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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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Related in: MedlinePlus

Serial sectioning transmission electron microscopy of 2N1K cells. An example of a 2N1K cell with just one basal body pair and one flagellum is given for PLK RNAi (A) and PLKty (ka)-expressing (B) cell lines. Top left panels show a low-magnification image [1840× (A) and 2200× (B)] to illustrate the 2N1K configuration and the lower seven panels are serial higher-power images [6300× (A) and 5000× (B)] of the kinetoplast region. Numbers indicate the order of the serial sections. Key structures in the cell are indicated with letters. N, nucleus; K, kinetoplast; bb, pro-basal body; BB, mature basal body; A, flagellar axoneme; FP, flagellar pocket.
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fig06: Serial sectioning transmission electron microscopy of 2N1K cells. An example of a 2N1K cell with just one basal body pair and one flagellum is given for PLK RNAi (A) and PLKty (ka)-expressing (B) cell lines. Top left panels show a low-magnification image [1840× (A) and 2200× (B)] to illustrate the 2N1K configuration and the lower seven panels are serial higher-power images [6300× (A) and 5000× (B)] of the kinetoplast region. Numbers indicate the order of the serial sections. Key structures in the cell are indicated with letters. N, nucleus; K, kinetoplast; bb, pro-basal body; BB, mature basal body; A, flagellar axoneme; FP, flagellar pocket.

Mentions: To confirm the delay in basal body duplication, and to exclude the possibility that changes in PLK expression levels had led to a decrease in the protein recognized by the BBA4 antibody, PLK RNAi cells and PLKty (ka)-overexpressing cells were examined by serial sectioning transmission electron microscopy (Fig. 6), which confirmed the presence of 2N1K cells with only one basal body pair.


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)

Serial sectioning transmission electron microscopy of 2N1K cells. An example of a 2N1K cell with just one basal body pair and one flagellum is given for PLK RNAi (A) and PLKty (ka)-expressing (B) cell lines. Top left panels show a low-magnification image [1840× (A) and 2200× (B)] to illustrate the 2N1K configuration and the lower seven panels are serial higher-power images [6300× (A) and 5000× (B)] of the kinetoplast region. Numbers indicate the order of the serial sections. Key structures in the cell are indicated with letters. N, nucleus; K, kinetoplast; bb, pro-basal body; BB, mature basal body; A, flagellar axoneme; FP, flagellar pocket.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169650&req=5

fig06: Serial sectioning transmission electron microscopy of 2N1K cells. An example of a 2N1K cell with just one basal body pair and one flagellum is given for PLK RNAi (A) and PLKty (ka)-expressing (B) cell lines. Top left panels show a low-magnification image [1840× (A) and 2200× (B)] to illustrate the 2N1K configuration and the lower seven panels are serial higher-power images [6300× (A) and 5000× (B)] of the kinetoplast region. Numbers indicate the order of the serial sections. Key structures in the cell are indicated with letters. N, nucleus; K, kinetoplast; bb, pro-basal body; BB, mature basal body; A, flagellar axoneme; FP, flagellar pocket.
Mentions: To confirm the delay in basal body duplication, and to exclude the possibility that changes in PLK expression levels had led to a decrease in the protein recognized by the BBA4 antibody, PLK RNAi cells and PLKty (ka)-overexpressing cells were examined by serial sectioning transmission electron microscopy (Fig. 6), which confirmed the presence of 2N1K cells with only one basal body pair.

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
Related in: MedlinePlus