Direct role of dynein motor in stable kinetochore-microtubule attachment, orientation, and alignment

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Abstract
Cytoplasmic dynein has been implicated in diverse mitotic functions, several involving its association with kinetochores. Much of the supporting evidence comes from inhibition of dynein regulatory factors. To obtain direct insight into kinetochore dynein function, we expressed a series of dynein tail fragments, which we find displace motor-containing dynein heavy chain (HC) from kinetochores without affecting other subunits, regulatory factors, or microtubule binding proteins. Cells with bipolar mitotic spindles progress to late prometaphase-metaphase at normal rates. However, the dynein tail, dynactin, Mad1, and BubR1 persist at the aligned kinetochores, which is consistent with a role for dynein in self-removal and spindle assembly checkpoint inactivation. Kinetochore pairs also show evidence of misorientation relative to the spindle equator and abnormal oscillatory behavior. Further, kinetochore microtubule bundles are severely destabilized at reduced temperatures. Dynein HC RNAi and injection of anti-dynein antibody in MG132-arrested metaphase cells produced similar effects. These results identify a novel function for the dynein motor in stable microtubule attachment and maintenance of kinetochore orientation during metaphase chromosome alignment.