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A more recent version of this article appeared on October 1, 2002
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Submitted on March 7, 2002
Revised on June 19, 2002
Accepted on July 8, 2002
1 SPIRE fellow, University of North Carolina, Department of Biology, 607 Fordham Hall, CB#3280, Chapel Hill, NC 27599-3280
2 University of North Carolina, Department of Biology, 607 Fordham Hall, CB#3280, Chapel Hill, NC 27599-3280
* Corresponding author. E-mail address: ktphd{at}email.unc.edu.
The spindle checkpoint monitors microtubule attachment and tension at kinetochores to ensure proper chromosome segregation. Previously, PtK1 cells in hypothermic conditions (23°C) were shown to have a pronounced mitotic delay, despite having normal numbers of kinetochore microtubules. At 23°C, we found that PtK1 cells remained in metaphase for an average of 101 minutes, compared to 21 minutes for cells at 37°C. The metaphase delay at 23°C was abrogated by injection of Mad2 inhibitors, showing that Mad2 and the spindle checkpoint were responsible for the prolonged metaphase. Live cell imaging showed that kinetochore Mad2 became undetectable soon after chromosome congression. Measurements of the stretch between sister kinetochores at metaphase found a 24% decrease in tension at 23°C, and metaphase kinetochores at 23°C exhibited higher levels of 3F3/2, Bub1, and BubR1 compared to 37°C. Microinjection of anti-BubR1 antibody abolished the metaphase delay at 23°C, indicating that the higher kinetochore levels of BubR1 may contribute to the delay. Disrupting both Mad2 and BubR1 function induced anaphase with the same timing as single inhibitions, suggesting that these checkpoint genes function in the same pathway. We conclude that reduced tension at kinetochores with a full complement of kinetochore microtubules induces a checkpoint dependent metaphase delay associated with elevated amounts of kinetochore 3F3/2, Bub1, and BubR1 labeling.
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