Trypanosoma brucei Polo‐like kinase is essential for basal body duplication, kDNA segregation and cytokinesis

Abstract

SummaryPolo-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.

Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

“…Cells with an unseparated enlarged kinetoplast and a detached daughter flagellum became apparent at later timepoints, suggesting that kinetoplast and basal body segregation are also affected by sphingolipid depletion. Presumably, these cells did not initiate cytokinesis, which heavily depends on correct mitochondrial DNA segregation and flagellar attachment (LaCount et al, 2002;Robinson and Gull, 1991), but re-replicated their DNA, as has previously been reported (Hammarton et al, 2007;LaCount et al, 2002). This uncoupling of mitosis and cytokinesis is consistent with reports showing that the cytokinesis checkpoint present in other eukaryotes is not functional in trypanosomes (Ellis et al, 2004;Grellier et al, 1999;Ngo et al, 1998;Rothberg et al, 2006).…”

“…A similar phenotype in T. brucei has recently been reported for cells overexpressing the Polo-like kinase TbPLK (Hammarton et al, 2007). This also correlated with the presence of cells with two flagella emerging from a single flagellar pocket observed by SEM.…”

“…One report indicates that the Polo-like kinase TbPLK localizes to the cleavage furrow in T. brucei (Kumar and Wang, 2006), although a subsequent study suggests that its localization is cytoplasmic (Hammarton et al, 2007). Our phenotype suggests that perturbation of this and other kinases is responsible in part for the defects in kinetoplast and basal body migration (Kumar and Wang, 2006).…”

Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

“…Cells with a single-unit kinetoplast (1N1K and 1N1K div ) represented 70% of the TbID-PTP population, with distributions similar to those previously reported in other karyotype analyses (14,16). Cells with a single-unit kDNA (1N1K), no TdT signal, and no obvious POLID-PTP foci (Fig.…”

“…Additionally, kDNA segregation and positioning are dependent on bb movement and separation. Failure to segregate the bb results in impaired network segregation, providing additional evidence for the link between bb segregation and kDNA division (16,40). A filament system called the tripartite attachment complex (TAC) physically links the bb and the kDNA (38,41).…”