Generating the spindle assembly checkpoint signal at the kinetochore
To avoid missegregation of chromosomes during mitosis cells employ a surveillance
mechanism termed Spindle assembly checkpoint that senses the lack of
tension/attachment on the kinetochores and consequently blocks anaphase onset by
inhibiting an E3 ubiquitin ligase called anaphase-promoting complex. The roles of two
kinases- BubR1 and Mps1, implicated in spindle assembly checkpoint were investigated.
A checkpoint complex containing BubR1 and Bub3 has been purified from mitotic
human cells. BubR1 directly interacts with Cdc20 and inhibits the activity of APC in
vitro,much more efficiently than Mad2. Surprisingly, the kinase activity of BubR1 or
association with Bub3 is not required for the inhibition of APCCdc20. Furthermore, BubR1
restores the mitotic arrest in Cdc20-overexpressing cells treated with nocodazole. Mps1
is a dual specificity kinase that localizes to kinetochores in mitosis. Depletion of Mps1
by RNAi leads to the abrogation of spindle assembly checkpoint.
The kinetochore proteins involved in the recruitment of checkpoint proteins and the
generation of wait-anaphase signal have not been identified. Kinetochores also provide
the attachment sites for spindle microtubules and are required for the alignment of
chromosomes at the metaphase plate (chromosome congression). Components of the
conserved Ndc80 complex have been implicated in both these function. To better
understand the function of the Ndc80 complex, we have identified two novel subunits of
the human Ndc80 complex, termed human Spc25 (hSpc25) and human Spc24 (hSpc24),
using an immuno-affinity approach. Human Spc25 interacts with Hec1 (human Ndc80)
throughout the cell cycle and localizes to kinetochores during mitosis. RNAi-mediated
depletion of hSpc25 in HeLa cells causes aberrant mitosis followed by cell death, a
phenotype similar to that of cells depleted for Hec1. Loss of hSpc25 also causes multiple
spindle aberrations, including elongated, multipolar, and fractured spindles. In the
absence of hSpc25, Mad1 and Hec1 fail to localize to kinetochores during mitosis
whereas the kinetochore localization of Bub1 and BubR1 is largely unaffected.
Interestingly, the kinetochore localization of Mad1 in cells with a compromised Ndc80
function is restored upon microtubule depolymerization. Thus, hSpc25 is an essential
kinetochore component that plays a significant role in proper execution of mitotic events.