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TbUNC119 and its binding protein complex are essential for propagation, motility, and morphogenesis of Trypanosoma brucei procyclic form cells.

Ohshima S, Ohashi-Suzuki M, Miura Y, Yabu Y, Okada N, Ohta N, Suzuki T - PLoS ONE (2010)

Bottom Line: Through in silico analysis, we found 88 gene products which were highly similar to C. elegans UNC proteins and categorized them as TbCEUN (T. brucei gene products which have high similarity to C. elegansUNC proteins).However, knock-down analysis of both TbUNC119 and its binding protein (TbUNC119BP) which was found by yeast two-hybrid analysis showed characteristic phenotypes, including reduced motility, morphological change (extended cell shape), and cellular apoptosis.Based on the observed phenotypes, possible function of the TbUNC119 and TbUNC119BP is discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Core Laboratory, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.

ABSTRACT
Flagellum-mediated motility of Trypanosoma brucei is considered to be essential for the parasite to complete stage development in the tsetse fly vector, while the mechanism by which flagellum-mediated motility is controlled are not fully understood. We thus compared T. brucei whole gene products (amino acid sequence) with Caenorhabditis elegans UNC (uncoordinated) proteins, in order to find uncharacterized motility-related T. brucei genes. Through in silico analysis, we found 88 gene products which were highly similar to C. elegans UNC proteins and categorized them as TbCEUN (T. brucei gene products which have high similarity to C. elegansUNC proteins). Approximately two thirds of the 88 TbCEUN gene products were kinesin-related molecules. A gene product highly similar to C. elegans UNC119 protein was designated as TbUNC119. RNAi-mediated depletion of TbUNC119 showed no apparent phenotype. However, knock-down analysis of both TbUNC119 and its binding protein (TbUNC119BP) which was found by yeast two-hybrid analysis showed characteristic phenotypes, including reduced motility, morphological change (extended cell shape), and cellular apoptosis. Based on the observed phenotypes, possible function of the TbUNC119 and TbUNC119BP is discussed.

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Double knock-down analysis of TbUNC119 and TbUNC119BP.A. Growth of TbUNC119-TbUNC119BP double knock-down procyclic form cells. Three TbUNC119 and TbUNC119BP double knock-down inducible cell lines were cloned (TbUNC119+TbUNC119BP-1, TbUNC119+TbUNC119BP-2 and TbUNC119+TbUNC119BP-3). RNAi-mediated gene knock-down was induced with tetracycline. (Tet+) denotes RNAi-induced cells. The growth rates of the double knock-down cells were significantly reduced, and the cells could not grow after 12 days of RNAi induction. The inset in A shows TbUNC119 and TbUNC119BP RT-PCR results with tubulin as a control. B. Motility assay of TbUNC119-TbUNC119BP double knock-down cells. Double knock-down cells and parental 29-13 cells were assayed for motility 7 days after RNAi induction or without RNAi induction. Thirty seconds of motion was captured for each cell. Motility traces for 50 cells in each group were generated using VIDEOPOINT 2.5 software. Cells that were not tracked for the full 30 s were not used for the analysis. Motility of the RNAi-induced double knock-down cells was significantly reduced (*: P<0.01). C. Time-course motility assay of TbUNC119-TbUNC119BP double knock-down cells. Double knock-down cells were assayed for motility 0, 2, 5, 7 and 9 days after RNAi induction or without RNAi induction. Thirty seconds of motion was captured for each cell. Motility traces for 50 cells in each group were generated using VIDEOPOINT 2.5 software. Cells that were not tracked for the full 30 s were not used for the analysis. Motilities of the double knock-down cells 7 days and 9 days after RNAi induction were significantly reduced (*: P<0.01).
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pone-0015577-g004: Double knock-down analysis of TbUNC119 and TbUNC119BP.A. Growth of TbUNC119-TbUNC119BP double knock-down procyclic form cells. Three TbUNC119 and TbUNC119BP double knock-down inducible cell lines were cloned (TbUNC119+TbUNC119BP-1, TbUNC119+TbUNC119BP-2 and TbUNC119+TbUNC119BP-3). RNAi-mediated gene knock-down was induced with tetracycline. (Tet+) denotes RNAi-induced cells. The growth rates of the double knock-down cells were significantly reduced, and the cells could not grow after 12 days of RNAi induction. The inset in A shows TbUNC119 and TbUNC119BP RT-PCR results with tubulin as a control. B. Motility assay of TbUNC119-TbUNC119BP double knock-down cells. Double knock-down cells and parental 29-13 cells were assayed for motility 7 days after RNAi induction or without RNAi induction. Thirty seconds of motion was captured for each cell. Motility traces for 50 cells in each group were generated using VIDEOPOINT 2.5 software. Cells that were not tracked for the full 30 s were not used for the analysis. Motility of the RNAi-induced double knock-down cells was significantly reduced (*: P<0.01). C. Time-course motility assay of TbUNC119-TbUNC119BP double knock-down cells. Double knock-down cells were assayed for motility 0, 2, 5, 7 and 9 days after RNAi induction or without RNAi induction. Thirty seconds of motion was captured for each cell. Motility traces for 50 cells in each group were generated using VIDEOPOINT 2.5 software. Cells that were not tracked for the full 30 s were not used for the analysis. Motilities of the double knock-down cells 7 days and 9 days after RNAi induction were significantly reduced (*: P<0.01).

Mentions: We next investigated whether double knock-down of TbUNC119 and TbUNC119BP lead to changes in T. brucei phenotypes. Genes for TbUNC119 and TbUNC119BP were cloned into the knock-down vector with which T. brucei cells were transformed. RT-PCR analysis 7 days after RNAi induction resulted in marked reduction of both TbUNC119 and TbUNC119BP transcripts (Fig. 4A). Western blot analysis also showed marked reduction of TbUNC119 in double knock-down cells 7 days after RNAi induction (Fig. S1). The growth rate of RNAi-induced double knock-down cells was significantly reduced, and the cells were unable to grow after 12 days of RNAi induction (Fig. 4A).


TbUNC119 and its binding protein complex are essential for propagation, motility, and morphogenesis of Trypanosoma brucei procyclic form cells.

Ohshima S, Ohashi-Suzuki M, Miura Y, Yabu Y, Okada N, Ohta N, Suzuki T - PLoS ONE (2010)

Double knock-down analysis of TbUNC119 and TbUNC119BP.A. Growth of TbUNC119-TbUNC119BP double knock-down procyclic form cells. Three TbUNC119 and TbUNC119BP double knock-down inducible cell lines were cloned (TbUNC119+TbUNC119BP-1, TbUNC119+TbUNC119BP-2 and TbUNC119+TbUNC119BP-3). RNAi-mediated gene knock-down was induced with tetracycline. (Tet+) denotes RNAi-induced cells. The growth rates of the double knock-down cells were significantly reduced, and the cells could not grow after 12 days of RNAi induction. The inset in A shows TbUNC119 and TbUNC119BP RT-PCR results with tubulin as a control. B. Motility assay of TbUNC119-TbUNC119BP double knock-down cells. Double knock-down cells and parental 29-13 cells were assayed for motility 7 days after RNAi induction or without RNAi induction. Thirty seconds of motion was captured for each cell. Motility traces for 50 cells in each group were generated using VIDEOPOINT 2.5 software. Cells that were not tracked for the full 30 s were not used for the analysis. Motility of the RNAi-induced double knock-down cells was significantly reduced (*: P<0.01). C. Time-course motility assay of TbUNC119-TbUNC119BP double knock-down cells. Double knock-down cells were assayed for motility 0, 2, 5, 7 and 9 days after RNAi induction or without RNAi induction. Thirty seconds of motion was captured for each cell. Motility traces for 50 cells in each group were generated using VIDEOPOINT 2.5 software. Cells that were not tracked for the full 30 s were not used for the analysis. Motilities of the double knock-down cells 7 days and 9 days after RNAi induction were significantly reduced (*: P<0.01).
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pone-0015577-g004: Double knock-down analysis of TbUNC119 and TbUNC119BP.A. Growth of TbUNC119-TbUNC119BP double knock-down procyclic form cells. Three TbUNC119 and TbUNC119BP double knock-down inducible cell lines were cloned (TbUNC119+TbUNC119BP-1, TbUNC119+TbUNC119BP-2 and TbUNC119+TbUNC119BP-3). RNAi-mediated gene knock-down was induced with tetracycline. (Tet+) denotes RNAi-induced cells. The growth rates of the double knock-down cells were significantly reduced, and the cells could not grow after 12 days of RNAi induction. The inset in A shows TbUNC119 and TbUNC119BP RT-PCR results with tubulin as a control. B. Motility assay of TbUNC119-TbUNC119BP double knock-down cells. Double knock-down cells and parental 29-13 cells were assayed for motility 7 days after RNAi induction or without RNAi induction. Thirty seconds of motion was captured for each cell. Motility traces for 50 cells in each group were generated using VIDEOPOINT 2.5 software. Cells that were not tracked for the full 30 s were not used for the analysis. Motility of the RNAi-induced double knock-down cells was significantly reduced (*: P<0.01). C. Time-course motility assay of TbUNC119-TbUNC119BP double knock-down cells. Double knock-down cells were assayed for motility 0, 2, 5, 7 and 9 days after RNAi induction or without RNAi induction. Thirty seconds of motion was captured for each cell. Motility traces for 50 cells in each group were generated using VIDEOPOINT 2.5 software. Cells that were not tracked for the full 30 s were not used for the analysis. Motilities of the double knock-down cells 7 days and 9 days after RNAi induction were significantly reduced (*: P<0.01).
Mentions: We next investigated whether double knock-down of TbUNC119 and TbUNC119BP lead to changes in T. brucei phenotypes. Genes for TbUNC119 and TbUNC119BP were cloned into the knock-down vector with which T. brucei cells were transformed. RT-PCR analysis 7 days after RNAi induction resulted in marked reduction of both TbUNC119 and TbUNC119BP transcripts (Fig. 4A). Western blot analysis also showed marked reduction of TbUNC119 in double knock-down cells 7 days after RNAi induction (Fig. S1). The growth rate of RNAi-induced double knock-down cells was significantly reduced, and the cells were unable to grow after 12 days of RNAi induction (Fig. 4A).

Bottom Line: Through in silico analysis, we found 88 gene products which were highly similar to C. elegans UNC proteins and categorized them as TbCEUN (T. brucei gene products which have high similarity to C. elegansUNC proteins).However, knock-down analysis of both TbUNC119 and its binding protein (TbUNC119BP) which was found by yeast two-hybrid analysis showed characteristic phenotypes, including reduced motility, morphological change (extended cell shape), and cellular apoptosis.Based on the observed phenotypes, possible function of the TbUNC119 and TbUNC119BP is discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Core Laboratory, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.

ABSTRACT
Flagellum-mediated motility of Trypanosoma brucei is considered to be essential for the parasite to complete stage development in the tsetse fly vector, while the mechanism by which flagellum-mediated motility is controlled are not fully understood. We thus compared T. brucei whole gene products (amino acid sequence) with Caenorhabditis elegans UNC (uncoordinated) proteins, in order to find uncharacterized motility-related T. brucei genes. Through in silico analysis, we found 88 gene products which were highly similar to C. elegans UNC proteins and categorized them as TbCEUN (T. brucei gene products which have high similarity to C. elegansUNC proteins). Approximately two thirds of the 88 TbCEUN gene products were kinesin-related molecules. A gene product highly similar to C. elegans UNC119 protein was designated as TbUNC119. RNAi-mediated depletion of TbUNC119 showed no apparent phenotype. However, knock-down analysis of both TbUNC119 and its binding protein (TbUNC119BP) which was found by yeast two-hybrid analysis showed characteristic phenotypes, including reduced motility, morphological change (extended cell shape), and cellular apoptosis. Based on the observed phenotypes, possible function of the TbUNC119 and TbUNC119BP is discussed.

Show MeSH
Related in: MedlinePlus