The N-terminal domain of DDA3 regulates the spindle-association of the microtubule depolymerase Kif2a and controls the mitotic function of DDA3

DDA3 is a microtubule-associated protein that controls chromosome congression and segregation by regulating the dynamics of the mitotic spindle. Depletion of DDA3 alters spindle structure, generates unaligned chromosomes at metaphase, and delays the mitotic progression. DDA3 interacts with the microtubule depolymerase Kif2a and controls the association of Kif2a to the mitotic spindle and the dynamic turnover of microtubules in the spindle. To understand the function and regulation of DDA3, we analyzed its domain structure and found that the C-terminal domain of DDA3 directly binds to microtubules in vitro and associates with the mitotic spindle in vivo. The N-terminal domain of DDA3 does not interact with microtubules, but acts dominant negatively over the wild-type protein. Ectopic expression of this domain prevents the endogenous DDA3 from association with the spindle and results in a high frequency of unaligned chromosomes in metaphase cells, a phenotype similar to that in metaphase cells depleted of DDA3. Mechanistically, expression of N-terminal DDA3 reduces the amount of spindle-associated Kif2a and increases the spindle microtubule density, pheno-copying those in DDA3-depleted cells. We conclude that DDA3 has a distinct domain structure. The C-terminal domain confers its ability to associate with the mitotic spindle, while the regulatory N-terminal domain controls the microtubule-binding by the C-terminal domain and determines the cellular activity of the DDA3 protein.