Generating the spindle assembly checkpoint signal at the kinetochore

by Bharadwaj, Rajnish.

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 vi 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. vii