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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

Oleate feeding stimulates lipid droplet formation in procyclic T. brucei cells.Staining of lipid droplets with nile red (A) or BODIPY 493/503 (B) was as detailed in experimental procedures. Myriocin treatment (0.5 µM for 24 h) was included for comparison to a previous report [36]. An example of several experiments is shown.
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pone-0114628-g001: Oleate feeding stimulates lipid droplet formation in procyclic T. brucei cells.Staining of lipid droplets with nile red (A) or BODIPY 493/503 (B) was as detailed in experimental procedures. Myriocin treatment (0.5 µM for 24 h) was included for comparison to a previous report [36]. An example of several experiments is shown.

Mentions: After oleate/BSA feeding of procyclic trypanosomes for 2–3 days the number of nile red stained LDs increased (Fig. 1A), as previously shown upon drug treatment with myriocin [35] and included here for reference (Fig. 1A). Whereas myriocin treatment led to a cytokinesis phenotype [35], feeding with oleate/BSA did not change the growth rate. The effect of oleate feeding was quantified by counting the number of nile red stained LDs per cell in stacks of confocal laser scanning images. The average number of LDs per cell increased almost 5-fold compared to unfed cells (Fig. 2A). The histogram in Fig. 2B shows the bell-shaped, apparently normal, distribution of the LD numbers per cell in the populations. The maximum number of LDs that a single cell can build up, nine LDs in oleate fed cells in our experiments with strain AnTat1.1, may depend on cell clone-specific properties like uptake capacity and growth rate. A similar argument applies to the average number of lipid droplets in unfed cells that is also likely to depend on the batch of FCS and the amount of fatty acids (FAs) contained within. As a routine assay to quantify LDs in T. brucei, we optimized flow cytometry after BODIPY 493/503 staining. The microscopic picture upon BODIPY 493/503 staining is not different from nile red staining (Fig. 1B). Yet, nile red has wide and overlapping emission spectra when bound to polar and nonpolar lipids, whereas BODIPY 493/503 accumulates more specifically in the nonpolar lipophilic environment in LDs [20]. Flow cytometry integrates the fluorescence signal of the whole cell, and therefore low background from membrane lipid staining is essential for LD quantification by flow cytometry. The validity of the flow cytometric assay was demonstrated by an increase of the fluorescence signal between the unfed and oleate fed cells (Fig. 2C), that was very close (4.6-fold) to the increase determined by microscopic LD counting (4.7-fold, Fig. 2A). The TAG content of cells incubated with or without oleate was also directly quantified by thin layer chromatography (TLC) (Fig. 2D), again resulting in the very same increase (4.6-fold). The perfect quantitative correlation of LD numbers, flow cytometry and TAG analysis upon oleate feeding, strongly suggests that oleate uptake results in TAG storage in LDs. The TAG species in oleate fed and unfed cells were then analyzed by mass spectrometry. A high number of 96 TAG species were resolved and identified (S1 Figure). Such a high number of TAG species has already been observed in serum and butter [37], [38]. In both conditions the 54∶2,3,4 TAG species were by far the predominant species and were significantly increased upon oleate feeding (Fig. 3A). As oleate is a C18 fatty acid with one unsaturated double bond, the predominant 54∶3 TAG species provides evidence that at least part of the oleate taken up is esterified with glycerol for storage in lipid droplets. To directly follow incorporation of oleate into TAGs, we performed a labeling experiment with [14C]-oleate (Fig. 3B). Procyclic trypanosomes were cultured in the presence of radiolabeled oleate up to 8 hours. Samples were collected during this uptake time course and labeled lipid species were separated by TLC and quantified using a phosphor imager. Oleate was incorporated into TAG as well as into phospholipids (PPL) in a time-dependent manner.


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)

Oleate feeding stimulates lipid droplet formation in procyclic T. brucei cells.Staining of lipid droplets with nile red (A) or BODIPY 493/503 (B) was as detailed in experimental procedures. Myriocin treatment (0.5 µM for 24 h) was included for comparison to a previous report [36]. An example of several experiments is shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4262433&req=5

pone-0114628-g001: Oleate feeding stimulates lipid droplet formation in procyclic T. brucei cells.Staining of lipid droplets with nile red (A) or BODIPY 493/503 (B) was as detailed in experimental procedures. Myriocin treatment (0.5 µM for 24 h) was included for comparison to a previous report [36]. An example of several experiments is shown.
Mentions: After oleate/BSA feeding of procyclic trypanosomes for 2–3 days the number of nile red stained LDs increased (Fig. 1A), as previously shown upon drug treatment with myriocin [35] and included here for reference (Fig. 1A). Whereas myriocin treatment led to a cytokinesis phenotype [35], feeding with oleate/BSA did not change the growth rate. The effect of oleate feeding was quantified by counting the number of nile red stained LDs per cell in stacks of confocal laser scanning images. The average number of LDs per cell increased almost 5-fold compared to unfed cells (Fig. 2A). The histogram in Fig. 2B shows the bell-shaped, apparently normal, distribution of the LD numbers per cell in the populations. The maximum number of LDs that a single cell can build up, nine LDs in oleate fed cells in our experiments with strain AnTat1.1, may depend on cell clone-specific properties like uptake capacity and growth rate. A similar argument applies to the average number of lipid droplets in unfed cells that is also likely to depend on the batch of FCS and the amount of fatty acids (FAs) contained within. As a routine assay to quantify LDs in T. brucei, we optimized flow cytometry after BODIPY 493/503 staining. The microscopic picture upon BODIPY 493/503 staining is not different from nile red staining (Fig. 1B). Yet, nile red has wide and overlapping emission spectra when bound to polar and nonpolar lipids, whereas BODIPY 493/503 accumulates more specifically in the nonpolar lipophilic environment in LDs [20]. Flow cytometry integrates the fluorescence signal of the whole cell, and therefore low background from membrane lipid staining is essential for LD quantification by flow cytometry. The validity of the flow cytometric assay was demonstrated by an increase of the fluorescence signal between the unfed and oleate fed cells (Fig. 2C), that was very close (4.6-fold) to the increase determined by microscopic LD counting (4.7-fold, Fig. 2A). The TAG content of cells incubated with or without oleate was also directly quantified by thin layer chromatography (TLC) (Fig. 2D), again resulting in the very same increase (4.6-fold). The perfect quantitative correlation of LD numbers, flow cytometry and TAG analysis upon oleate feeding, strongly suggests that oleate uptake results in TAG storage in LDs. The TAG species in oleate fed and unfed cells were then analyzed by mass spectrometry. A high number of 96 TAG species were resolved and identified (S1 Figure). Such a high number of TAG species has already been observed in serum and butter [37], [38]. In both conditions the 54∶2,3,4 TAG species were by far the predominant species and were significantly increased upon oleate feeding (Fig. 3A). As oleate is a C18 fatty acid with one unsaturated double bond, the predominant 54∶3 TAG species provides evidence that at least part of the oleate taken up is esterified with glycerol for storage in lipid droplets. To directly follow incorporation of oleate into TAGs, we performed a labeling experiment with [14C]-oleate (Fig. 3B). Procyclic trypanosomes were cultured in the presence of radiolabeled oleate up to 8 hours. Samples were collected during this uptake time course and labeled lipid species were separated by TLC and quantified using a phosphor imager. Oleate was incorporated into TAG as well as into phospholipids (PPL) in a time-dependent manner.

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