GFP tagging reveals human Polo-like kinase 1 at the kinetochore/centromereregion of mitotic chromosomes

Polo-like kinases (Plks) have been implicated in various aspects of M-phase progression in organisms ranging from yeast to man. In vertebrates, Plks p articipate in centrosome maturation and spindle assembly, as well as the ac tivation of the Cdk1/cyclin B complex. Moreover, Plks are required for the destruction of mitotic cyclins, indicating that they play an important role in the regulation of the ubiquitin-dependent proteolytic degradation machi nery that controls exit from M-phase. Here, we have fused Green Fluorescent Protein (GFP) to the N-terminus of human Plk1, and expressed this chimeric construct in human cells. We found that GFP-Plk1 associates with centrosom es, the equatorial spindle midzone and the postmitotic bridge of dividing c ells, confirming and extending previous results obtained with conventional immunofluorescence microscopy. In addition, however, we observed fluorescen ce emanating from the midbody between dividing cells, and from discrete dot s associated with mitotic chromosomes. This latter staining pattern being r eminiscent of centromeres, we performed double-labeling experiments with an tibodies against the centromeric marker CENP-B, and reexamined the subcellu lar localization of endogenous Plk1 using different fixation procedures. Ou r data clearly show that both GFP-tagged Plk1 and endogenous Plk1 associate with the kinetochore/centromere region of human mitotic chromosomes. This novel localization of Plk1 suggests that substrates and/or regulators of Pl ks may be found among kinetochore-associated proteins with important functi ons in chromosome segregation and/or spindle checkpoint mechanisms.