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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 2N1K and 2N2K cells. A and B. The relative positioning of the nuclei and single kinetoplast in 2N1K cells generated by PLK RNAi clone B7 (A) and PLKty (ka)-overexpressing (B) cell lines over time following induction. The order of N and K is given from the anterior to the posterior end of the cell. The numbers of cells where the kinetoplast appeared to overlay one of the nuclei are also given. C and D. DAPI/DIC merged images of 2N1K or >2N1K cells from PLK RNAi and PLKty (ka)-overexpressing cell lines respectively. Arrowheads point to enlarged/elongated kinetoplasts. The black bars represent 10 μm. E. DAPI/DIC merged image of a wild-type 2N2K cell. Arrowheads point to kinetoplasts. The black bar represents 10 μm. F and G. The arrangement of nuclei and kinetoplasts in 2N2K cells generated by PLK RNAi clone B7 (F) and PLKty (ka)-overexpressing (G) cell lines over time. The order of N and K is given from the anterior to the posterior end of the cell. The inset in (F) shows a 2N2K cell with the arrangement NKKN, while the two cell images in (G) show 2N2K cells with a NNKK arrangement. Arrowheads point to kinetoplasts.
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fig03: Analysis of 2N1K and 2N2K cells. A and B. The relative positioning of the nuclei and single kinetoplast in 2N1K cells generated by PLK RNAi clone B7 (A) and PLKty (ka)-overexpressing (B) cell lines over time following induction. The order of N and K is given from the anterior to the posterior end of the cell. The numbers of cells where the kinetoplast appeared to overlay one of the nuclei are also given. C and D. DAPI/DIC merged images of 2N1K or >2N1K cells from PLK RNAi and PLKty (ka)-overexpressing cell lines respectively. Arrowheads point to enlarged/elongated kinetoplasts. The black bars represent 10 μm. E. DAPI/DIC merged image of a wild-type 2N2K cell. Arrowheads point to kinetoplasts. The black bar represents 10 μm. F and G. The arrangement of nuclei and kinetoplasts in 2N2K cells generated by PLK RNAi clone B7 (F) and PLKty (ka)-overexpressing (G) cell lines over time. The order of N and K is given from the anterior to the posterior end of the cell. The inset in (F) shows a 2N2K cell with the arrangement NKKN, while the two cell images in (G) show 2N2K cells with a NNKK arrangement. Arrowheads point to kinetoplasts.

Mentions: The 2N1K cells produced by downregulation of PLK by RNAi, and by overexpression of PLKty (ka) in procyclic T. brucei were further investigated. The position of the single kinetoplast in 2N1K cells relative to the two nuclei was scored for each cell line. In PLK RNAi cell lines, the kinetoplast was located predominantly between the two nuclei (Figs 1E and 3A), whereas in the PLKty (ka)-overexpressing cell line, it was predominantly found at the posterior end of the parasite (Figs 2G and 3B). 2N1K and >2N1K cells with an enlarged and/or elongated kinetoplast were also observed (Fig. 3C and D), suggesting that the kinetoplast divides in these cells (albeit with delayed timing, as the nuclei had already completed one or more mitoses in these cells). To corroborate this, the positions of the two kinetoplasts in 2N2K cells in these cell lines were also investigated. In wild-type procyclic cells, the nuclei and kinetoplasts in a 2N2K cell are arranged N–K–N–K (anterior to posterior, Fig. 3E). In PLK RNAi cell lines, however, over 30% of 2N2K cells displayed an N–K–K–N distribution 29 h post induction (n = 71; Fig. 3F), while in cell lines overexpressing PLKty (ka), over 35% of 2N2K cells had an N–N–K–K distribution at 18 h post induction (n > 65; Fig. 3G). These data are consistent with a model where the level of downregulation of PLK or overexpression of PLKty (ka) obtained in this study delays (rather than completely inhibits) kinetoplast division. Downregulation and overexpression of PLK result in the single undivided kinetoplast being positioned differentially relative to the two nuclei, and subsequent kinetoplast division and segregation would account for the abnormal 2N2K cells observed.


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 2N1K and 2N2K cells. A and B. The relative positioning of the nuclei and single kinetoplast in 2N1K cells generated by PLK RNAi clone B7 (A) and PLKty (ka)-overexpressing (B) cell lines over time following induction. The order of N and K is given from the anterior to the posterior end of the cell. The numbers of cells where the kinetoplast appeared to overlay one of the nuclei are also given. C and D. DAPI/DIC merged images of 2N1K or >2N1K cells from PLK RNAi and PLKty (ka)-overexpressing cell lines respectively. Arrowheads point to enlarged/elongated kinetoplasts. The black bars represent 10 μm. E. DAPI/DIC merged image of a wild-type 2N2K cell. Arrowheads point to kinetoplasts. The black bar represents 10 μm. F and G. The arrangement of nuclei and kinetoplasts in 2N2K cells generated by PLK RNAi clone B7 (F) and PLKty (ka)-overexpressing (G) cell lines over time. The order of N and K is given from the anterior to the posterior end of the cell. The inset in (F) shows a 2N2K cell with the arrangement NKKN, while the two cell images in (G) show 2N2K cells with a NNKK arrangement. Arrowheads point to kinetoplasts.
© Copyright Policy
Related In: Results  -  Collection

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

fig03: Analysis of 2N1K and 2N2K cells. A and B. The relative positioning of the nuclei and single kinetoplast in 2N1K cells generated by PLK RNAi clone B7 (A) and PLKty (ka)-overexpressing (B) cell lines over time following induction. The order of N and K is given from the anterior to the posterior end of the cell. The numbers of cells where the kinetoplast appeared to overlay one of the nuclei are also given. C and D. DAPI/DIC merged images of 2N1K or >2N1K cells from PLK RNAi and PLKty (ka)-overexpressing cell lines respectively. Arrowheads point to enlarged/elongated kinetoplasts. The black bars represent 10 μm. E. DAPI/DIC merged image of a wild-type 2N2K cell. Arrowheads point to kinetoplasts. The black bar represents 10 μm. F and G. The arrangement of nuclei and kinetoplasts in 2N2K cells generated by PLK RNAi clone B7 (F) and PLKty (ka)-overexpressing (G) cell lines over time. The order of N and K is given from the anterior to the posterior end of the cell. The inset in (F) shows a 2N2K cell with the arrangement NKKN, while the two cell images in (G) show 2N2K cells with a NNKK arrangement. Arrowheads point to kinetoplasts.
Mentions: The 2N1K cells produced by downregulation of PLK by RNAi, and by overexpression of PLKty (ka) in procyclic T. brucei were further investigated. The position of the single kinetoplast in 2N1K cells relative to the two nuclei was scored for each cell line. In PLK RNAi cell lines, the kinetoplast was located predominantly between the two nuclei (Figs 1E and 3A), whereas in the PLKty (ka)-overexpressing cell line, it was predominantly found at the posterior end of the parasite (Figs 2G and 3B). 2N1K and >2N1K cells with an enlarged and/or elongated kinetoplast were also observed (Fig. 3C and D), suggesting that the kinetoplast divides in these cells (albeit with delayed timing, as the nuclei had already completed one or more mitoses in these cells). To corroborate this, the positions of the two kinetoplasts in 2N2K cells in these cell lines were also investigated. In wild-type procyclic cells, the nuclei and kinetoplasts in a 2N2K cell are arranged N–K–N–K (anterior to posterior, Fig. 3E). In PLK RNAi cell lines, however, over 30% of 2N2K cells displayed an N–K–K–N distribution 29 h post induction (n = 71; Fig. 3F), while in cell lines overexpressing PLKty (ka), over 35% of 2N2K cells had an N–N–K–K distribution at 18 h post induction (n > 65; Fig. 3G). These data are consistent with a model where the level of downregulation of PLK or overexpression of PLKty (ka) obtained in this study delays (rather than completely inhibits) kinetoplast division. Downregulation and overexpression of PLK result in the single undivided kinetoplast being positioned differentially relative to the two nuclei, and subsequent kinetoplast division and segregation would account for the abnormal 2N2K cells observed.

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