Abstract
Polo-like kinases (PLKs) are conserved eukaryotic cell cycle regulators, which play multiple roles, particularly during mitosis. The function of Trypanosoma brucei PLK was investigated in procyclic and bloodstream-form parasites. In procyclic trypanosomes, RNA interference (RNAi) of PLK, or overexpression of TY1-epitope-tagged PLK (PLKty), but not overexpression of a kinase-dead variant, resulted in the accumulation of cells that had divided their nucleus but not their kinetoplast (2N1K cells). Analysis of basal bodies and flagella in these cells suggested the defect in kinetoplast division arose because of an inhibition of basal body duplication, which occurred when PLK expression levels were altered. Additionally, a defect in kDNA replication was observed in the 2N1K cells. However, the 2N1K cells obtained by each approach were not equivalent. Following PLK depletion, the single kinetoplast was predominantly located between the two divided nuclei, while in cells overexpressing PLKty, the kinetoplast was mainly found at the posterior end of the cell, suggesting a role for PLK kinase activity in basal body and kinetoplast migration. PLK RNAi in bloodstream trypanosomes also delayed kinetoplast division, and was further observed to inhibit furrow ingression during cytokinesis. Notably, no additional roles were detected for trypanosome PLK in mitosis, setting this protein kinase apart from its counterparts in other eukaryotes.
Highlights
Polo-like kinases (PLKs) are evolutionarily conserved serine/threonine protein kinases, which play multiple essential cell cycle roles, during mitosis
Following PLK depletion, the single kinetoplast was predominantly located between the two divided nuclei, while in cells overexpressing PLKty, the kinetoplast was mainly found at the posterior end of the cell, suggesting a role for PLK kinase activity in basal body and kinetoplast migration
Induction of PLK RNA interference (RNAi) was achieved by the addition of tetracycline to the culture medium, and resulted in a growth defect visible from 48 h post induction (Fig. 1A), which was accompanied by a 30–60% downregulation of PLK mRNA, as demonstrated by Northern blotting (Fig. 1B)
Summary
Polo-like kinases (PLKs) are evolutionarily conserved serine/threonine protein kinases, which play multiple essential cell cycle roles, during mitosis. The founding member of the PLK family, POLO, was identified in Drosophila melanogaster (Sunkel and Glover, 1988) as a protein kinase required for spindle assembly during mitosis. Plk functions in multiple cell cycle events such as centrosome duplication/maturation, spindle assembly, the G2/M phase and metaphase/anaphase transitions, cytokinesis and the DNA damage response (Dai, 2005), and the multitude of Plk substrates reflects this. Vertebrate Plk is found at the centrosomes and kinetochore during late S phase/early G2 phase but relocates to the spindle midzone during the metaphase/anaphase transition and to the midbody during cytokinesis. Cdc (the POLO homologue in Saccharomyces cerevisiae) localizes to the spindle pole bodies in G1 and to the bud neck in late G2 phase, remaining at these sites until late mitosis, and does not translocate to the spindle midzone
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