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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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The morphology of TbUNC119-TbUNC119BP double knock-down cells.A. Fluorescent differential interference contrast image of TbUNC119-TbUNC119BP double knock-down procyclic form cells. TbUNC119-TbUNC119BP double knock-down cells 7 days after induction (b) and RNAi-uninduced control cells (a) were labeled with DAPI. Note that the posterior end and the kinetoplast were extended in the RNAi-induced double knock-down cells. The phenotype was clearly observed from 5 days after RNAi induction in double knock-down cells. Bar = 20 µm. B. Scanning electron microscopy of TbUNC119-TbUNC119BP double knock-down procyclic form cells. Double knock-down cells 7 days after RNAi induction (b–d) and RNAi-uninduced control cell (a) were used for the analysis. Bar = 10 µm.
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pone-0015577-g006: The morphology of TbUNC119-TbUNC119BP double knock-down cells.A. Fluorescent differential interference contrast image of TbUNC119-TbUNC119BP double knock-down procyclic form cells. TbUNC119-TbUNC119BP double knock-down cells 7 days after induction (b) and RNAi-uninduced control cells (a) were labeled with DAPI. Note that the posterior end and the kinetoplast were extended in the RNAi-induced double knock-down cells. The phenotype was clearly observed from 5 days after RNAi induction in double knock-down cells. Bar = 20 µm. B. Scanning electron microscopy of TbUNC119-TbUNC119BP double knock-down procyclic form cells. Double knock-down cells 7 days after RNAi induction (b–d) and RNAi-uninduced control cell (a) were used for the analysis. Bar = 10 µm.

Mentions: While counting the numbers of double knock-down cells, we observed abnormally extended trypanosomes in each time point clearly from 5 days after RNAi induction, which were consistent with flow cytometry results (Fig. 5). Fluorescent microscopy and scanning electron microscopy analyses revealed that the phenotype was caused by extension mainly in the region between the posterior end and the kinetoplast, while knock-down had no significant effect on the length between the kinetoplast and the nucleus, or between the nucleus and the anterior end (Fig. 6).


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)

The morphology of TbUNC119-TbUNC119BP double knock-down cells.A. Fluorescent differential interference contrast image of TbUNC119-TbUNC119BP double knock-down procyclic form cells. TbUNC119-TbUNC119BP double knock-down cells 7 days after induction (b) and RNAi-uninduced control cells (a) were labeled with DAPI. Note that the posterior end and the kinetoplast were extended in the RNAi-induced double knock-down cells. The phenotype was clearly observed from 5 days after RNAi induction in double knock-down cells. Bar = 20 µm. B. Scanning electron microscopy of TbUNC119-TbUNC119BP double knock-down procyclic form cells. Double knock-down cells 7 days after RNAi induction (b–d) and RNAi-uninduced control cell (a) were used for the analysis. Bar = 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0015577-g006: The morphology of TbUNC119-TbUNC119BP double knock-down cells.A. Fluorescent differential interference contrast image of TbUNC119-TbUNC119BP double knock-down procyclic form cells. TbUNC119-TbUNC119BP double knock-down cells 7 days after induction (b) and RNAi-uninduced control cells (a) were labeled with DAPI. Note that the posterior end and the kinetoplast were extended in the RNAi-induced double knock-down cells. The phenotype was clearly observed from 5 days after RNAi induction in double knock-down cells. Bar = 20 µm. B. Scanning electron microscopy of TbUNC119-TbUNC119BP double knock-down procyclic form cells. Double knock-down cells 7 days after RNAi induction (b–d) and RNAi-uninduced control cell (a) were used for the analysis. Bar = 10 µm.
Mentions: While counting the numbers of double knock-down cells, we observed abnormally extended trypanosomes in each time point clearly from 5 days after RNAi induction, which were consistent with flow cytometry results (Fig. 5). Fluorescent microscopy and scanning electron microscopy analyses revealed that the phenotype was caused by extension mainly in the region between the posterior end and the kinetoplast, while knock-down had no significant effect on the length between the kinetoplast and the nucleus, or between the nucleus and the anterior end (Fig. 6).

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