SUMMARY Proper bipolar attachment of sister kinetochores to the mitotic spindle is critical for accurate chromosome segregation in mitosis. Here we show an essential role of the formin mDia3 in achieving metaphase chromosome alignment. This function is independent of mDia3 actin nucleation activity, but is attributable to EB1-binding by mDia3. Furthermore, the microtubule binding FH2 domain of mDia3 is phosphorylated by Aurora B kinase in vitro, and cells expressing the nonphosphorylatable mDia3 mutant cannot position chromosomes at the metaphase plate. Purified recombinant mDia3 phosphorylated by Aurora B exhibits reduced ability to bind microtubules and stabilize microtubules against cold-induced disassembly in vitro. Cells expressing the phosphomimetic mDia3 mutant do not form stable kinetochore microtubule fibers; despite they are able to congress chromosomes to the metaphase plate. These findings reveal a key role for mDia3 and its regulation by Aurora B phosphorylation in achieving proper stable kinetochore microtubule attachment.

The state of CENP-E–dependent BubR1 autophosphorylation in response to spindle microtubule capture regulates kinetochore function and accurate chromosome segregation.

The accurate segregation of chromosomes in mitosis requires the stable attachment of microtubules to kinetochores. The details of this complex and dynamic process are poorly understood. In this study, we report the interaction of a kinetochore-associated mitotic checkpoint kinase, BubR1, with two microtubule plus end–associated proteins, adenomatous polyposis coli (APC) and EB1, providing a potential link in stable kinetochore microtubule attachment. Using immunodepletion from and antibody addition to Xenopus laevis egg extracts, we show that BubR1 and its kinase activity are essential for positioning chromosomes at the metaphase plate. BubR1 associates with APC and EB1 in egg extracts, and the complex formation is necessary for metaphase chromosome alignment. Using purified components, BubR1 directly phosphorylates APC and forms a ternary complex with APC and microtubules. These findings support a model in which BubR1 kinase may directly regulate APC function involved in stable kinetochore microtubule attachment.

Stable attachment of kinetochores to spindle microtubules is essential for accurate chromosome segregation. We have shown that a kinetochore-associated formin protein, mDia3, contributes to the generation of stable kinetochore-microtubule attachment. The published report reviewed here shows an essential role of mDia3 in achieving metaphase chromosome alignment, and this function is directly regulated by Aurora B phosphorylation. Aurora B is a central component during the capture of spindle microtubules by kinetochores, in which it selectively eliminates incorrect attachments by phosphorylating a group of microtubule binding proteins at kinetochores to reduce their microtubule binding affinity. Here, we discuss the roles of Aurora B kinase and its substrates in achieving proper kinetochore-microtubule attachment.