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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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RNAi of PLK in bloodstream-form T. brucei. A. Representative cumulative growth curves of bloodstream PLK RNAi clones 7 and 15, passaged to maintain the cell density between 105 and 106 cells ml−1, in the presence and absence of 1 μg ml−1 tetracycline (tet). B. Northern blot of RNA prepared from PLK RNAi clones up to 12 h post induction. The blots were probed with part of the PLK ORF (top), stripped and re-probed with the actin ORF (bottom), as a loading control. The relative intensities of the signals obtained for PLK for each clone over time, normalized to the actin signals, are given. C. Western blot analysis of PLK RNAi cell lines where one PLK allele has been replaced with PLKty. Data for one pool and one clone derived from each original clone are shown. Cell lines were grown in the presence or absence of tetracycline (tet) for 6 h before cell lysates were prepared for SDS-PAGE and Western blotting with anti-TY antibody (top). Approximately 1.5 × 106 cell equivalents were loaded per lane, and equal loading was confirmed by Coomassie blue staining of a replica gel (bottom). D. DAPI staining of nuclei and kinetoplasts for clone 7 and the RNAi host cell line (13–90), induced with tetracycline. E. Classification of 2N2K cells according to the stage of cytokinesis (clone 7 and 13–90). Example images of cells in the different stages of cytokinesis are given. F. Abnormal nucleus-kinetoplast configurations in detail as revealed by DAPI staining for clone 7, induced with tetracycline. Unclass, unclassifiable. G. DIC and DAPI images of abnormal cells. The black bar represents 10 μm. H. Flow cytometry analysis of PLK RNAi cells (clone 7) at the time points indicated. The ploidies of the peaks are shown.
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fig07: RNAi of PLK in bloodstream-form T. brucei. A. Representative cumulative growth curves of bloodstream PLK RNAi clones 7 and 15, passaged to maintain the cell density between 105 and 106 cells ml−1, in the presence and absence of 1 μg ml−1 tetracycline (tet). B. Northern blot of RNA prepared from PLK RNAi clones up to 12 h post induction. The blots were probed with part of the PLK ORF (top), stripped and re-probed with the actin ORF (bottom), as a loading control. The relative intensities of the signals obtained for PLK for each clone over time, normalized to the actin signals, are given. C. Western blot analysis of PLK RNAi cell lines where one PLK allele has been replaced with PLKty. Data for one pool and one clone derived from each original clone are shown. Cell lines were grown in the presence or absence of tetracycline (tet) for 6 h before cell lysates were prepared for SDS-PAGE and Western blotting with anti-TY antibody (top). Approximately 1.5 × 106 cell equivalents were loaded per lane, and equal loading was confirmed by Coomassie blue staining of a replica gel (bottom). D. DAPI staining of nuclei and kinetoplasts for clone 7 and the RNAi host cell line (13–90), induced with tetracycline. E. Classification of 2N2K cells according to the stage of cytokinesis (clone 7 and 13–90). Example images of cells in the different stages of cytokinesis are given. F. Abnormal nucleus-kinetoplast configurations in detail as revealed by DAPI staining for clone 7, induced with tetracycline. Unclass, unclassifiable. G. DIC and DAPI images of abnormal cells. The black bar represents 10 μm. H. Flow cytometry analysis of PLK RNAi cells (clone 7) at the time points indicated. The ploidies of the peaks are shown.

Mentions: RNAi was next used to investigate the function of PLK in the bloodstream form of T. brucei. PLK RNAi was induced in two independent clones by the addition of tetracycline to the culture medium and resulted in a rapid growth arrest (visible from 4 h post induction) (Fig. 7A) that was accompanied by a significant decrease (up to 80% reduction) in PLK mRNA as determined by Northern blot analysis (Fig. 7B). To confirm that PLK protein levels also decreased following induction of the RNAi, one of the PLK alleles in each of the PLK RNAi clones was replaced with a modified PLK gene encoding PLKty under the control of the endogenous promoter. This allowed the detection of PLK in the RNAi cell lines without the need for a specific anti-PLK antibody. Western blot analysis of PLK RNAi: PLKty pools and clones derived from each original RNAi clone demonstrated a decrease (estimated 50–75% reduction) in PLKty protein levels 6 h following induction (Fig. 7C). A similar approach to confirm protein knockdown following RNAi induction in procyclic cells was, however, unsuccessful as PLK RNAi: PLKty procyclic cell lines could not be generated.


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)

RNAi of PLK in bloodstream-form T. brucei. A. Representative cumulative growth curves of bloodstream PLK RNAi clones 7 and 15, passaged to maintain the cell density between 105 and 106 cells ml−1, in the presence and absence of 1 μg ml−1 tetracycline (tet). B. Northern blot of RNA prepared from PLK RNAi clones up to 12 h post induction. The blots were probed with part of the PLK ORF (top), stripped and re-probed with the actin ORF (bottom), as a loading control. The relative intensities of the signals obtained for PLK for each clone over time, normalized to the actin signals, are given. C. Western blot analysis of PLK RNAi cell lines where one PLK allele has been replaced with PLKty. Data for one pool and one clone derived from each original clone are shown. Cell lines were grown in the presence or absence of tetracycline (tet) for 6 h before cell lysates were prepared for SDS-PAGE and Western blotting with anti-TY antibody (top). Approximately 1.5 × 106 cell equivalents were loaded per lane, and equal loading was confirmed by Coomassie blue staining of a replica gel (bottom). D. DAPI staining of nuclei and kinetoplasts for clone 7 and the RNAi host cell line (13–90), induced with tetracycline. E. Classification of 2N2K cells according to the stage of cytokinesis (clone 7 and 13–90). Example images of cells in the different stages of cytokinesis are given. F. Abnormal nucleus-kinetoplast configurations in detail as revealed by DAPI staining for clone 7, induced with tetracycline. Unclass, unclassifiable. G. DIC and DAPI images of abnormal cells. The black bar represents 10 μm. H. Flow cytometry analysis of PLK RNAi cells (clone 7) at the time points indicated. The ploidies of the peaks are shown.
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fig07: RNAi of PLK in bloodstream-form T. brucei. A. Representative cumulative growth curves of bloodstream PLK RNAi clones 7 and 15, passaged to maintain the cell density between 105 and 106 cells ml−1, in the presence and absence of 1 μg ml−1 tetracycline (tet). B. Northern blot of RNA prepared from PLK RNAi clones up to 12 h post induction. The blots were probed with part of the PLK ORF (top), stripped and re-probed with the actin ORF (bottom), as a loading control. The relative intensities of the signals obtained for PLK for each clone over time, normalized to the actin signals, are given. C. Western blot analysis of PLK RNAi cell lines where one PLK allele has been replaced with PLKty. Data for one pool and one clone derived from each original clone are shown. Cell lines were grown in the presence or absence of tetracycline (tet) for 6 h before cell lysates were prepared for SDS-PAGE and Western blotting with anti-TY antibody (top). Approximately 1.5 × 106 cell equivalents were loaded per lane, and equal loading was confirmed by Coomassie blue staining of a replica gel (bottom). D. DAPI staining of nuclei and kinetoplasts for clone 7 and the RNAi host cell line (13–90), induced with tetracycline. E. Classification of 2N2K cells according to the stage of cytokinesis (clone 7 and 13–90). Example images of cells in the different stages of cytokinesis are given. F. Abnormal nucleus-kinetoplast configurations in detail as revealed by DAPI staining for clone 7, induced with tetracycline. Unclass, unclassifiable. G. DIC and DAPI images of abnormal cells. The black bar represents 10 μm. H. Flow cytometry analysis of PLK RNAi cells (clone 7) at the time points indicated. The ploidies of the peaks are shown.
Mentions: RNAi was next used to investigate the function of PLK in the bloodstream form of T. brucei. PLK RNAi was induced in two independent clones by the addition of tetracycline to the culture medium and resulted in a rapid growth arrest (visible from 4 h post induction) (Fig. 7A) that was accompanied by a significant decrease (up to 80% reduction) in PLK mRNA as determined by Northern blot analysis (Fig. 7B). To confirm that PLK protein levels also decreased following induction of the RNAi, one of the PLK alleles in each of the PLK RNAi clones was replaced with a modified PLK gene encoding PLKty under the control of the endogenous promoter. This allowed the detection of PLK in the RNAi cell lines without the need for a specific anti-PLK antibody. Western blot analysis of PLK RNAi: PLKty pools and clones derived from each original RNAi clone demonstrated a decrease (estimated 50–75% reduction) in PLKty protein levels 6 h following induction (Fig. 7C). A similar approach to confirm protein knockdown following RNAi induction in procyclic cells was, however, unsuccessful as PLK RNAi: PLKty procyclic cell lines could not be generated.

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