, at each time point, retained at least 50% of the fluorescence intensity measured 15 minutes prior to anaphase onset. n≥30 cells per genotype. https://doi.org/10.1371/journal.pgen.1010707.g004 We monitored the localization of Bub3-3mCherry during mitosis, using two different methods of activating the spindle checkpoint, through nocodazole addition, which disrupts kinetochore-microtubule attachments, and through depletion of Cdc6, which prevents replication of sister chromatids. We observed three phenotypes. First, Bub3-3mCherry dispersed from the kinetochore prior to anaphase onset. Second, Bub3-3mCherry dispersed in the same five-minute time interval in which anaphase onset occurred. Third, Bub3-3mCherry stayed localized during anaphase onset, not dispersing until after anaphase onset. When haploid cells were treated with nocodazole in mitosis, 64% retained Bub3-3mCherry at anaphase onset, as scored by separase biosensor cleavage (Fig 4B). These results supported previous findings of mitotic slippage in the presence of nocodazole [28]. Similarly, we observed that 68% of diploid cells depleted for Cdc6 retained Bub3-3mCherry at the kinetochore at anaphase onset, suggesting that most mitotic cells underwent mitotic slippage after prolonged checkpoint activation (Fig 4A and 4B). We performed similar experiments in meiosis, using either nocodazole to disrupt kinetochore-microtubule attachments or deletion of MEK1 to decrease crossovers between homologous chromosomes. Strikingly, with nocodazole treatment, 58% of cells dispersed Bub3-3mCherry from the kinetochore prior to anaphase I onset (Fig 4C and 4D). In mek1Δ cells, Bub3-3mCherry localized to kinetochore foci, but also to the spindle, especially at anaphase onset (Fig 4D and 4E). Similar to the nocodazole-treated cells, the focus of Bub3 at the kinetochore dispersed from the kinetochore prior to anaphase I onset in 54% of mek1Δ cells. Although, we note that there is still a population on the microtubules. To further analyze our results, we measured the fluorescence intensity of both kinetochore-localized Bub3-3mcherry and the separase biosensor foci every five minutes, from 15 minutes prior to anaphase until 10 minutes after anaphase. We plotted the percentage of cells that retained at least half of the fluorescence measured at 15 minutes prior to anaphase. In Cdc6-dp mitotic cells, 87% of cells retained at least half of their localized Bub3 fluorescence at anaphase onset (Fig 4F). In contrast, only 38% of mek1Δ cells retained at least half of their Bub3 fluorescence intensity at anaphase I onset (Fig 4G). These results are consistent with the conclusion that cells in mitosis primarily undergo mitotic slippage, while cells in meiosis I primarily silence the checkpoint at the kinetochore after a prolonged delay. We also monitored Bub3-eGFP kinetochore localization in cells expressing mCherry-Tub1, scoring anaphase I onset as the time at which spindle elongation occurred in wildtype and mek1Δ cells (S4A Fig). Similar to wildtype, most mek1Δ cells dispersed Bub3-eGFP from the kinetochore prior to anaphase I onset (S4B Fig). Although both Cdc6-depletion and loss of MEK1 prevent chromosomes from achieving biorientation in either mitosis or meiosis, respectively, our results suggest that unlike mitotic cells, most meiosis I cells underwent inappropriate spindle checkpoint silencing after a delay. We next wondered if the checkpoint in meiosis II more closely resembled that of meiosis I or mitosis. To disrupt kinetochore tension in meiosis II, we deleted SPO12. Previous experiments showed that spo12Δ cells undergo meiosis I normally but fail to duplicate SPBs in meiosis II, producing two half-spindles or one weak spindle in between the two old SPBs [96–98]. We then monitored the cleavage of the centromeric Rec8-GFP at anaphase II. Similar to previous findings, the spo12Δ cells were delayed in cohesin cleavage, with an average duration from cohesin cleavage in anaphase I to cohesin cleavage in anaphase II of 156 ± 54 minutes (average ± SD), which is 93 minutes longer than that observed in wildtype cells (S5A–S5C Fig) [98]. The delay was dependent on the spindle checkpoint because disruption of MAD3 resulted in a decreased time to anaphase II onset. We conclude that the spindle checkpoint delay is also shorter in meiosis II compared to mitosis. One hypothesis for a faster anaphase II onset in meiosis II compared to mitosis is that precocious APC/C activation occurs through the meiosis-specific co-activator the APC/C, Ama1, which is active at the end of meiosis II [98]. Ama1 is impervious to the spindle checkpoint, which inhibits the APC/C coactivator Cdc20. To test this hypothesis, we deleted AMA1 in spo12Δ cells and measured the duration of anaphase II onset. The spo12Δ ama1Δ cells had a delay with a duration similar to that of spo12Δ cells (S5C Fig). Therefore, the shorter duration of the spindle checkpoint delay in meiosis II compared to mitosis was not dependent on Ama1. To determine if the meiosis II cells underwent mitotic slippage or checkpoint silencing, we monitored Bub3-3mcherry kinetochore localization at anaphase II onset. Surprisingly, we found that 87% of cells retained Bub3-3mcherry at the kinetochore at anaphase II onset, suggesting that they underwent mitotic slippage (S5D and S5E Fig). Similarly, 83% of spo12Δ ama1Δ cells retained Bub3-3mcherry at the kinetochore at anaphase II onset. Therefore, in meiosis II, slippage was not dependent on the activation of APC/CAma1. Overall, we conclude that meiosis I has a mechanism for spindle checkpoint silencing after a prolonged delay, whereas cells in mitosis or meiosis II undergo mitotic slippage.

[END]
---
[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010707

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.