Metaphase

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 (Mad2^O) and a closed conformation (Mad2^C).

1. Mad1 and Mad2^C form a tetramer that binds unattached kinetochores via the Mad1 subunit.
2. Mad2^C of the kinetochore-bound tetramer can transiently bind Free Mad2^O.
3. This transient interaction causes Mad2^O to bind and inactivate Cdc20, and to convert from Mad2^O→Mad2^C.
4. Mad2^C-Cdc20 transiently interacts with additional free Mad2^O.
5. This causes Mad2^O to bind and inactivate additional Cdc20, forming a second Mad2^C-Cdc20.
6. The cycle repeats and free Mad2^O is quickly converted to Mad2^C-Cdc20.
7. Binding of microtubules to the kinetochore displaces Mad1-Mad2^C tetramers.
8. Free tetrameric Mad2^C cannot bind Mad2^O. Instead, free tetrameric Mad2^C binds p31^comet.
9. p31 then binds the Mad2^C of the Mad2^C-Cdc20 complexes, resulting in release of active Cdc20.

Just a few Mad1-Mad2^C tetramers bound to kinetochores can generate enough Mad2^C-Cdc20 to overcome p31 activity. Once all kinetochores have attached to microtubules (thus releasing all Mad1-Mad2^C tetramers), p31 activity predominates and active Cdc20 is released from Mad2^C. 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.