Dissociation of Cytokinesis Initiation from Mitotic Control in a Eukaryote

Abstract

ABSTRACT Cytokinesis is initiated only after mitotic exit in eukaryotes. However, in the insect (procyclic) form of an ancient protist, Trypanosoma brucei , a blockade at the G 2 /M checkpoint results in an enrichment of anucleate cells (zoids), suggesting separated regulations between mitosis and cytokinesis (X. Tu and C. C. Wang, J. Biol. Chem. 279: 20519-20528, 2004). Polo-like kinases (Plks) are known to play critical roles in controlling both mitosis and cytokinesis. A single Plk homologue in T. brucei , Tb PLK , was found to be capable of complementing the Plk (Cdc5) functions in Saccharomyces cerevisiae , thus raising the question of how it may function in the trypanosome with cytokinesis dissociated from mitosis. Depletion of TbPLK in the procyclic form of T. brucei by RNA interference resulted in growth arrest with accumulation of multiple nuclei, kinetoplasts, basal bodies, and flagella in approximately equal numbers among individual cells. There were, however, few zoids detectable, indicating inhibited cytokinesis with unblocked mitosis and kinetoplast segregation. TbPLK is thus apparently involved only in initiating cytokinesis in T. brucei . Overexpression of TbPLK in the trypanosome did not affect cell growth, but 13% of the resulting population was in the zoid form, suggesting runaway cytokinesis. An immunofluorescence assay indicated that TbPLK was localized in a chain of likely flagellum attachment zones in the cytoskeleton. In a dividing cell, a new line of such zones appeared closely paralleling the existing one, which could constitute the cleavage furrow. An exposed region of TbPLK at the anterior tip of the cell may provide the trigger of cytokinesis. Taken together, our results revealed a novel mechanism of cytokinesis initiation in the trypanosome that may serve as a useful model for further in-depth investigations.