Mad2 and BubR1 Function in a Single Checkpoint Pathway that Responds to a Loss of Tension

Abstract

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.

Partial downregulation of MAD1 causes spindle checkpoint inactivation and aneuploidy, but does not confer resistance towards taxol

“…Normal levels of Mad2 but not of Mad1 are required for normal mitotic timing Previous studies have indicated that the spindle checkpoint regulates the timing of a normal mitosis in the absence of damage (Taylor and McKeon, 1997;Gorbsky et al, 1998;Shannon et al, 2002;Meraldi et al, 2004). Moreover, a kinetochore independent function of Mad2 and BubR1, but not of other Mad or Bub proteins, appears to be required for the mitotic timer function (Meraldi et al, 2004).…”

The spindle checkpoint, aneuploidy, and cancer

“…Multiple attempts have been made in different organisms to uncouple microtubule attachment at the kinetochores from the subsequent development of tension across the kinetochores. Treatment of mammalian cells with microtubule toxins, such as taxol, vinblastine or noscapine at low concentrations or, in the case of PtK cells, exposure to hypothermia, alters microtubule dynamics in such a way that kinetochore microtubule attachment remains largely unaffected, but the tension across the paired kinetochores is absent (Waters et al, 1998;Hoffman et al, 2001;Skoufias et al, 2001;Shannon et al, 2002;Zhou et al, 2002). These cells show a prolonged mitotic arrest under these conditions, suggesting that microtubule attachment per se might not be sufficient to silence the checkpoint.…”

Detection and Correction of Merotelic Kinetochore Orientation by Aurora B and its Partners

“…[13][14][15] Syntelic orientation is observed rarely in untreated mitotic cells 16 and it is still a matter of debate if this mis-attachment can activate the spindle checkpoint in mitotic cells. However, in syntelically-oriented chromosomes the centromere is under reduced tension and tension is thought to play an important role in spindle checkpoint activation both in meiotic [17][18][19][20] and mitotic [21][22][23] cells. In addition, evidence from budding yeast would suggest that syntelic attachments can be detected by the mitotic spindle checkpoint.…”