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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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Overexpression of PLKty in procyclic T. brucei. A. Western blot analysis of PLKty (ka)- and PLKty (kd)-expressing cells. Cell lines were grown in the presence or absence of tetracycline (tet) (t = 37 h) before cell lysates were prepared for SDS-PAGE and Western blotting with anti-TY antibody (top). Approximately 106 cell equivalents were loaded per lane, and equal loading was confirmed by Coomassie blue staining of a replica gel (bottom). B. Kinase assays. PLKty (ka or kd) was immunoprecipitated from cell lysates with anti-TY antibody, and used in a radioactive kinase assay with alpha- or beta-casein as substrate. C. Immunofluorescence of PLKty (ka)-expressing cells (t = 18 h). Left: DIC image; middle: FITC channel, anti-TY antibody; right: DAPI channel. The black bar represents 10 μm. D. Representative cumulative growth curves of PLKty (ka) and PLKty (kd) cell lines, passaged to maintain the cell density between 106 and 107 cells ml−1, in the presence or absence of tetracycline (tet). E. DAPI staining of PLKty (ka) cells, following induction with tetracycline. F. Abnormal cell types in detail, as revealed by DAPI staining for PLKty (ka)-expressing cell lines. Unclass, unclassifiable. G. DAPI/DIC merged images of abnormal cells. The N–K configuration of each cell is given, and the arrow points to a dividing nucleus in a 1N*1K cell. The black bar represents 10 μm.
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fig02: Overexpression of PLKty in procyclic T. brucei. A. Western blot analysis of PLKty (ka)- and PLKty (kd)-expressing cells. Cell lines were grown in the presence or absence of tetracycline (tet) (t = 37 h) before cell lysates were prepared for SDS-PAGE and Western blotting with anti-TY antibody (top). Approximately 106 cell equivalents were loaded per lane, and equal loading was confirmed by Coomassie blue staining of a replica gel (bottom). B. Kinase assays. PLKty (ka or kd) was immunoprecipitated from cell lysates with anti-TY antibody, and used in a radioactive kinase assay with alpha- or beta-casein as substrate. C. Immunofluorescence of PLKty (ka)-expressing cells (t = 18 h). Left: DIC image; middle: FITC channel, anti-TY antibody; right: DAPI channel. The black bar represents 10 μm. D. Representative cumulative growth curves of PLKty (ka) and PLKty (kd) cell lines, passaged to maintain the cell density between 106 and 107 cells ml−1, in the presence or absence of tetracycline (tet). E. DAPI staining of PLKty (ka) cells, following induction with tetracycline. F. Abnormal cell types in detail, as revealed by DAPI staining for PLKty (ka)-expressing cell lines. Unclass, unclassifiable. G. DAPI/DIC merged images of abnormal cells. The N–K configuration of each cell is given, and the arrow points to a dividing nucleus in a 1N*1K cell. The black bar represents 10 μm.

Mentions: The effect of overexpressing PLK in procyclic T. brucei was investigated. PLK was tagged with the TY-1 epitope tag at its N-terminus. The tag was introduced at the N-terminus in order to avoid a possible deleterious interaction of the epitope tag with the PBD at the C-terminus of the protein. Site-directed mutagenesis was used to generate a kinase-dead version by mutating asparagine 169 in the kinase catalytic domain to alanine (N169A). The PLKty sequences were cloned into pHD675 (Biebinger et al., 1997) and transfected into procyclic strain 427 pHD449 (Wirtz and Clayton, 1995), allowing PLKty to be expressed under the control of a tetracycline-inducible promoter. Inducible expression of approximately equivalent levels of PLKty (kinase active, ka) and PLKty (kinase dead, kd) was confirmed by Western blotting of the appropriate cell lysates with the anti-TY (BB2) antibody (Fig. 2A). Kinase assays were performed on PLKty (ka) and PLKty (kd) immunoprecipitated from tetracycline-induced trypanosome cell lysates. PLKty (ka), but not PLKty (kd), showed activity towards alpha-casein, and to a lesser extent, towards beta-casein (Fig. 2B), indicating that the TY-1 epitope tag had not interfered with the kinase activity of PLK, and that the N169A mutation had, as expected, abrogated the kinase activity of PLK. Neither kinase showed activity against myelin basic protein or histone H1 (data not shown).


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)

Overexpression of PLKty in procyclic T. brucei. A. Western blot analysis of PLKty (ka)- and PLKty (kd)-expressing cells. Cell lines were grown in the presence or absence of tetracycline (tet) (t = 37 h) before cell lysates were prepared for SDS-PAGE and Western blotting with anti-TY antibody (top). Approximately 106 cell equivalents were loaded per lane, and equal loading was confirmed by Coomassie blue staining of a replica gel (bottom). B. Kinase assays. PLKty (ka or kd) was immunoprecipitated from cell lysates with anti-TY antibody, and used in a radioactive kinase assay with alpha- or beta-casein as substrate. C. Immunofluorescence of PLKty (ka)-expressing cells (t = 18 h). Left: DIC image; middle: FITC channel, anti-TY antibody; right: DAPI channel. The black bar represents 10 μm. D. Representative cumulative growth curves of PLKty (ka) and PLKty (kd) cell lines, passaged to maintain the cell density between 106 and 107 cells ml−1, in the presence or absence of tetracycline (tet). E. DAPI staining of PLKty (ka) cells, following induction with tetracycline. F. Abnormal cell types in detail, as revealed by DAPI staining for PLKty (ka)-expressing cell lines. Unclass, unclassifiable. G. DAPI/DIC merged images of abnormal cells. The N–K configuration of each cell is given, and the arrow points to a dividing nucleus in a 1N*1K cell. The black bar represents 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169650&req=5

fig02: Overexpression of PLKty in procyclic T. brucei. A. Western blot analysis of PLKty (ka)- and PLKty (kd)-expressing cells. Cell lines were grown in the presence or absence of tetracycline (tet) (t = 37 h) before cell lysates were prepared for SDS-PAGE and Western blotting with anti-TY antibody (top). Approximately 106 cell equivalents were loaded per lane, and equal loading was confirmed by Coomassie blue staining of a replica gel (bottom). B. Kinase assays. PLKty (ka or kd) was immunoprecipitated from cell lysates with anti-TY antibody, and used in a radioactive kinase assay with alpha- or beta-casein as substrate. C. Immunofluorescence of PLKty (ka)-expressing cells (t = 18 h). Left: DIC image; middle: FITC channel, anti-TY antibody; right: DAPI channel. The black bar represents 10 μm. D. Representative cumulative growth curves of PLKty (ka) and PLKty (kd) cell lines, passaged to maintain the cell density between 106 and 107 cells ml−1, in the presence or absence of tetracycline (tet). E. DAPI staining of PLKty (ka) cells, following induction with tetracycline. F. Abnormal cell types in detail, as revealed by DAPI staining for PLKty (ka)-expressing cell lines. Unclass, unclassifiable. G. DAPI/DIC merged images of abnormal cells. The N–K configuration of each cell is given, and the arrow points to a dividing nucleus in a 1N*1K cell. The black bar represents 10 μm.
Mentions: The effect of overexpressing PLK in procyclic T. brucei was investigated. PLK was tagged with the TY-1 epitope tag at its N-terminus. The tag was introduced at the N-terminus in order to avoid a possible deleterious interaction of the epitope tag with the PBD at the C-terminus of the protein. Site-directed mutagenesis was used to generate a kinase-dead version by mutating asparagine 169 in the kinase catalytic domain to alanine (N169A). The PLKty sequences were cloned into pHD675 (Biebinger et al., 1997) and transfected into procyclic strain 427 pHD449 (Wirtz and Clayton, 1995), allowing PLKty to be expressed under the control of a tetracycline-inducible promoter. Inducible expression of approximately equivalent levels of PLKty (kinase active, ka) and PLKty (kinase dead, kd) was confirmed by Western blotting of the appropriate cell lysates with the anti-TY (BB2) antibody (Fig. 2A). Kinase assays were performed on PLKty (ka) and PLKty (kd) immunoprecipitated from tetracycline-induced trypanosome cell lysates. PLKty (ka), but not PLKty (kd), showed activity towards alpha-casein, and to a lesser extent, towards beta-casein (Fig. 2B), indicating that the TY-1 epitope tag had not interfered with the kinase activity of PLK, and that the N169A mutation had, as expected, abrogated the kinase activity of PLK. Neither kinase showed activity against myelin basic protein or histone H1 (data not shown).

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