Crm1 binds to its export cargo in complexes that also contain Ran-GTP. Blocking the formation of these Crm1-cargo-Ran-GTP complexes, either by preventing cargo binding or by starving cells of Ran-GTP, resulted in no Ran-GAP1/Ran-BP2 kinetochore recruitment, which was bad news for dividing cells. Crm1-blocked cells rarely established mature kinetochore fiber attachments between kinetochores and spindle poles. The microtubules within kinetochore fibers failed to end discretely and attach to kinetochores at their plus ends. Instead, microtubule bundles that extended far past where they should end lay along centromeric DNA.
Subjecting the Crm1-blocked cells to cold treatment—a common method used to depolymerize microtubules that are not assembled into mature kinetochore fibers—broke down the majority of microtubule attachments to kinetochores, indicating that kinetochore fiber stability had been compromised. These compromised cells still held tension on their kinetochores, however, so force can be exerted on the kinetochores even when the fibers are not properly assembled or stably attached.
How Crm1 binds to kinetochores during mitosis and the function of Ran-BP2 and Ran-GAP1 in mature kinetochore fiber attachment remain to be determined. “Ran-GAP1 and Ran-BP2 can be detected on kinetochores about the time that the kinetochores make their first microtubule attachments,” says Dasso. “We are intrigued by the idea that Ran-GAP1/Ran-BP2 may be important for converting those initial attachments into mature kinetochore fibers. We now want to understand how these fibers are built, and whether Ran-BP2 and Ran-GAP1 [function] in this process.”