New Insights into the Role of BubR1 in Mitosis and Beyond - International Review of Cell and Molecular Biology - page 232

Taylor et al., 1998 ), but in other cell types only after NEB ( Hoffman et al.,
2001 ). As determined by immunogold labeling and electron microscopy,
BubR1 localized to the outer and inner plates during prometaphase but only
at the outer kinetochore plate during metaphase ( Jablonski et al., 1998 ). Dro-
sophila GFP-BubR1 was reported to similarly redistribute ( Buffin et al., 2005 )
upon attachment to the spindle.
Kinetochore-associated BubR1 declines upon MT attachment. But is it
the simple fact of attachment or must the chromosomes be under bipolar
tension for BubR1 to decline? The literature is not consistent on the ques-
tion. It was initially reported that BubR1 protein is distributed symmetrically
between the two sister kinetochores of mono-oriented chromosomes in
prometaphase ( Jablonski et al., 1998; Taylor et al., 1998 ) when presumably
only one kinetochore is attached, and that kinetochore levels of BubR1 do
not increase upon microtubule depolymerization with nocodazole ( Taylor
et al., 2001 ). This behavior would normally indicate that BubR1 levels did
not change as a function of kinetochore attachment or tension. In contrast,
Mad2 in these conditions rapidly declines uponMT attachment and is nearly
completely gone from bioriented kinetochores ( Hoffman et al., 2001;
Howell et al., 2001 ).
These early studies, however, were performed with antibodies on fixed
cells. Howell et al. (2004) , monitoring GFP-BubR1 in living cells, con-
cluded that BubR1 levels were maximal on unattached kinetochores,
declined fivefold upon attachment, and declined a further fivefold upon
alignment on the metaphase plate. GFP-tagged BubR1 in Drosophila cells
( Buffin et al., 2005 ) behaved in much the same way. These results would
suggest that there exists a pool of kinetochore BubR1 that is sensitive to
MT binding, and a second pool that is sensitive to kinetochore tension.
Skoufias et al. (2001) treated HeLa cells with a low dose of vinblastine,
which suppresses microtubule dynamics, but nevertheless allows chromo-
somes to align on the metaphase plate, with reduced interkinetochore ten-
sion. BubR1 was detected at high levels on these aligned but relaxed
kinetochores while Mad2 was absent. Similarly, in Drosophila S2 cells,
BubR1 localization at kinetochores was reportedly insensitive to microtu-
bule attachment but was dependent on loss of tension ( Logarinho et al.,
2004 ). These observations as well as others led to the idea that the SAC
may have two parallel activating pathways, one being responsive to lack
of K-MT attachment (mediated by Mad2) and the other to lack of kineto-
chore tension (monitored by BubR1) ( Pinsky and Biggins, 2005; Zhou
et al., 2002 ). However, interpretation of the data is complicated by the fact