Tethering Sister Centromeres to Each Other Suggests the Spindle Checkpoint Detects Stretch within the Kinetochore

The spindle checkpoint ensures that newly born cells receive one copy of each chromosome by preventing chromosomes from segregating until they are all correctly attached to the spindle. The checkpoint monitors tension to distinguish between correctly aligned chromosomes and those with both sisters attached to the same spindle pole. Tension arises when sister kinetochores attach to and are pulled toward opposite poles, stretching the chromatin around centromeres and elongating kinetochores. We distinguished between two hypotheses for where the checkpoint monitors tension: between the kinetochores, by detecting alterations in the distance between them, or by responding to changes in the structure of the kinetochore itself. To distinguish these models, we inhibited chromatin stretch by tethering sister chromatids together by binding a tetrameric form of the Lac repressor to arrays of the Lac operator located on either side of a centromere. Inhibiting chromatin stretch did not activate the spindle checkpoint; these cells entered anaphase at the same time as control cells that express a dimeric version of the Lac repressor, which cannot cross link chromatids, and cells whose checkpoint has been inactivated. There is no dominant checkpoint inhibition when sister kinetochores are held together: cells expressing the tetrameric Lac repressor still arrest in response to microtubule-depolymerizing drugs. Tethering chromatids together does not disrupt kinetochore function; chromosomes are successfully segregated to opposite poles of the spindle. Our results indicate that the spindle checkpoint does not monitor inter-kinetochore separation, thus supporting the hypothesis that tension is measured within the kinetochore

Chromosomal attachments set length and microtubule number in the Saccharomyces cerevisiae mitotic spindle

The length of the mitotic spindle varies among different cell types. A simple model for spindle length regulation requires balancing two forces: pulling, due to micro­tubules that attach to the chromosomes at their kinetochores, and pushing, due to interactions between microtubules that emanate from opposite spindle poles. In the budding yeast Saccharomyces cerevisiae, we show that spindle length scales with kinetochore number, increasing when kinetochores are inactivated and shortening on addition of synthetic or natural kinetochores, showing that kinetochore–microtubule interactions generate an inward force to balance forces that elongate the spindle. Electron microscopy shows that manipulating kinetochore number alters the number of spindle microtubules: adding extra kinetochores increases the number of spindle microtubules, suggesting kinetochore-based regulation of microtubule number

Inhibition of chromatin stretch does not delay mitotic progression.

<p>(<b>A</b>) To assay the progression of cells through mitosis, asynchronous populations were treated with alpha factor to arrest cells in G1. Cells were released from G1 and allowed to proceed synchronously through mitosis and into the next cell cycle. Samples were collected every 10 minutes and scored for mitotic progression (<b>B</b>) Cartoon of how the spindle checkpoint can arrest cells in metaphase, delaying anaphase. Anaphase (purple box) is scored in mitotic progression assays by the separation of GFP-labeled chromatids (green dots) into mother and daughter cells. (<b>C</b>) Cells expressing the tetrameric Lac repressor do not delay mitosis compared to control cells expressing the dimeric Lac repressor. Both strains peak in anaphase 60–70 minutes after release from G1 (no statistical difference between populations). The essential spindle checkpoint component Mad2 was deleted in cells expressing the dimeric or tetrameric Lac repressor, and all four strains move through mitosis on the same time scale. There was no statistical significance between any of the four strains at any one of the time points, suggesting that neither form of the Lac repressor delayed mitosis due to checkpoint activation. More than 100 cells were scored for anaphase for each strain in each experiment; error bars represent the standard deviation of at least 3 independent trials.</p

Strains used in this study.

<p>All strains are derivatives of <i>Saccharomyces cerevisiae</i> W303 with the following auxotrophic genotypes: <i>ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1</i>.</p

Cells with tethered centromeres can activate the spindle checkpoint.

<p>(<b>A</b>) To assay spindle checkpoint activation of cells in the presence of microtubule-depolymerizing drugs, cells were synchronized in G1 with alpha factor, and released into media containing microtubule-depolymerizing drugs (benomyl and nocodazole). Samples were collected every 60 minutes and scored for the large-budded phenotype indicative of checkpoint activation. (<b>B</b>) The ability of both control (dimeric) and tethered (tetrameric) cells to activate the spindle checkpoint was assayed by scoring the percent of cells arrested as large-budded cells, and compared to cells that do not possess a functional checkpoint and cannot arrest (<i>mad2Δ</i>). Cells expressing either the dimeric or tetrameric Lac repressor arrested in the drugs, indicating that the spindle checkpoint was functional in both strains. 200 cells scored for large-budded phenotype; error bars represent the standard deviation of 3 independent trials. (<b>C</b>) To assay the ability of cells to recover from spindle checkpoint activation induced by microtubule-depolymerizing drugs, cells were synchronized in G1 with alpha factor, released into media containing benomyl and nocodazole for 90 minutes then washed and transferred to media without drugs. Samples were collected every 10 minutes and scored for anaphase (GFP dots in both mother and daughter cells) (<b>D</b>) The ability of both control and tethered strains to recover from spindle checkpoint activation was assayed by transiently treating cells with microtubule-depolymerizing drugs. Both strains arrested in the presence of the drugs and did not enter anaphase until after the drugs were washed out at T = 90 minutes (red arrow). 100 cells were scored for anaphase for each strain in each experiment; error bars represent the standard deviation of at least 3 independent trials.</p

Tetrameric Lac repressor inhibits sister chromatid stretching.

<p>(<b>A</b>) To measure the stretching of chromatids, asynchronous populations were treated with alpha factor to arrest cells in G1. Cells were released from G1 into a metaphase arrest generated by depletion of Cdc20, an essential co-activator of the Anaphase Promoting Complex; samples were taken every 30 minutes and scored for separated chromatids (n>100). (<b>B</b>) Both versions of the repressor are fused to GFP to visualize sister chromatids. Separated chromatids appear as two GFP dots, and one GFP dot is categorized as no separation. Scale bar is 3 µm. (<b>C</b>) Inter-kinetochore separation is not inhibited if a LacO array is placed on only one side of the centromere. Both control, dimeric repressor (GFP-LacI₂) cells and tetrameric repressor (GFP-LacI₄) cells reach maximum percent separation 60 minutes after release from G1; there is no statistical difference between dimer and tetramer cells at all time points. (<b>D</b>) Centromere separation is inhibited if the tetrameric form of the Lac repressor (GFP-LacI₄) is expressed and LacO arrays are placed on both sides of the centromere. Control cells expressing the dimeric repressor (GFP-LacI₂) reach maximum stretching 60 minutes post-release from G1; the tetrameric strain has fewer cells with visibly stretched chromatids at all time points in metaphase arrest (<i>p</i><0.005, Student's <i>t</i>-test). More than 100 cells were scored for GFP dots for each strain in each experiment; error bars represent standard deviation across 3 independent trials.</p

Holding sister centromeres close together does not disrupt chromosome segregation.

<p>(<b>A</b>) To determine if the tetrameric Lac repressor disrupts kinetochore assembly or error correction mechanisms, cells were grown in YP +2% raffinose at the permissive temperature and synchronized in G1 with alpha factor. Cells were washed and released into an anaphase arrest at the restrictive temperature in either YP +2% glucose or +2% galactose; anaphase arrest was induced by the <i>cdc15-2</i> temperature sensitive allele. The segregation of GFP-labeled chromatids was assessed three hours after release from G1. (<b>B</b>) DAPI staining was used to confirm anaphase arrest in <i>cdc15-2</i> cells grown at the restrictive temperature. Anaphase cells contain a DNA mass in both the mother and daughter cell; 99±1.5% of scored cells had a DAPI staining pattern corresponding to anaphase. (<b>C</b>) Correctly segregated chromosomes were scored as cells in which both the mother and daughter cell received a copy of the chromosome (one GFP dot in each cell). Chromosome mis-segregation was scored as cells in which both copies of the chromosome were located in one cell. (<b>D</b>) A conditional centromere was used as a positive control for chromosome mis-segregation. When grown in glucose, the <i>GAL1</i> promoter placed upstream of <i>CEN3</i> is turned off and the centromere is functional; however, when grown in galactose, the <i>GAL1</i> promoter is turned on and the centromere is disrupted by the transcriptional machinery. (<b>E</b>) Cells expressing the tetrameric repressor do not have increased rates of chromosome mis-segregation compared to those expressing the dimeric repressor or cells with their conditional centromere turned on. However, there was a large and statistically significant difference between these three strains and cells with their conditional centromeres turned off, which were as likely to mis-segregate sister chromatids as they were to segregate them properly. 200 cells scored for correct chromosome segregation for each strain in each experiment; error bars are the standard deviation of 3 independent trials.</p

The maize Divergent spindle-1 (dv1) gene encodes a kinesin-14A motor protein required for meiotic spindle pole organization

The classic maize mutant divergent spindle-1 (dv1) causes failures in meiotic spindle assembly and a decrease in pollen viability. By analyzing two independent dv1 alleles we demonstrate that this phenotype is caused by mutations in a member of the kinesin-14A subfamily, a class of C-terminal, minus-end directed microtubule motors. Further analysis demonstrates that defects in early spindle assembly are rare, but that later stages of spindle organization promoting the formation of finely focused spindle poles are strongly dependent on Dv1. Anaphase is error-prone in dv1 lines but not severely so, and the majority of cells show normal chromosome segregation. Live-cell imaging of wild type and mutant plants carrying CFP-tagged β-tubulin confirm that meiosis in dv1 lines fails primarily at the pole-sharpening phase of spindle assembly. These data indicate that plant kinesin-14A proteins help to enforce bipolarity by focusing spindle poles and that this stage of spindle assembly is not required for transition through the spindle checkpoint but improves the accuracy of chromosome segregation

Frequent Spindle Assembly Errors Require Structural Rearrangement to Complete Meiosis in Zea mays

The success of an organism is contingent upon its ability to faithfully pass on its genetic material. In the meiosis of many species, the process of chromosome segregation requires that bipolar spindles be formed without the aid of dedicated microtubule organizing centers, such as centrosomes. Here, we describe detailed analyses of acentrosomal spindle assembly and disassembly in time-lapse images, from live meiotic cells of Zea mays. Microtubules organized on the nuclear envelope with a perinuclear ring structure until nuclear envelope breakdown, at which point microtubules began bundling into a bipolar form. However, the process and timing of spindle assembly was highly variable, with frequent assembly errors in both meiosis I and II. Approximately 61% of cells formed incorrect spindle morphologies, with the most prevalent being tripolar spindles. The erroneous spindles were actively rearranged to bipolar through a coalescence of poles before proceeding to anaphase. Spindle disassembly occurred as a two-state process with a slow depolymerization, followed by a quick collapse. The results demonstrate that maize meiosis I and II spindle assembly is remarkably fluid in the early assembly stages, but otherwise proceeds through a predictable series of events