Metaphase

By Levi Clancy for Student Reader on
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Metaphase checkpoint

How does the metaphase checkpoint prevent sister chromatid separation at the onset of anaphase until every kinetochore has become associated with spindle microtubules? In the spindle assembly checkpoint (aka metaphase checkpoint), mitotic arrest deficient 2 (aka Mad2) blocks metaphase until every single kinetochore has properly attached to spindle microtubules. Mad2 exists in an open conformation (Mad2O) and a closed conformation (Mad2C).

  1. Mad1 and Mad2C form a tetramer that binds unattached kinetochores via the Mad1 subunit.

  2. Mad2C of the kinetochore-bound tetramer can transiently bind Free Mad2O.

  3. This transient interaction causes Mad2O to bind and inactivate Cdc20, and to convert from Mad2O→Mad2C.

  4. Mad2C-Cdc20 transiently interacts with additional free Mad2O.

  5. This causes Mad2O to bind and inactivate additional Cdc20, forming a second Mad2C-Cdc20.

  6. The cycle repeats and free Mad2O is quickly converted to Mad2C-Cdc20.

  7. Binding of microtubules to the kinetochore displaces Mad1-Mad2C tetramers.

  8. Free tetrameric Mad2C cannot bind Mad2O. Instead, free tetrameric Mad2C binds p31comet.

  9. p31 then binds the Mad2C of the Mad2C-Cdc20 complexes, resulting in release of active Cdc20.

Just a few Mad1-Mad2C tetramers bound to kinetochores can generate enough Mad2C-Cdc20 to overcome p31 activity. Once all kinetochores have attached to microtubules (thus releasing all Mad1-Mad2C tetramers), p31 activity predominates and active Cdc20 is released from Mad2C. The active Cdc20 binds APC/C, and the APC/C-Cdc20 degrades securin, the inhibitor of separase. Free separase digests the Kleisin subunit of cohesin, breaking open the Smc1-Smc3-Kleisin ring and allowing sister chromatids to separate. Cohesin is a Smc1/Smc3/Kleisin heterotrimer that holds together sister chromatids, with Kleisin acting like a clasp.