ANTIBODY MICROINJECTION REVEALS AN ESSENTIAL ROLE FOR HUMAN POLO-LIKEKINASE-1 (PLK1) IN THE FUNCTIONAL MATURATION OF MITOTIC CENTROSOMES

Mammalian polo-like kinase 1 (Plk1) is structurally related to the pol o gene product of Drosophila melanogaster, Cdc5p of Saccharomyces cere visiae, and plo1(+) of Schizosaccharomyces pombe, a newly emerging fam ily of serine-threonine kinases implicated in cell cycle regulation, B ased on data obtained for its putative homologues in invertebrates and yeasts, human Plk1 is suspected to regulate some fundamental aspect(s ) of mitosis, but no direct experimental evidence in support of this h ypothesis has previously been reported. In this study, we have used a cell duplication, microinjection assay to investigate the in vivo func tion of Plk1 in both immortalized (HeLa) and nonimmortalized (Hs68) hu man cells. Injection of anti-Plk1 antibodies (Plk1(+)) at various stag es of the cell cycle had no effect on the kinetics of DNA replication but severely impaired the ability of cells to divide. Analysis of Plk1 (+)-injected, mitotically arrested HeLa cells by fluorescence microsco py revealed abnormal distributions of condensed chromatin and monoastr al microtubule arrays that were nucleated from duplicated but unsepara ted centrosomes. Most strikingly, centrosomes in Plk1(+)-injected cell s were drastically reduced in size, and the accumulation of both gamma -tubulin and MPM-2 immunoreactivity was impaired. These data indicate that Plk1 activity is necessary for the functional maturation of centr osomes in late G2/early prophase and, consequently, for the establishm ent of a bipolar spindle, Additional roles for Plk1 at later stages of mitosis are not excluded, although injection of Plk1(+) after the com pletion of spindle formation did not interfere with cytokinesis. Injec tion of Plk1(+) into nonimmortalized Hs68 cells produced qualitatively similar phenotypes, but the vast majority of the injected Hs68 cells arrested as single, mononucleated cells in G2, This latter observation hints at the existence, in nonimmortalized cells, of a centrosome-mat uration checkpoint sensitive to the impairment of Plk1 function.