Limits...
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.

Show MeSH

Related in: MedlinePlus

Knock-down analysis of TbUNC119.A. Growth of TbUNC119 knock-down procyclic form cells. Three TbUNC119 knock-down inducible cell lines were cloned (TbUNC119-1, TbUNC119-2 and TbUNC119-3). RNAi-mediated gene knock-down was induced with tetracycline. (Tet+) denotes RNAi-induced cells. There was no significant difference in the growth of the cell lines with or without RNAi induction. The inset shows TbUNC119 RT-PCR results with tubulin as a control. B. Motility assay of TbUNC119 knock-down cells. TbUNC119 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. There was no significant difference in the motility of the cell lines with or without RNAi induction.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3008729&req=5

pone-0015577-g001: Knock-down analysis of TbUNC119.A. Growth of TbUNC119 knock-down procyclic form cells. Three TbUNC119 knock-down inducible cell lines were cloned (TbUNC119-1, TbUNC119-2 and TbUNC119-3). RNAi-mediated gene knock-down was induced with tetracycline. (Tet+) denotes RNAi-induced cells. There was no significant difference in the growth of the cell lines with or without RNAi induction. The inset shows TbUNC119 RT-PCR results with tubulin as a control. B. Motility assay of TbUNC119 knock-down cells. TbUNC119 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. There was no significant difference in the motility of the cell lines with or without RNAi induction.

Mentions: To analyze the physiological function of TbUNC119, we first attempted to delete TbUNC119 genes with hygromycin-resistant and neomycin-resistant markers. However no transformants were obtained. Thus we then performed RNAi-mediated gene knock-down analysis. For these studies, we used T. brucei procyclic 29-13 cells, in which RNAi can be induced by the addition of tetracycline. RT-PCR analysis 7 days after RNAi induction revealed marked reduction of TbUNC119 transcripts (Fig. 1A). Western blot analysis also showed marked reduction of TbUNC119 in TbUNC119 knock-down cells 7 days after RNAi induction (Fig. S1). However, there was no significant change in growth between RNAi-induced TbUNC119 knock-down cells and RNAi-uninduced control cells (Fig. 1A). A motility assay also showed no significant change between the RNAi-induced TbUNC119 knock-down cells and RNAi-uninduced control cells (Fig. 1B).


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)

Knock-down analysis of TbUNC119.A. Growth of TbUNC119 knock-down procyclic form cells. Three TbUNC119 knock-down inducible cell lines were cloned (TbUNC119-1, TbUNC119-2 and TbUNC119-3). RNAi-mediated gene knock-down was induced with tetracycline. (Tet+) denotes RNAi-induced cells. There was no significant difference in the growth of the cell lines with or without RNAi induction. The inset shows TbUNC119 RT-PCR results with tubulin as a control. B. Motility assay of TbUNC119 knock-down cells. TbUNC119 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. There was no significant difference in the motility of the cell lines with or without RNAi induction.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3008729&req=5

pone-0015577-g001: Knock-down analysis of TbUNC119.A. Growth of TbUNC119 knock-down procyclic form cells. Three TbUNC119 knock-down inducible cell lines were cloned (TbUNC119-1, TbUNC119-2 and TbUNC119-3). RNAi-mediated gene knock-down was induced with tetracycline. (Tet+) denotes RNAi-induced cells. There was no significant difference in the growth of the cell lines with or without RNAi induction. The inset shows TbUNC119 RT-PCR results with tubulin as a control. B. Motility assay of TbUNC119 knock-down cells. TbUNC119 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. There was no significant difference in the motility of the cell lines with or without RNAi induction.
Mentions: To analyze the physiological function of TbUNC119, we first attempted to delete TbUNC119 genes with hygromycin-resistant and neomycin-resistant markers. However no transformants were obtained. Thus we then performed RNAi-mediated gene knock-down analysis. For these studies, we used T. brucei procyclic 29-13 cells, in which RNAi can be induced by the addition of tetracycline. RT-PCR analysis 7 days after RNAi induction revealed marked reduction of TbUNC119 transcripts (Fig. 1A). Western blot analysis also showed marked reduction of TbUNC119 in TbUNC119 knock-down cells 7 days after RNAi induction (Fig. S1). However, there was no significant change in growth between RNAi-induced TbUNC119 knock-down cells and RNAi-uninduced control cells (Fig. 1A). A motility assay also showed no significant change between the RNAi-induced TbUNC119 knock-down cells and RNAi-uninduced control cells (Fig. 1B).

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