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

Hammarton TC, Kramer S, Tetley L, Boshart M, Mottram JC - Mol. Microbiol. (2007)

Bottom Line: 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).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.Notably, no additional roles were detected for trypanosome PLK in mitosis, setting this protein kinase apart from its counterparts in other eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Infection and Immunity, Wellcome Centre for Molecular Parasitology, University of Glasgow, Biomedical Research Centre, 120 University Place, Glasgow G12 8TA, UK. t.hammarton@bio.gla.ac.uk

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.

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Analysis of kDNA intensity in 2N1K cells. The kDNA intensity was measured in 1N1K, 1N2K, 2N1K and >2N1K cells, and the graphs in (A) and (B) show the distributions obtained for PLK RNAi and PLKty (ka)-overexpressing clones, respectively, at the time points indicated. 1N2Kc: combined kDNA intensity values for each kinetoplast; 1N2Ks: kDNA intensity values plotted separately. Horizontal bars indicate the median value of data in each column (also see Table S1). For 1N1K cells, n = 50 in all cases; for the other cell types, n values are indicated on the graphs.
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fig04: Analysis of kDNA intensity in 2N1K cells. The kDNA intensity was measured in 1N1K, 1N2K, 2N1K and >2N1K cells, and the graphs in (A) and (B) show the distributions obtained for PLK RNAi and PLKty (ka)-overexpressing clones, respectively, at the time points indicated. 1N2Kc: combined kDNA intensity values for each kinetoplast; 1N2Ks: kDNA intensity values plotted separately. Horizontal bars indicate the median value of data in each column (also see Table S1). For 1N1K cells, n = 50 in all cases; for the other cell types, n values are indicated on the graphs.

Mentions: To investigate whether the single kinetoplasts in the 2N1K cells generated by depletion or overexpression of PLK had replicated their DNA (as would be expected in a post-mitotic cell), images of PLK RNAi and PLKty (ka)-expressing cells stained with DAPI were captured and the intensity of the kDNA signal in 1N1K, 1N2K and 2N1K cells was measured over time (Fig. 4 and Table S1). The intensities of the two kDNA networks in 1N2K cells were plotted either combined (1N2Kc) or separately (1N2Ks). As expected, in uninduced (control) populations, the median kDNA intensities of 1N1K cells were slightly higher than the 1N2Ks median intensity values, reflecting that kDNA synthesis had initiated in a subset of the 1N1K population, with the median 1N2Kc intensity values higher still. Following PLK RNAi, the kDNA intensities of 2N1K cells were analysed at 22 and 30 h post induction (Fig. 4A). At 22 h post induction, the kDNA intensities of the 2N1K cells were found to be significantly higher than the 1N2Ks values (W = 880, P = 0.02), but significantly lower than the 1N2Kc values (W = 406, P = 0.01), indicating that the kinetoplasts in the 2N1K cells had partially, but not fully replicated their DNA at this time point. However, at 30 h post induction, the kDNA intensities of the 2N1K cells were not significantly different from the 1N2Kc values (W = 497, P = 0.84), indicating that on average, the 2N1K cells had fully replicated their kDNA by this time point. kDNA replication was also able to continue in cells that did not divide (represented by cells with more than two nuclei but only one kinetoplast, >2N1K cells), and the kDNA intensities of the >2N1K cells at 30 h post induction were markedly higher than the 1N2Kc values (W = 237, P = 0.07). It was also observed that the kDNA intensities of the 1N1K cells increased over time following PLK RNAi induction, such that they were no longer significantly lower than the 1N2Kc values (W = 1732, P = 0.29) at 30 h post induction, reflecting the delayed kinetoplast division.


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

Hammarton TC, Kramer S, Tetley L, Boshart M, Mottram JC - Mol. Microbiol. (2007)

Analysis of kDNA intensity in 2N1K cells. The kDNA intensity was measured in 1N1K, 1N2K, 2N1K and >2N1K cells, and the graphs in (A) and (B) show the distributions obtained for PLK RNAi and PLKty (ka)-overexpressing clones, respectively, at the time points indicated. 1N2Kc: combined kDNA intensity values for each kinetoplast; 1N2Ks: kDNA intensity values plotted separately. Horizontal bars indicate the median value of data in each column (also see Table S1). For 1N1K cells, n = 50 in all cases; for the other cell types, n values are indicated on the graphs.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169650&req=5

fig04: Analysis of kDNA intensity in 2N1K cells. The kDNA intensity was measured in 1N1K, 1N2K, 2N1K and >2N1K cells, and the graphs in (A) and (B) show the distributions obtained for PLK RNAi and PLKty (ka)-overexpressing clones, respectively, at the time points indicated. 1N2Kc: combined kDNA intensity values for each kinetoplast; 1N2Ks: kDNA intensity values plotted separately. Horizontal bars indicate the median value of data in each column (also see Table S1). For 1N1K cells, n = 50 in all cases; for the other cell types, n values are indicated on the graphs.
Mentions: To investigate whether the single kinetoplasts in the 2N1K cells generated by depletion or overexpression of PLK had replicated their DNA (as would be expected in a post-mitotic cell), images of PLK RNAi and PLKty (ka)-expressing cells stained with DAPI were captured and the intensity of the kDNA signal in 1N1K, 1N2K and 2N1K cells was measured over time (Fig. 4 and Table S1). The intensities of the two kDNA networks in 1N2K cells were plotted either combined (1N2Kc) or separately (1N2Ks). As expected, in uninduced (control) populations, the median kDNA intensities of 1N1K cells were slightly higher than the 1N2Ks median intensity values, reflecting that kDNA synthesis had initiated in a subset of the 1N1K population, with the median 1N2Kc intensity values higher still. Following PLK RNAi, the kDNA intensities of 2N1K cells were analysed at 22 and 30 h post induction (Fig. 4A). At 22 h post induction, the kDNA intensities of the 2N1K cells were found to be significantly higher than the 1N2Ks values (W = 880, P = 0.02), but significantly lower than the 1N2Kc values (W = 406, P = 0.01), indicating that the kinetoplasts in the 2N1K cells had partially, but not fully replicated their DNA at this time point. However, at 30 h post induction, the kDNA intensities of the 2N1K cells were not significantly different from the 1N2Kc values (W = 497, P = 0.84), indicating that on average, the 2N1K cells had fully replicated their kDNA by this time point. kDNA replication was also able to continue in cells that did not divide (represented by cells with more than two nuclei but only one kinetoplast, >2N1K cells), and the kDNA intensities of the >2N1K cells at 30 h post induction were markedly higher than the 1N2Kc values (W = 237, P = 0.07). It was also observed that the kDNA intensities of the 1N1K cells increased over time following PLK RNAi induction, such that they were no longer significantly lower than the 1N2Kc values (W = 1732, P = 0.29) at 30 h post induction, reflecting the delayed kinetoplast division.

Bottom Line: 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).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.Notably, no additional roles were detected for trypanosome PLK in mitosis, setting this protein kinase apart from its counterparts in other eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Infection and Immunity, Wellcome Centre for Molecular Parasitology, University of Glasgow, Biomedical Research Centre, 120 University Place, Glasgow G12 8TA, UK. t.hammarton@bio.gla.ac.uk

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.

Show MeSH