Function of the kinesin-like motor protein CHO1 in cytokinesis of mammalian cells.

摘要

Cell division is largely dependent on the mechanical forces generated by motor proteins. CHO1 is a mammalian, plus-end directed kinesin-like motor protein of the MKLP1 subfamily. During mitosis, CHO1 associates with the spindle midzone and midbody matrix and therefore was implicated in spindle elongation during anaphase B. To analyze the function of the protein, we mutated the ATP-binding site of CHO1 and expressed it in mammalian cells. Mutant protein failed to accumulate at the midline of central spindles but had no effect on the segregation of chromosomes during anaphase B or the initiation of cytokinesis. Nevertheless, the completion of cytokinesis was severely affected. Immunofluorescence and electron microscopy indicated that the inhibition of cytokinesis could be due to the disorganization of the midbody matrix in the intercellular bridge. Depletion of endogenous CHO1 by RNAi also affected the formation of the midbody matrix, caused the disorganization of midzone microtubules, and resulted in ∼65% binucleation. Thus, CHO1 may not be required for karyokinesis, but it is essential for the proper midzone/midbody formation and cytokinesis in mammalian cells.; To extend the analysis of CHO1, a genetic rescue assay was developed in which the exogenous CHO1 restored the formation of the midbody matrix and rescued the cytokinesis in endogenous CHO1-depleted cells. Various deletion/mutation constructs of CHO1 were tested for their ability to rescue cytokinesis. In this study, both motor and stalk domains were shown to be required for the formation of the midzone/midbody region, while the tail domain was essential for the completion of cytokinesis after the midbody has formed. A region of 55 amino acids containing two nuclear localization signals and residing at the C terminus of the tail domain was found to be required for stabilization of the midbody matrix, since the deletion of this region caused a complete disintegration of the midbody matrix after its formation, leading to the fusion of daughter cells. Nuclear localization of CHO1 was also found to be important for the successful completion of cytokinesis. The data demonstrate that specific subdomains of CHO1 play different roles in cytokinesis to ensure the faithful and complete separation of daughter cells.