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that microtubule attachment and tension are interdependent. Clearly, an
unattached kinetochore cannot be under tension. Moreover, if a functional
SAC needs all the components at the kinetochore, it is sufficient to remove
one (such as Mad2) to inactivate it.
Photobleaching and recovery (FRAP) experiments of GFP-tagged
kinetochore proteins ( Howell et al., 2004 ) found evidence for two
populations of BubR1 with different dynamics on unattached kinetochores.
Half the bound BubR1 turned over with a T 1/2 residency at kinetochores of
3 s, a very high rate. The remainder turned over at a slightly slower rate with
a T 1/2 of 20 s and similar to rates of measured for Mad2 and Cdc20. Bub1, by
contrast, was more stable ( T 1/2
60 s, with a substantial fraction that did not
recover even after 5 min). Bub3 kinetics were similar to the “slow” popu-
lation of BubR1.
To summarize, it would appear that there are at least two populations of
BubR1 at unattached kinetochores. One is released upon MT attachment
and one released when kinetochores are under tension. It would be inter-
esting to knowwhether (and how) the two pools correspond to the fast turn-
over and slow turnover populations. Perhaps, the different populations of
BubR1 reflect its multiple roles at kinetochores, promoting K-MT attach-
ment (see below) in addition to its function in the SAC ( Elowe et al., 2007;
Huang et al., 2008; Matsumura et al., 2007 ).
By what mechanism is BubR1 removed from kinetochores following
MT attachment? Studies in both vertebrate cells and Drosophila have rev-
ealed that Mad1, Mad2, and the RZZ complex are removed from attached
kinetochores in a Dynein-dependent process and transported along K-fibers
to the spindle poles. This removal (called stripping or shedding) may con-
tribute to the mechanism shutting off the SAC ( Howell et al., 2001; Wojcik
et al., 2001 ). The situation with regard to BubR1 is somewhat less clear. As
mentioned above, BubR1 levels decline from their peak on unattached
kinetochores in two steps ( Howell et al., 2001, 2004 ). The initial rapid
decline upon MT attachment is reportedly blocked if Dynein activity is
perturbed ( Howell et al., 2001 ). But neither in vertebrate cells nor in
Drosophila neuroblasts can BubR1 particles be detected migrating along
K-fibers. Buffin et al. (2005) , studying living Drosophila neuroblasts, where
the shedding of fluorescent Mad2 and RZZ is very robust, saw no evidence
of GFP-BubR1 transport along K-fibers. Therefore, an alternative mecha-
nism may be responsible for the removal of BubR1 from the kinetochores.
Proteolytic degradation is one possibility, at least for kinetochores on the
metaphase plate (see Section 4.5 ).
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