Purification and characterization of microtubule motor proteins from mitotic HeLa cell

Microtubule (MT) motors play a critical role in mitosis of virtually all cell types. A system was developed for the purification and biochemical characterization of native, full length NIT motor proteins from mitotic HeLa cells to further understand their roles in cell division. Traditional kinesin was purified from mitotic HeLa cells and found to exist in two distinct forms. A 7 S kinesin complex comprised only of heavy chains was isolated and demonstrated to have robust MT gliding activity. In addition, a 9.5 S kinesin complex comprised of both heavy and light chains was identified and isolated, and this form lacked detectable MT gliding activity. These data suggest that the MT gliding activity of kinesin is negatively regulated by association with light chains in vivo. This is the first demonstration of a native kinesin complex lacking activity as a result of association with light chains.;The purification scheme for kinesin was expanded to purify additional kinesin-related proteins (KRPs) from mitotic HeLa cells. Centromere binding protein E(CENP-E), a KRP that localizes to the kinetochores of mitotic chromosomes, was purified and found to exhibit MT binding, but no MT gliding activity. This suggests that CENP-E's role in mitosis may be to tether chromosomes to polymerizing and depolymerizing MTs rather than to actively move along kinetochore MTs. In addition, the mitotic motor HSET, which is the human homolog of the C-terminal motor ncd, was purified and its motility characteristics were investigated. Finally, partial purification schemes were developed for the mitotic KRPs MKLP1 and hKLP2, which will aid in their future biochemical characterization.