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Triacylglycerol Storage in Lipid Droplets in Procyclic Trypanosoma brucei.

Allmann S, Mazet M, Ziebart N, Bouyssou G, Fouillen L, Dupuy JW, Bonneu M, Moreau P, Bringaud F, Boshart M - PLoS ONE (2014)

Bottom Line: TFEα1 is expressed in procyclic T. brucei and present in glycosomal proteomes, Unexpectedly, a TFEα1 gene knock-out mutant still expressed wild-type levels of previously reported NADP-dependent 3-hydroxyacyl-CoA dehydrogenase activity, and therefore, another gene encodes this enzymatic activity.Homozygous Δtfeα1/Δtfeα1 mutant cells show a normal growth rate and an unchanged glycosomal proteome in procyclic T. brucei.Also, the possibility remains that TAG catabolism is completely repressed by other carbon sources in culture media or developmentally activated in post-procyclic stages in the tsetse.

View Article: PubMed Central - PubMed

Affiliation: Fakultät für Biologie, Genetik, Ludwig-Maximilians-Universität München, Biozentrum, Martinsried, Germany.

ABSTRACT
Carbon storage is likely to enable adaptation of trypanosomes to nutritional challenges or bottlenecks during their stage development and migration in the tsetse. Lipid droplets are candidates for this function. This report shows that feeding of T. brucei with oleate results in a 4-5 fold increase in the number of lipid droplets, as quantified by confocal fluorescence microscopy and by flow cytometry of BODIPY 493/503-stained cells. The triacylglycerol (TAG) content also increased 4-5 fold, and labeled oleate is incorporated into TAG. Fatty acid carbon can thus be stored as TAG in lipid droplets under physiological growth conditions in procyclic T. brucei. β-oxidation has been suggested as a possible catabolic pathway for lipids in T. brucei. A single candidate gene, TFEα1 with coding capacity for a subunit of the trifunctional enzyme complex was identified. TFEα1 is expressed in procyclic T. brucei and present in glycosomal proteomes, Unexpectedly, a TFEα1 gene knock-out mutant still expressed wild-type levels of previously reported NADP-dependent 3-hydroxyacyl-CoA dehydrogenase activity, and therefore, another gene encodes this enzymatic activity. Homozygous Δtfeα1/Δtfeα1 mutant cells show a normal growth rate and an unchanged glycosomal proteome in procyclic T. brucei. The decay kinetics of accumulated lipid droplets upon oleate withdrawal can be fully accounted for by the dilution effect of cell division in wild-type and Δtfeα1/Δtfeα1 cells. The absence of net catabolism of stored TAG in procyclic T. brucei, even under strictly glucose-free conditions, does not formally exclude a flux through TAG, in which biosynthesis equals catabolism. Also, the possibility remains that TAG catabolism is completely repressed by other carbon sources in culture media or developmentally activated in post-procyclic stages in the tsetse.

No MeSH data available.


Related in: MedlinePlus

LD and TAG turnover in WT and Δtfeα1/Δtfeα1 cells.Cells were fed with oleate in glucose-rich SDM79 medium for three days, and after oleate withdrawal samples were taken at the time points indicated. (A) WT cells stained with BODIPY and analyzed by flow cytometry (left y-axis). Error bars represent the SEM of independent replicates (n = 3). The growth curve is given as dashed line (right y-axis). (B) Growth curve and sampling time points (arrows) for the experiments in panels (C) and (D). Total TAG content was determined in triplicate by HPTLC and densitometry in WT (C) and Δtfeα1/Δtfeα1 (D) cells. Error bars represent the SEM of independent replicates (n = 3). The calculated values (filled symbols) account for dilution of LDs or TAG content by cell division, based on the matched growth data.
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pone-0114628-g006: LD and TAG turnover in WT and Δtfeα1/Δtfeα1 cells.Cells were fed with oleate in glucose-rich SDM79 medium for three days, and after oleate withdrawal samples were taken at the time points indicated. (A) WT cells stained with BODIPY and analyzed by flow cytometry (left y-axis). Error bars represent the SEM of independent replicates (n = 3). The growth curve is given as dashed line (right y-axis). (B) Growth curve and sampling time points (arrows) for the experiments in panels (C) and (D). Total TAG content was determined in triplicate by HPTLC and densitometry in WT (C) and Δtfeα1/Δtfeα1 (D) cells. Error bars represent the SEM of independent replicates (n = 3). The calculated values (filled symbols) account for dilution of LDs or TAG content by cell division, based on the matched growth data.

Mentions: The fate of the accumulated LDs in oleate fed cells was then determined. We quantified the kinetics of LD decay upon oleate withdrawal and culture in normal SDM79 medium. The LD decay kinetic was first analyzed by flow cytometry with BODIPY 493/503 staining. After maximal feeding for 3 days, samples were collected over a period of 32 hours. We assumed that in a growing cell population the preformed lipid droplets are equally distributed to daughter cells and therefore calculated the expected fluorescent signal decrease using the population doubling time in the actual experiment, as derived from the growth curve in Fig. 6A. The thereby calculated decay kinetics is represented by filled squares in Fig. 6A. The fluorescence decrease measured from flow cytometry data (open circles) was identical with the calculated kinetic until a basal level was reached. Thus, dilution during cell divisions can fully account for the initial kinetics of LD decay down to basal level. The same kinetic experiment was performed with quantification of the total TAG content by TLC. The growth curve and sampling time points are shown in Fig. 6B and the TAG content kinetics in Fig. 6C, D. Again, a very similar decrease of calculated and experimentally determined TAG content is seen upon oleate withdrawal. Whereas the calculated dilution curve predicts very low TAG levels after several cell cycles, the experimental values return to the basal level maintained by the lipid uptake in normal medium and lipid synthesis. Importantly, the experimental values were never found below the calculated prediction. In summary, there is no net catabolism of the accumulated and stored TAGs, which does not however exclude balanced rates of lipid uptake and degradation in steady state conditions. The Δtfeα1/Δtfeα1 mutant was also analyzed in this experiment (Fig. 6D). The results were identical, and the kinetics for WT and Δtfeα1/Δtfeα1 were perfectly superimposed. This was expected if TFEα1 was not involved in lipid catabolism in procyclic trypanosomes.


Triacylglycerol Storage in Lipid Droplets in Procyclic Trypanosoma brucei.

Allmann S, Mazet M, Ziebart N, Bouyssou G, Fouillen L, Dupuy JW, Bonneu M, Moreau P, Bringaud F, Boshart M - PLoS ONE (2014)

LD and TAG turnover in WT and Δtfeα1/Δtfeα1 cells.Cells were fed with oleate in glucose-rich SDM79 medium for three days, and after oleate withdrawal samples were taken at the time points indicated. (A) WT cells stained with BODIPY and analyzed by flow cytometry (left y-axis). Error bars represent the SEM of independent replicates (n = 3). The growth curve is given as dashed line (right y-axis). (B) Growth curve and sampling time points (arrows) for the experiments in panels (C) and (D). Total TAG content was determined in triplicate by HPTLC and densitometry in WT (C) and Δtfeα1/Δtfeα1 (D) cells. Error bars represent the SEM of independent replicates (n = 3). The calculated values (filled symbols) account for dilution of LDs or TAG content by cell division, based on the matched growth data.
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Related In: Results  -  Collection

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pone-0114628-g006: LD and TAG turnover in WT and Δtfeα1/Δtfeα1 cells.Cells were fed with oleate in glucose-rich SDM79 medium for three days, and after oleate withdrawal samples were taken at the time points indicated. (A) WT cells stained with BODIPY and analyzed by flow cytometry (left y-axis). Error bars represent the SEM of independent replicates (n = 3). The growth curve is given as dashed line (right y-axis). (B) Growth curve and sampling time points (arrows) for the experiments in panels (C) and (D). Total TAG content was determined in triplicate by HPTLC and densitometry in WT (C) and Δtfeα1/Δtfeα1 (D) cells. Error bars represent the SEM of independent replicates (n = 3). The calculated values (filled symbols) account for dilution of LDs or TAG content by cell division, based on the matched growth data.
Mentions: The fate of the accumulated LDs in oleate fed cells was then determined. We quantified the kinetics of LD decay upon oleate withdrawal and culture in normal SDM79 medium. The LD decay kinetic was first analyzed by flow cytometry with BODIPY 493/503 staining. After maximal feeding for 3 days, samples were collected over a period of 32 hours. We assumed that in a growing cell population the preformed lipid droplets are equally distributed to daughter cells and therefore calculated the expected fluorescent signal decrease using the population doubling time in the actual experiment, as derived from the growth curve in Fig. 6A. The thereby calculated decay kinetics is represented by filled squares in Fig. 6A. The fluorescence decrease measured from flow cytometry data (open circles) was identical with the calculated kinetic until a basal level was reached. Thus, dilution during cell divisions can fully account for the initial kinetics of LD decay down to basal level. The same kinetic experiment was performed with quantification of the total TAG content by TLC. The growth curve and sampling time points are shown in Fig. 6B and the TAG content kinetics in Fig. 6C, D. Again, a very similar decrease of calculated and experimentally determined TAG content is seen upon oleate withdrawal. Whereas the calculated dilution curve predicts very low TAG levels after several cell cycles, the experimental values return to the basal level maintained by the lipid uptake in normal medium and lipid synthesis. Importantly, the experimental values were never found below the calculated prediction. In summary, there is no net catabolism of the accumulated and stored TAGs, which does not however exclude balanced rates of lipid uptake and degradation in steady state conditions. The Δtfeα1/Δtfeα1 mutant was also analyzed in this experiment (Fig. 6D). The results were identical, and the kinetics for WT and Δtfeα1/Δtfeα1 were perfectly superimposed. This was expected if TFEα1 was not involved in lipid catabolism in procyclic trypanosomes.

Bottom Line: TFEα1 is expressed in procyclic T. brucei and present in glycosomal proteomes, Unexpectedly, a TFEα1 gene knock-out mutant still expressed wild-type levels of previously reported NADP-dependent 3-hydroxyacyl-CoA dehydrogenase activity, and therefore, another gene encodes this enzymatic activity.Homozygous Δtfeα1/Δtfeα1 mutant cells show a normal growth rate and an unchanged glycosomal proteome in procyclic T. brucei.Also, the possibility remains that TAG catabolism is completely repressed by other carbon sources in culture media or developmentally activated in post-procyclic stages in the tsetse.

View Article: PubMed Central - PubMed

Affiliation: Fakultät für Biologie, Genetik, Ludwig-Maximilians-Universität München, Biozentrum, Martinsried, Germany.

ABSTRACT
Carbon storage is likely to enable adaptation of trypanosomes to nutritional challenges or bottlenecks during their stage development and migration in the tsetse. Lipid droplets are candidates for this function. This report shows that feeding of T. brucei with oleate results in a 4-5 fold increase in the number of lipid droplets, as quantified by confocal fluorescence microscopy and by flow cytometry of BODIPY 493/503-stained cells. The triacylglycerol (TAG) content also increased 4-5 fold, and labeled oleate is incorporated into TAG. Fatty acid carbon can thus be stored as TAG in lipid droplets under physiological growth conditions in procyclic T. brucei. β-oxidation has been suggested as a possible catabolic pathway for lipids in T. brucei. A single candidate gene, TFEα1 with coding capacity for a subunit of the trifunctional enzyme complex was identified. TFEα1 is expressed in procyclic T. brucei and present in glycosomal proteomes, Unexpectedly, a TFEα1 gene knock-out mutant still expressed wild-type levels of previously reported NADP-dependent 3-hydroxyacyl-CoA dehydrogenase activity, and therefore, another gene encodes this enzymatic activity. Homozygous Δtfeα1/Δtfeα1 mutant cells show a normal growth rate and an unchanged glycosomal proteome in procyclic T. brucei. The decay kinetics of accumulated lipid droplets upon oleate withdrawal can be fully accounted for by the dilution effect of cell division in wild-type and Δtfeα1/Δtfeα1 cells. The absence of net catabolism of stored TAG in procyclic T. brucei, even under strictly glucose-free conditions, does not formally exclude a flux through TAG, in which biosynthesis equals catabolism. Also, the possibility remains that TAG catabolism is completely repressed by other carbon sources in culture media or developmentally activated in post-procyclic stages in the tsetse.

No MeSH data available.


Related in: MedlinePlus