MAB3610 Sigma-AldrichAnti-Bub1 Antibody, clone 14H5

Importin-β is the main vector for interphase nuclear protein import and plays roles after nuclear envelope breakdown. Here we show that importin-β regulates multiple aspects of mitosis via distinct domains that interact with different classes of proteins in human cells. The C-terminal region (which binds importin-α) inhibits mitotic spindle pole formation. The central region (harboring nucleoporin-binding sites) regulates microtubule dynamic functions and interaction with kinetochores. Importin-β interacts through this region with NUP358/RANBP2, which in turn binds SUMO-conjugated RANGAP1 in nuclear pores. We show that this interaction continues after nuclear pore disassembly. Overexpression of importin-β, or of the nucleoporin-binding region, inhibited RANGAP1 recruitment to mitotic kinetochores, an event that is known to require microtubule attachment and the exportin CRM1. Co-expressing either importin-β-interacting RANBP2 fragments, or CRM1, restored RANGAP1 to kinetochores and rescued importin-β-dependent mitotic dynamic defects. These results reveal previously unrecognized importin-β functions at kinetochores exerted via RANBP2 and opposed by CRM1.

The spindle checkpoint that monitors kinetochore-microtubule attachment has been implicated in tumorigenesis; however, the relation between the spindle checkpoint and cell death remains obscure. In BUB1-deficient (but not MAD2-deficient) cells, conditions that activate the spindle checkpoint (i.e., cold shock or treatment with nocodazole, paclitaxel, or 17-AAG) induced DNA fragmentation during early mitosis. This mitotic cell death was independent of caspase activation; therefore, we named it caspase-independent mitotic death (CIMD). CIMD depends on p73, a homologue of p53, but not on p53. CIMD also depends on apoptosis-inducing factor and endonuclease G, which are effectors of caspase-independent cell death. Treatment with nocodazole, paclitaxel, or 17-AAG induced CIMD in cell lines derived from colon tumors with chromosome instability, but not in cells from colon tumors with microsatellite instability. This result was due to low BUB1 expression in the former cell lines. When BUB1 is completely depleted, aneuploidy rather than CIMD occurs. These results suggest that cells prone to substantial chromosome missegregation might be eliminated via CIMD.