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Autophagy in Trypanosoma brucei: amino acid requirement and regulation during different growth phases.

Schmidt RS, Bütikofer P - PLoS ONE (2014)

Bottom Line: We have generated multiple parasite cell lines stably expressing green fluorescent protein- or hemagglutinin-tagged forms of the autophagy marker proteins, TbAtg8.1 and TbAtg8.2, in T. brucei procyclic forms to establish a trypanosome system for quick and reliable determination of autophagy under different culture conditions using flow cytometry.We found that starvation-induced autophagy in T. brucei can be inhibited by addition of a single amino acid, histidine, to the incubation buffer.In addition, we show that autophagy is induced when parasites enter stationary growth phase in culture and that their capacity to undergo starvation-induced autophagy decreases with increasing cell density.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland; Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.

ABSTRACT
Autophagy in the protozoan parasite, Trypanosoma brucei, may be involved in differentiation between different life cycle forms and during growth in culture. We have generated multiple parasite cell lines stably expressing green fluorescent protein- or hemagglutinin-tagged forms of the autophagy marker proteins, TbAtg8.1 and TbAtg8.2, in T. brucei procyclic forms to establish a trypanosome system for quick and reliable determination of autophagy under different culture conditions using flow cytometry. We found that starvation-induced autophagy in T. brucei can be inhibited by addition of a single amino acid, histidine, to the incubation buffer. In addition, we show that autophagy is induced when parasites enter stationary growth phase in culture and that their capacity to undergo starvation-induced autophagy decreases with increasing cell density.

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Processing of GFP-TbAtg8 during parasite growth in culture.T. brucei procyclic forms expressing GFP-TbAtg8.1 were cultured in mid-log phase for several days, then diluted to 3×105 cells/mL at day 0 and kept in culture without further dilution for 7 days. (A) Parasite density (mean values ± standard deviations from three independent cultures). (B) Each day, GFP-TbAtg8.1 fluorescence was determined by flow cytometry before (•) and after (▪) starvation for 2 hours in gHBSS and expressed relative to the values obtained at day 1 (upper panel). The differences between GFP-TbAtg8.1 fluorescence before and after starvation were plotted to obtain a number for the capacity of parasites to undergo autophagy. The values are expressed relative to the difference observed on day 1. (C) Immunoblot of parasite lysates (5×106 cell equivalents per lane) before (−) and after (+) starvation probed with antibodies against GFP (lower panel) or eukaryotic elongation factor 1A (upper panel, used as loading control). (D) Quantification by fluorescence microscopy of GFP-TbAtg8.1 puncta per cell and cells containing puncta in parasites before (•) and after (▪) starvation. At each time point, at least 100 cells per experimental condition were counted. Error bars indicate standard deviations from three independent experiments.
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pone-0093875-g008: Processing of GFP-TbAtg8 during parasite growth in culture.T. brucei procyclic forms expressing GFP-TbAtg8.1 were cultured in mid-log phase for several days, then diluted to 3×105 cells/mL at day 0 and kept in culture without further dilution for 7 days. (A) Parasite density (mean values ± standard deviations from three independent cultures). (B) Each day, GFP-TbAtg8.1 fluorescence was determined by flow cytometry before (•) and after (▪) starvation for 2 hours in gHBSS and expressed relative to the values obtained at day 1 (upper panel). The differences between GFP-TbAtg8.1 fluorescence before and after starvation were plotted to obtain a number for the capacity of parasites to undergo autophagy. The values are expressed relative to the difference observed on day 1. (C) Immunoblot of parasite lysates (5×106 cell equivalents per lane) before (−) and after (+) starvation probed with antibodies against GFP (lower panel) or eukaryotic elongation factor 1A (upper panel, used as loading control). (D) Quantification by fluorescence microscopy of GFP-TbAtg8.1 puncta per cell and cells containing puncta in parasites before (•) and after (▪) starvation. At each time point, at least 100 cells per experimental condition were counted. Error bars indicate standard deviations from three independent experiments.

Mentions: Autophagy has been shown to affect cell growth in various organisms. In Arabidopsis, disruption of autophagy lead to earlier chlorosis under starvation conditions and to earlier leaf senescence under normal conditions [42]. In addition, it was shown to be involved in life-span extension in Caenorhabditis elegans[43] and increased longevity in S. cerevisiae in the non-dividing state [44]. In the protozoan parasite Entamoeba invadens, the number of Atg8-associated structures changed significantly during growth [45]. Based on these reports, we investigated if GFP-TbAtg8.1 puncta formation and processing may also be affected during growth of T. brucei procyclic forms under standard culture conditions. Parasites were cultured for 7 consecutive days without dilution (Fig. 8A) and GFP-TbAtg8.1 fluorescence was determined by flow cytometry. The results show that fluorescence intensity decreased with increasing cell density (Fig. 8B, upper panel), indicating that parasites underwent autophagy as the cell density increased. Subsequently, parasites were challenged during late logarithmic and stationary growth phase by starving for 2 hours in gHBSS to determine their ability to cope with additional stress. The results show that starvation led to a further decrease in GFP-TbAtg8.1 fluorescence (Fig. 8B, upper panel), however, the starvation-induced reduction in fluorescence intensity decreased with increasing cell density. Using the difference in signal intensity before and after starvation as parameter to express the capacity/ability of parasites to perform starvation-induced autophagy (Fig. 8C, lower panel) showed that the ability of T. brucei procyclic forms to undergo autophagy sharply decreased at day 4, i.e. when parasite cultures reached maximal density and entered stationary phase. A similar change in autophagic behavior was also seen when counting the number of GFP-TbAtg8.1 puncta per cell (Fig. 8D, upper panel) or the number of cells containing puncta (Fig. 8D, lower panel). In parallel, protein levels were analyzed by SDS-PAGE and immunoblotting, showing that GFP-TbAtg8.1 decreased with increasing cell density, and after starvation (Fig. 8C).


Autophagy in Trypanosoma brucei: amino acid requirement and regulation during different growth phases.

Schmidt RS, Bütikofer P - PLoS ONE (2014)

Processing of GFP-TbAtg8 during parasite growth in culture.T. brucei procyclic forms expressing GFP-TbAtg8.1 were cultured in mid-log phase for several days, then diluted to 3×105 cells/mL at day 0 and kept in culture without further dilution for 7 days. (A) Parasite density (mean values ± standard deviations from three independent cultures). (B) Each day, GFP-TbAtg8.1 fluorescence was determined by flow cytometry before (•) and after (▪) starvation for 2 hours in gHBSS and expressed relative to the values obtained at day 1 (upper panel). The differences between GFP-TbAtg8.1 fluorescence before and after starvation were plotted to obtain a number for the capacity of parasites to undergo autophagy. The values are expressed relative to the difference observed on day 1. (C) Immunoblot of parasite lysates (5×106 cell equivalents per lane) before (−) and after (+) starvation probed with antibodies against GFP (lower panel) or eukaryotic elongation factor 1A (upper panel, used as loading control). (D) Quantification by fluorescence microscopy of GFP-TbAtg8.1 puncta per cell and cells containing puncta in parasites before (•) and after (▪) starvation. At each time point, at least 100 cells per experimental condition were counted. Error bars indicate standard deviations from three independent experiments.
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Related In: Results  -  Collection

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pone-0093875-g008: Processing of GFP-TbAtg8 during parasite growth in culture.T. brucei procyclic forms expressing GFP-TbAtg8.1 were cultured in mid-log phase for several days, then diluted to 3×105 cells/mL at day 0 and kept in culture without further dilution for 7 days. (A) Parasite density (mean values ± standard deviations from three independent cultures). (B) Each day, GFP-TbAtg8.1 fluorescence was determined by flow cytometry before (•) and after (▪) starvation for 2 hours in gHBSS and expressed relative to the values obtained at day 1 (upper panel). The differences between GFP-TbAtg8.1 fluorescence before and after starvation were plotted to obtain a number for the capacity of parasites to undergo autophagy. The values are expressed relative to the difference observed on day 1. (C) Immunoblot of parasite lysates (5×106 cell equivalents per lane) before (−) and after (+) starvation probed with antibodies against GFP (lower panel) or eukaryotic elongation factor 1A (upper panel, used as loading control). (D) Quantification by fluorescence microscopy of GFP-TbAtg8.1 puncta per cell and cells containing puncta in parasites before (•) and after (▪) starvation. At each time point, at least 100 cells per experimental condition were counted. Error bars indicate standard deviations from three independent experiments.
Mentions: Autophagy has been shown to affect cell growth in various organisms. In Arabidopsis, disruption of autophagy lead to earlier chlorosis under starvation conditions and to earlier leaf senescence under normal conditions [42]. In addition, it was shown to be involved in life-span extension in Caenorhabditis elegans[43] and increased longevity in S. cerevisiae in the non-dividing state [44]. In the protozoan parasite Entamoeba invadens, the number of Atg8-associated structures changed significantly during growth [45]. Based on these reports, we investigated if GFP-TbAtg8.1 puncta formation and processing may also be affected during growth of T. brucei procyclic forms under standard culture conditions. Parasites were cultured for 7 consecutive days without dilution (Fig. 8A) and GFP-TbAtg8.1 fluorescence was determined by flow cytometry. The results show that fluorescence intensity decreased with increasing cell density (Fig. 8B, upper panel), indicating that parasites underwent autophagy as the cell density increased. Subsequently, parasites were challenged during late logarithmic and stationary growth phase by starving for 2 hours in gHBSS to determine their ability to cope with additional stress. The results show that starvation led to a further decrease in GFP-TbAtg8.1 fluorescence (Fig. 8B, upper panel), however, the starvation-induced reduction in fluorescence intensity decreased with increasing cell density. Using the difference in signal intensity before and after starvation as parameter to express the capacity/ability of parasites to perform starvation-induced autophagy (Fig. 8C, lower panel) showed that the ability of T. brucei procyclic forms to undergo autophagy sharply decreased at day 4, i.e. when parasite cultures reached maximal density and entered stationary phase. A similar change in autophagic behavior was also seen when counting the number of GFP-TbAtg8.1 puncta per cell (Fig. 8D, upper panel) or the number of cells containing puncta (Fig. 8D, lower panel). In parallel, protein levels were analyzed by SDS-PAGE and immunoblotting, showing that GFP-TbAtg8.1 decreased with increasing cell density, and after starvation (Fig. 8C).

Bottom Line: We have generated multiple parasite cell lines stably expressing green fluorescent protein- or hemagglutinin-tagged forms of the autophagy marker proteins, TbAtg8.1 and TbAtg8.2, in T. brucei procyclic forms to establish a trypanosome system for quick and reliable determination of autophagy under different culture conditions using flow cytometry.We found that starvation-induced autophagy in T. brucei can be inhibited by addition of a single amino acid, histidine, to the incubation buffer.In addition, we show that autophagy is induced when parasites enter stationary growth phase in culture and that their capacity to undergo starvation-induced autophagy decreases with increasing cell density.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland; Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.

ABSTRACT
Autophagy in the protozoan parasite, Trypanosoma brucei, may be involved in differentiation between different life cycle forms and during growth in culture. We have generated multiple parasite cell lines stably expressing green fluorescent protein- or hemagglutinin-tagged forms of the autophagy marker proteins, TbAtg8.1 and TbAtg8.2, in T. brucei procyclic forms to establish a trypanosome system for quick and reliable determination of autophagy under different culture conditions using flow cytometry. We found that starvation-induced autophagy in T. brucei can be inhibited by addition of a single amino acid, histidine, to the incubation buffer. In addition, we show that autophagy is induced when parasites enter stationary growth phase in culture and that their capacity to undergo starvation-induced autophagy decreases with increasing cell density.

Show MeSH
Related in: MedlinePlus