Analysis of Bub3 spindle checkpoint function inXenopusegg extracts

The spindle checkpoint delays the onset of anaphase if there are any defects in the interactions between spindle microtubules and kinetochores. This checkpoint has been reconstituted in vitro in Xenopus egg extracts, and here we use antibodies to Xenopus Bub3 (XBub3) to show that this protein is required for both the activation and the maintenance of a spindle checkpoint arrest in egg extracts. We detect two forms of XBub3 in egg extracts and find both to be complexed with the XBub1 and XBubR1 kinases. Only one form of XBub3 is apparent in Xenopus tissue culture (XTC) cells, and localisation studies reveal that, unlike the Mad proteins, which are concentrated at the nuclear periphery, XBub3 is diffusely localised throughout the nucleus during interphase. During early prophase it is recruited to kinetochores, where it remains until chromosomes align at the metaphase plate. We discuss the mechanism by which our alpha-XBub3 antibodies interfere with the checkpoint and possible roles for XBub3 in the spindle checkpoint pathway.