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Vol. 13, Issue 10, 3706-3719, October 2002
and
*SPIRE fellow 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 min, compared with 21 min 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 with 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.
Department of Biology, University of
North Carolina, Chapel Hill, North Carolina 27599-3280
Corresponding author. E-mail address:
ktphd{at}emailunc.edu.
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