Generating the Spindle Assembly Checkpoint Signal at the Kinetochore

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Title: Generating the Spindle Assembly Checkpoint Signal at the Kinetochore
Author: Bharadwaj, Rajnish
Abstract: 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 .
URI: http : / /hdl .handle .net /2152 .5 /268
Date: 2004-08-19


Generating the Spindle Assembly Checkpoint Signal at the Kinetochore. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /268 .

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