Function of a minus-end-directed kinesin-like motor protein in mammalian cells

CHO2 is a mammalian minus-end-directed kinesin-like motor protein present i n interphase centrosomes/nuclei and mitotic spindle fibers/poles. Expressio n of HA- or GFP-tagged subfragments in transfected CHO cells revealed the p resence of the nuclear localization site at the N-terminal tail, This domai n becomes associated with spindle fibers during mitosis, indicating that th e tail is capable of interaction with microtubules in vivo. While the centr al stalk diffusely distributes in the entire cytoplasm of cells, the motor domain co-localizes with microtubules throughout the cell cycle, which is e liminated by mutation of the ATP-binding consensus motif from GKT to AAA. O verexpression of the full-length CHO2 causes mitotic arrest and spindle abn ormality, The effect of protein expression was first seen around the polar region where microtubule tended to be bundled together. A higher level of p rotein expression induces more elongated spindles which eventually become d isorganized by loosing the structural integrity between microtubule bundles . Live cell observation demonstrated that GFP-labeled microtubule bundles u nderwent continuous changes in their relative position to one another throu gh repeated attachment and detachment at one end; this results in the forma tion of irregular number of microtubule focal points in mitotic arrested ce lls. Thus the primary action of CHO2 appears to cross-link microtubules and move toward the minus-end direction to maintain association of the microtu bule end at the pole. In contrast to the full-length of CHO2, overexpressio n of neither truncated nor mutant polypeptides resulted in significant effe cts on mitosis and mitotic spindles, suggesting that the function of CHO2 i n mammalian cells may be redundant with other motor molecules during cell d ivision.