Abstract
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.
Highlights
The spindle checkpoint delays the onset of chromosome segregation until all chromosomes have attached their two sister kinetochores to microtubules emanating from opposite poles [5,6]; it is activated by unattached kinetochores [5,7] and lack of tension at the kinetochore [8,9]
We inhibited the ability of pericentric chromatin to stretch by tethering sister centromeres to each other, and we asked whether the resulting reduction in interkinetochore separation artificially activated the spindle checkpoint
There was no statistical difference between cells grown in glucose, cells with GFP-LacI2, and cells with GFP-LacI4, but all were significantly different from cells grown in galactose (p#0.003, Student’s t-test). These results indicate that the presence of tetrameric Lac repressor does not disrupt kinetochore assembly or interfere with the correction of erroneous attachments, suggesting that the reduction in the fraction of metaphase-arrested cells with 2 GFP dots (Figure 2D) represents chromosomes that are correctly attached to opposite poles but cannot stretch apart due to the tethering effect of the tetrameric Lac repressor
Summary
To ensure proper division of chromosomes, eukaryotes have evolved the spindle checkpoint, which monitors the kinetochore, a large multi-protein complex that assembles on centromeric DNA and attaches microtubules to chromosomes. In Saccharomyces cerevisiae, the budding yeast, the kinetochore consists of over 65 proteins that are assembled on the conserved 125 bp centromere [4]. The spindle checkpoint delays the onset of chromosome segregation until all chromosomes have attached their two sister kinetochores to microtubules emanating from opposite poles (bi-orientation) [5,6]; it is activated by unattached kinetochores [5,7] and lack of tension at the kinetochore [8,9]. The checkpoint regulates the transition between metaphase, when the pairs of sister chromatids are aligned equidistant from the two poles, and anaphase, when the sisters split apart and are pulled to opposite poles
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
