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Identification of a Wee1-like kinase gene essential for procyclic Trypanosoma brucei survival.

Boynak NY, Rojas F, D'Alessio C, Vilchez Larrea SC, Rodriguez V, Ghiringhelli PD, Téllez-Iñón MT - PLoS ONE (2013)

Bottom Line: Abnormal phenotypes showing an increase in the percentage of cells with 1N0K, 0N1K and 2N1K were observed in these RNAi cell lines.Using parasites with a synchronized cell cycle, we demonstrated that TbWee1 is linked to the G2/M phase.Our results provide evidence for the existence of a functional Wee1 in T. brucei with a potential role in cell division at G2/M.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres", (INGEBI-CONICET), Buenos Aires, Argentina.

ABSTRACT
Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases (CDKs). Activation of the cyclin B-cdc2 kinase complex is a pivotal step in mitotic initiation and the tyrosine kinase Wee1 is a key regulator of cell cycle sequence during G2/M transition and inhibits mitotic entry by phosphorylating the inhibitory tyrosine 15 on the cdc2 M-phase-inducing kinase. Wee1 degradation is essential for the exit from the G2 phase. In trypanosomatids, little is known about the genes that regulate cyclin B-cdc2 complexes at the G2/M transition of their cell cycle. Although canonical tyrosine kinases are absent in the genome of trypanosomatids, phosphorylation on protein tyrosine residues has been reported in Trypanosoma brucei. Here, we characterized a Wee1-like protein kinase gene from T. brucei. Expression of TbWee1 in a Schizosaccharomyces pombe strain for Wee1 inhibited cell division and caused cell elongation. This demonstrates the lengthening of G2, which provided cells with extra time to grow before dividing. The Wee1-like protein kinase was expressed in the procyclic and bloodstream proliferative slender forms of T. brucei and the role of Wee1 in cell cycle progression was analyzed by generating RNA interference cell lines. In the procyclic form of T. brucei, the knock-down of TbWee1 expression by RNAi led to inhibition of parasite growth. Abnormal phenotypes showing an increase in the percentage of cells with 1N0K, 0N1K and 2N1K were observed in these RNAi cell lines. Using parasites with a synchronized cell cycle, we demonstrated that TbWee1 is linked to the G2/M phase. We also showed that TbWee1 is an essential gene necessary for proper cell cycle progression and parasite growth in T. brucei. Our results provide evidence for the existence of a functional Wee1 in T. brucei with a potential role in cell division at G2/M.

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Rescue of a Schyzosaccharomyces pombe Wee1 mutant by TbWee1.(A) S. pombe ∆Wee1 mutants were transformed with the pREP3x vector or with pREP3x in which S. pombe Wee1 or TbWee1 was cloned. Fission yeast were cultured on solid media in the presence (+thia) or absence (-thia) of 15 µM thiamine. Lower panel: S. pombe cells expressing wild type Wee1. (B) DAPI staining of S. pombe cells transformed with pREP3x TbWee1 grown in the absence of thiamine. Average cell size in S. pombe yeasts complemented with TbWee1 was 16,3 µm, with 55% of the population in the 10-15 µm range. Average cell size in control S. pombe yeasts was 10,4 µm, with 57% of the population in the 5-10 µm range. n=100. Bar= 100 µm.
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pone-0079364-g004: Rescue of a Schyzosaccharomyces pombe Wee1 mutant by TbWee1.(A) S. pombe ∆Wee1 mutants were transformed with the pREP3x vector or with pREP3x in which S. pombe Wee1 or TbWee1 was cloned. Fission yeast were cultured on solid media in the presence (+thia) or absence (-thia) of 15 µM thiamine. Lower panel: S. pombe cells expressing wild type Wee1. (B) DAPI staining of S. pombe cells transformed with pREP3x TbWee1 grown in the absence of thiamine. Average cell size in S. pombe yeasts complemented with TbWee1 was 16,3 µm, with 55% of the population in the 10-15 µm range. Average cell size in control S. pombe yeasts was 10,4 µm, with 57% of the population in the 5-10 µm range. n=100. Bar= 100 µm.

Mentions: ∆Wee1 mutants expressing TbWee1 exhibited cell cycle arrest manifested by an increase in cell length after washing out thiamine repression from the cell culture. This same phenotype was observed when Wee1 from S. pombe was overexpressed in the same cells (Figure 4A). This long-cell phenotype was not seen when expression of either Spwee1 or TbWee1 was repressed with thiamine or when ΔWee1 cells were transformed with and empty vector (Figure 4A). Moreover, over-expression of TbWee1 caused S. pombe cells to elongate and reach a size even larger than that of wild-type cells, indicating that the effect of TbWee1 on the growth of ∆Wee1 cells is dose-dependent (Figure 4A).


Identification of a Wee1-like kinase gene essential for procyclic Trypanosoma brucei survival.

Boynak NY, Rojas F, D'Alessio C, Vilchez Larrea SC, Rodriguez V, Ghiringhelli PD, Téllez-Iñón MT - PLoS ONE (2013)

Rescue of a Schyzosaccharomyces pombe Wee1 mutant by TbWee1.(A) S. pombe ∆Wee1 mutants were transformed with the pREP3x vector or with pREP3x in which S. pombe Wee1 or TbWee1 was cloned. Fission yeast were cultured on solid media in the presence (+thia) or absence (-thia) of 15 µM thiamine. Lower panel: S. pombe cells expressing wild type Wee1. (B) DAPI staining of S. pombe cells transformed with pREP3x TbWee1 grown in the absence of thiamine. Average cell size in S. pombe yeasts complemented with TbWee1 was 16,3 µm, with 55% of the population in the 10-15 µm range. Average cell size in control S. pombe yeasts was 10,4 µm, with 57% of the population in the 5-10 µm range. n=100. Bar= 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3818516&req=5

pone-0079364-g004: Rescue of a Schyzosaccharomyces pombe Wee1 mutant by TbWee1.(A) S. pombe ∆Wee1 mutants were transformed with the pREP3x vector or with pREP3x in which S. pombe Wee1 or TbWee1 was cloned. Fission yeast were cultured on solid media in the presence (+thia) or absence (-thia) of 15 µM thiamine. Lower panel: S. pombe cells expressing wild type Wee1. (B) DAPI staining of S. pombe cells transformed with pREP3x TbWee1 grown in the absence of thiamine. Average cell size in S. pombe yeasts complemented with TbWee1 was 16,3 µm, with 55% of the population in the 10-15 µm range. Average cell size in control S. pombe yeasts was 10,4 µm, with 57% of the population in the 5-10 µm range. n=100. Bar= 100 µm.
Mentions: ∆Wee1 mutants expressing TbWee1 exhibited cell cycle arrest manifested by an increase in cell length after washing out thiamine repression from the cell culture. This same phenotype was observed when Wee1 from S. pombe was overexpressed in the same cells (Figure 4A). This long-cell phenotype was not seen when expression of either Spwee1 or TbWee1 was repressed with thiamine or when ΔWee1 cells were transformed with and empty vector (Figure 4A). Moreover, over-expression of TbWee1 caused S. pombe cells to elongate and reach a size even larger than that of wild-type cells, indicating that the effect of TbWee1 on the growth of ∆Wee1 cells is dose-dependent (Figure 4A).

Bottom Line: Abnormal phenotypes showing an increase in the percentage of cells with 1N0K, 0N1K and 2N1K were observed in these RNAi cell lines.Using parasites with a synchronized cell cycle, we demonstrated that TbWee1 is linked to the G2/M phase.Our results provide evidence for the existence of a functional Wee1 in T. brucei with a potential role in cell division at G2/M.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres", (INGEBI-CONICET), Buenos Aires, Argentina.

ABSTRACT
Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases (CDKs). Activation of the cyclin B-cdc2 kinase complex is a pivotal step in mitotic initiation and the tyrosine kinase Wee1 is a key regulator of cell cycle sequence during G2/M transition and inhibits mitotic entry by phosphorylating the inhibitory tyrosine 15 on the cdc2 M-phase-inducing kinase. Wee1 degradation is essential for the exit from the G2 phase. In trypanosomatids, little is known about the genes that regulate cyclin B-cdc2 complexes at the G2/M transition of their cell cycle. Although canonical tyrosine kinases are absent in the genome of trypanosomatids, phosphorylation on protein tyrosine residues has been reported in Trypanosoma brucei. Here, we characterized a Wee1-like protein kinase gene from T. brucei. Expression of TbWee1 in a Schizosaccharomyces pombe strain for Wee1 inhibited cell division and caused cell elongation. This demonstrates the lengthening of G2, which provided cells with extra time to grow before dividing. The Wee1-like protein kinase was expressed in the procyclic and bloodstream proliferative slender forms of T. brucei and the role of Wee1 in cell cycle progression was analyzed by generating RNA interference cell lines. In the procyclic form of T. brucei, the knock-down of TbWee1 expression by RNAi led to inhibition of parasite growth. Abnormal phenotypes showing an increase in the percentage of cells with 1N0K, 0N1K and 2N1K were observed in these RNAi cell lines. Using parasites with a synchronized cell cycle, we demonstrated that TbWee1 is linked to the G2/M phase. We also showed that TbWee1 is an essential gene necessary for proper cell cycle progression and parasite growth in T. brucei. Our results provide evidence for the existence of a functional Wee1 in T. brucei with a potential role in cell division at G2/M.

Show MeSH
Related in: MedlinePlus