Biochem. J. (2009) 417, 685–694 (Printed in Great Britain)
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doi:10.1042/BJ20080167
Glycosylphosphatidylinositol-specific phospholipase C regulates transferrin endocytosis in the African trypanosome Sandesh SUBRAMANYA*, C. Frank HARDIN*, Dietmar STEVERDING† and Kojo MENSA-WILMOT*1 *Department of Cellular Biology, University of Georgia, 724 Biological Sciences Building, Athens, GA 30602, U.S.A., and †BioMedical Research Centre, School of Medicine, Health Policy and Practice, University of East Anglia, Norwich NR4 7TJ, U.K.
GPI-PLC (glycosylphosphatidylinositol-specific phospholipase C) is expressed in bloodstream-form Trypanosoma brucei, a protozoan that causes human African trypanosomiasis. Loss of genes encoding GPI-PLC reduces the virulence of a pleomorphic strain of the parasite, for reasons that are not clear. In the present paper, we report that GPI-PLC stimulates endocytosis of transferrin by 300–500 %. Surprisingly, GPI-PLC is not detected at endosomes, suggesting that the enzyme does not interact directly with the endosomal machinery. We therefore hypothesized that a diffusible product of the GPI-PLC enzyme reaction [possibly DAG (diacylglycerol)] mediated the biological effects of the protein. Two sets of data support this assertion. First, a catalytically inactive Q81L mutant of GPI-PLC, expressed in a GPI-PLC-null background, had no effect on endocytosis, indicating that enzyme activity is essential for the protein to
stimulate endocytosis. Secondly, the exogenous DAGs OAG (1-oleyl-2-acetyl-sn-glycerol) and DMG (dimyristoylglycerol) independently stimulated endocytosis of transferrin. Furthermore, the DAG mimic PMA, a phorbol ester, also activated endocytosis in T. brucei. DAG-stimulated endocytosis is a novel pathway in the trypanosome. We surmise that (i) GPI-PLC regulates transferrin endocytosis in T. brucei, (ii) GPI-PLC is a signalling enzyme, and (iii) DAG is a second messenger for GPI-PLC. We propose that regulation of endocytosis is a physiological function of GPI-PLC in bloodstream T. brucei.
INTRODUCTION
being pursued (reviewed in [12]), little is known about smallmolecule regulators of the pathway in T. brucei. GPI-PLC [GPI (glycosylphosphatidylinositol)-specific phospholipase C] is expressed specifically in bloodstream T. brucei, which can differentiate to insect stage (procyclic) cells. During transformation of the parasite, GPI-PLC contributes to the release of VSG (variant surface glycoprotein) from the plasma membrane of the parasite [13,14], although the bulk of the enzyme has not been localized to the plasma membrane [15]. In non-differentiating bloodstream-form T. brucei, GPI-PLC is a virulence factor. In a mouse model of human African trypanosomiasis, the enzyme contributes to the virulence of a pleomorphic strain of T. brucei [16]. In the present paper, we report a new physiological function of GPI-PLC as a signalling enzyme that stimulates endocytosis in T. brucei. Furthermore, we demonstrate that exogenously added DAG modulates endocytosis in the parasite. DAG-regulated endocytosis is a novel pathway in T. brucei. We surmise that the importance of GPI-PLC in the virulence of T. brucei involves its contributions to the endocytosis of transferrin, a growth factor for the parasite [17].
Endocytosis in eukaryotes is important for uptake of some nutrients (e.g. iron, and cholesterol esters), maintenance of cell volume and for modulation of cell signalling (reviewed in [1]). Lipids (e.g. polyphosphoinositides) regulate various steps of endocytosis. DAG (diacylglycerol) is an intracellular second messenger that can be produced by phospholipases C. Phorbol esters (e.g. PMA) mimic the biological actions of DAG [2], and are frequently used in biochemical studies of DAG signalling. Trypanosoma brucei resides in blood and lymphatic tissue, and causes human African trypanosomiasis. As an extracellular parasite, T. brucei depends on endocytosis to take up some nutrients from host blood [3,4]. For example, transferrin, which is essential for the acquisition of iron by the parasite, is endocytosed by a unique receptor [4–6]. Endocytosis is also important for clearing of anti-parasite antibody from the plasma membrane of T. brucei [7]. The presence of anti-parasite antibody on the plasma membrane may lead to phagocytosis and killing of the trypanosome by macrophages [7]. Endocytosis is essential for viability and, by extension, virulence of T. brucei [8,9]. In contrast with its beneficial effects on the physiology of T. brucei, endocytosis of a host-derived anti-parasite toxin (e.g. trypanosome lytic factor or tumour necrosis factor) can kill the parasite (reviewed in [10,11]). Therefore survival of the parasite in host blood probably requires a delicate balance of endocytic pathways. Whereas studies of the core endocytosis machinery are
Key words: diacylglycerol (DAG), endocytosis, glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC), signalling, transferrin, Trypanosoma brucei.
MATERIALS AND METHODS Cell type/strains
Culture-adapted T. brucei 427 was a gift from Professor C. C. Wang (Department of Pharmaceutical Chemistry, University
Abbreviations used: AB, assay buffer; BBS/G, bicine-buffered saline with glucose; BCA, bicinchoninic acid; BCIP, 5-bromo-4-chloroindol-3-yl phosphate; BiP, immunoglobulin heavy-chain-binding protein; DAG, diacylglycerol; DAPI, 4 ,6-diamidino-2-phenylindole; DMG, dimyristoyl-sn-glycerol; ESAG, expression-site-associated gene; GPI, glycosylphosphatidylinositol; GPI-PLC, GPI-specific phospholipase C; IMDM, Iscove’s modified Dulbecco’s medium; NBT, Nitro Blue Tetrazolium; NP40, Nonidet P40; OAG, 1-oleyl-2-acetyl-sn-glycerol; RFU, relative fluorescence units; TfR, transferrin receptor; VSG, variant surface glycoprotein. 1 To whom correspondence should be addressed (email
[email protected]). c The Authors Journal compilation c 2009 Biochemical Society
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of California, San Francisco, San Francisco, CA, U.S.A.). Bloodstream-form T. brucei RUMP528 (GPI-PLC−/− ) [18] was provided by Professor George Cross (Laboratory of Molecular Parasitology, Rockefeller University, New York, NY, U.S.A.). Bloodstream T. brucei were cultured in HMI-9 medium [19]. Materials
Restriction enzymes were from New England Biolabs. NP40 (Nonidet P40), hygromycin and phleomycin were from Calbiochem. Plasmid purification kits were from Qiagen. SDS, Triton X-100, BCIP (5-bromo-4-chloroindol-3-yl phosphate) and NBT (Nitro Blue Tetrazolium) were from Bio-Rad Laboratories. Fetal bovine serum and newborn calf serum were from Hyclone. Leupeptin, aprotinin and alkaline phosphatase-conjugated goat anti-rabbit secondary antibody were from Roche. Antibody against VSG221 was provided by Professor George Cross. Alexa Fluor® 488-conjugated goat anti-rabbit/anti-mouse IgG, Alexa Fluor® 594-conjugated goat anti-rabbit/anti-mouse IgG, transferrin–Alexa Fluor® 594 and dextran–Texas Red were purchased from Molecular Probes. BCA (bicinchoninic acid) protein detection kit and DMSO were purchased from Pierce. Immobilon P membrane was obtained from Millipore. PMA, its analogue 4α-PMA and all other reagents were from Sigma. Plasmid construction
The GPI-PLC expression plasmid pLew-GPIPLC was obtained by insertion of a GPI-PLC coding region (from pUTK-GPIPLC [20,21]) into a T. brucei expression plasmid pLew82 in place of a luciferase gene [22]. The GPI-PLC DNA insert was generated by PCR using a lz-acc KCR4 forward primer (5 -TAAAAGCTTTTAACACAGGAGGCAGACCATGTTTGGTGGT-3 ) and KCR5 reverse primer (5 -TATGTGGATCCTTATGACCTTGCGGTTTGGTT-3 ), with pUTK-GPIPLC as a template [21,23]. The PCR product was digested with BamHI and HindIII, gelpurified, ligated into pLew82 [22] and transfected into Escherichia coli XL10 cells. DNA encoding a Q81L mutant of GPIPLC (Q81L_GPIPLC) was excised from pUTK constructs [21] and subcloned into pLew82. DNA transfection of a mutant GPI-PLC −/− T. brucei strain
Bloodstream form T. brucei RUMP528 (GPI-PLC−/− ) was refractory to transformation using standard procedures. To transfect DNA into T. brucei RUMP528, major modifications of published protocols were implemented. Cells were grown to a density of 2 × 106 cells/ml, centrifuged at 5000 g for 5 min at room temperature (22 ◦C), washed once with PBS (136 mM NaCl, 27 mM KCl, 5.3 mM Na2 HPO4 and 1.7 mM KH2 PO4 ), and washed once with Cytomix. The cell pellet was resuspended in 400 μl of Cytomix to a final density of 2.5 × 108 cells/ml. Plasmid DNA (100 μg) that had been purified using Qiagen-tip 500 gravity-flow columns (Qiagen), linearized with NotI and dissolved in 100 μl of TE (10 mM Tris/HCl and 1 mM EDTA, pH 8.0) was added to the cell suspension. Cells were electroporated in 4-mm-pathlength cuvettes with a Bio-Rad Gene Pulser II at 1.6 kV, 50 μF timing and 360 . Cells were cloned immediately after electroporation as follows: from the electroporated cell suspension, 170, 17 or 2 μl was added to 12 ml of pre-warmed HMI-9 medium in 75 cm2 culture flasks. Hygromycin (5 μg/ml, final concentration) and G418 (2.5 μg/ml, final concentration) were added to the three flasks, which were incubated for 16 h at 37 ◦C before phleomycin (final concentration of 1 μg/ml) was added. Each diluted cell suspension was divided c The Authors Journal compilation c 2009 Biochemical Society
into 24-well plates (1 ml/well) and incubated at 37 ◦C for 1– 2 weeks. Only cell lines obtained from dilutions that yielded less than eight wells (out of 24) with parasites were propagated. Stable transfectants were maintained in HMI-9 medium containing all three antibiotics (above). During passage, cells that reached lateexponential phase (2 × 106 cells/ml) were diluted back to 2 × 104 cells/ml in HMI-9 medium. GPI-PLC enzyme assay
T. brucei expressing various GPI-PLC mutants were induced for 16 h with tetracycline (1 μg/ml), harvested (107 cells) and washed once with PBS. The cell pellet was lysed in 200 μl of HLB (hypotonic lysis buffer) (10 mM sodium phosphate and 1 mM EDTA, pH 8.0) containing protease inhibitors [2.1 mM leupeptin, 0.1 mM tosyl-lysylchloromethane (‘TLCK’) and 0.4 unit of aprotinin]. This lysate was incubated for 20 min on ice and centrifuged at 14 000 g for 20 min at 4 ◦C. The pellet was solubilized in 100 μl of AB (assay buffer; 1 % NP40, 5 mM EDTA and 50 mM Tris/HCl, pH 8.0). Then, 5 μl of the above detergent extract was added to 25 μl of AB containing 2 μg of 3 H-labelled membrane-form VSG on ice. The mixture was incubated at 37 ◦C for 15 min, before the [3 H]dimyristoylglycerol released from 3 H-labelled membrane-form VSG was extracted with water-saturated n-butanol and quantified by scintillation counting. Protein concentration of cell lysates was determined using a BCA assay. Fluorescence microscopy
The reagents and methods used to detect intracellular GPI-PLC and endocytosed fluorescent transferrin were as described in [15,20]. Endosomal compartments were identified after uptake of the fluorescent marker FM4-64. For endocytosis, cells at a density of 106 /ml were pelleted gently by centrifugation at 2000 g for 2 min and resuspended in 500 μl of serum-free HMI-9 medium containing 10 μM FM4-64. Cells were incubated at 37 ◦C for 10 min before fixation, permeabilization and visualization (see below). Exponential-phase cells (106 /ml) were harvested, washed once with PBS (pH 7.4) and fixed in paraformaldehyde (400 μl of a 2 % solution in PBS) for 8 min on ice, before being quenched with 1 ml of 0.5 M glycine in PBS for 5 min at room temperature. Cells were washed once in PBS, settled on poly-L-lysine-coated coverslips for 30 min, permeabilized with methanol (pre-chilled at − 20 ◦C) for 2 min at 4 ◦C and washed twice with PBS. Nonspecific protein-adsorption sites were blocked with 1 % (w/v) BSA in PBS for 30 min at room temperature. To detect GPIPLC, anti-GPI-PLC monoclonal antibody 2A6-6 [24] (100 μl of a 1:1500 dilution in blocking solution) was used. In order to reduce the background from non-specific antibody adsorption, 2A6-6 (150 μl of a 1:1200 dilution) was pre-adsorbed on coverslips containing fixed and permeabilized T. brucei RUMP528 cells (GPI-PLC−/− ) for 15 min at room temperature. The coverslips were removed carefully, and the pre-adsorbed antibody solution was recovered for GPI-PLC detection. Cells were incubated with the pre-absorbed primary antibody for 1 h at room temperature and washed as follows: once with PBS, twice with high-salt buffer (PBS containing 500 mM NaCl) and twice with PBS. The cells were then incubated with Alexa Fluor® 594-conjugated goat anti-mouse IgG (200 μl of a 1:2000 dilution) for 1 h at room temperature in the dark. Coverslips were washed twice with PBS before mounting on slides. The cell nucleus and kinetoplast (mitochondrial) DNA were stained with 10 μM DAPI (4 ,6-diamidino-2-phenylindole) in
Glycosylphosphatidylinositol-specific phospholipase C
CitiFluor anti-fade reagent (CitiFluor Ltd). Cells were viewed with a fluorescence microscope (Leica DMIRBE). Images were captured with an interline chip-cooled CCD (charge-coupled device) camera (Orca 9545; Hamamatsu) and processed with Openlab 3.1.2 software (Improvision). SDS/PAGE and Western blotting
To detect GPI-PLC, an aliquot (10 μl) of solubilized membrane fractions (from 107 cells) was heated at 90 ◦C for 2 min after the addition of an equal volume of 2.5× SDS/PAGE sample buffer [25 % (v/v) glycerol, 5 % (w/v) SDS, 5 % (v/v) 2-mercaptoethanol, 0.05 % Bromophenol Blue and 0.05 M Tris/HCl, pH 6.8]. Total membrane proteins were resolved by SDS/PAGE (12 % minigel), and electrotransferred on to Immobilon P using a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad). GPI-PLC on Immobilon P was detected by Western blotting with anti-GPI-PLC (R18B3) antibody [20]. Enzymelinked secondary antibody was detected with BCIP and NBT. TfR (transferrin receptor) Western blotting was performed as follows. Bloodstream-form T. brucei 427 (GPI-PLC+/+ ) or T. brucei RUMP528 (GPI-PLC−/− ) were pelleted and lysed in 3× SDS/PAGE sample buffer. Proteins in 2 × 106 cell equivalents (per lane) were resolved by SDS/PAGE (14 % gels), transferred on to Immobilon P membrane and blocked with 5 % (w/v) nonfat dried skimmed milk powder and 0.1 % Tween 20 in PBS. The membrane was incubated with anti-TfR antibody (diluted 1:10000) [6] followed by goat anti-rabbit antibody conjugated to alkaline phosphatase (diluted 1:1000), after which colour was developed with BCIP/NBT staining. In a control experiment, T. brucei BiP (immunoglobulin heavy-chain-binding protein) was detected using a similar protocol except that anti-BiP antibody (a gift from Professor Jay Bangs, Medical Microbiology and Immunology, University of Wisconsin, Madison, WI, U.S.A.) [25] was used with 2 × 105 cell equivalents of protein per lane. Endocytosis assays
For internalization of dextran–Texas Red or transferrin–Alexa Fluor® 594, bloodstream-form T. brucei was cultured to a density of 106 cells/ml. Cells (5 × 107 ) were pelleted, rinsed with buffer containing BBS/G (bicine-buffered saline with glucose: 50 mM bicine, 50 mM NaCl, 5 mM KCl and 1 % glucose, pH 7.4), and centrifuged at 1400 g for 5 min at room temperature. The cell pellet was resuspended in ice-cold serum-free IMDM (Iscove’s modified Dulbecco’s medium), and the cell suspension was stored on ice for 10 min. Dextran–Texas Red was added to a final concentration of 10 μg/ml, and the cell suspension was incubated at 37 ◦C. Transferrin–Alexa Fluor® 594 was added at 25 μg/ml (final concentration). At different time intervals, aliquots of cells (5 × 106 ) were withdrawn and pelleted at 5000 g for 5 min at 4 ◦C. The supernatant was discarded, and the cell pellet was washed five times with ice-cold BBS/G containing 2 % (w/v) sodium azide (5000 g for 3 min at 4 ◦C). The final pellet was resuspended in 100 μl of the same buffer (ice-cold) and deposited into 96-well plates that were kept on ice at all times. Fluorescence of the resuspended cells was measured with a fluorimeter (Fluostar Galaxy, BMG Labtech), with excitation at 594 nm and emission at 612 nm. The instrument was initially set to measure the maximum gain in all wells of the 96-well plate to determine the peak fluorescence. Fluorescence in RFU (relative fluorescence units) was read against a blank that consisted of cells that did not receive the fluorescent endocytosis cargo. Values determined for the blank were subtracted from readings of the experimental samples, before plotting the RFU for each sample. Each set of assays was
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performed in triplicate. Each set of data was repeated multiple times with similar results, a representative of which is presented in the Figures. Endocytic membrane recycling assay with FM4-64
To monitor membrane recycling within the exo/endo-cytic system, T. brucei 427 (wild-type) and T. brucei RUMP528 (GPI-PLC−/− ) were harvested and washed once with BBS/G. FM4-64 (10 μM final concentration) was added to cells in 1 ml of ice-cold serumfree IMDM. After incubation for 30 min at 37 ◦C to allow internalization of the dye, cells (2 × 106 ) were washed once in BBS/G at 4 ◦C and incubated at 37 ◦C. At specified time intervals, aliquots (containing 4 × 106 cells) were washed once in BBS/G and centrifuged at 5000 g for 5 min at 4 ◦C, and the supernatant was saved. To the supernatant, CHAPS was added (final concentration of 1 %). The cell pellet was washed three times and resuspended in 100 μl of BBS/G. FM4-64 fluorescence associated with cells or released into the medium was quantified with a fluorimeter (excitation at 520 nm and emission at 670 nm). Each data point was acquired in triplicate. RFU were obtained as described for endocytosis assays (above). This protocol was modified from an assay developed by Riezman and colleagues [26]. RESULTS Transferrin endocytosis assay: the effect of metabolic poisons
We monitored a biological function of GPI-PLC in T. brucei by quantifying cell-associated transferrin. For this reason, it was important to evaluate whether or not the binding of transferrin to the parasite resulted in endocytosis of the ligand. In general, endocytosis is an energy-requiring cellular process. Therefore we envisaged that interference with energy metabolism in T. brucei would inhibit the endocytosis of transferrin. Bloodstream T. brucei relies almost exclusively on glycolysis for energy production. 2-Deoxyglucose antagonizes glucose metabolism [27]. Sodium azide is a metabolic poison [28,29] that kills T. brucei [30]. Therefore we used 2-deoxyglucose and sodium azide independently to evaluate the energy-dependence of transferrin uptake. Finally, we tested (at the single-cell level) whether cellassociated transferrin was intracellular and could be co-localized with lysosomes, the terminal station of the endocytic pathway. Uptake of transferrin was blocked by both sodium azide (Figure 1A) and 2-deoxyglucose (Figure 1B), indicating that endocytosis of the ligand was energy-dependent. Thus transferrin was not merely associating with its receptor on the plasma membrane, because binding of transferrin to its receptor does not require energy. Internalization of transferrin was confirmed in singlecell assays in which the intracellular movement of the ligand was tracked microscopically in a time-course study. At early times (i.e. less than 10 min) after addition of transferrin to the parasite, the ligand was associated with intracellular structures, presumably endosomes, found between the nucleus and the kinetoplast; transferrin was not concentrated on the plasma membrane and did not co-localize with the lysosome (identified with anti-p67 antibody). At 15 min and later, most of the transferrin co-localized with p67 (Figure 1C), signifying delivery of the ligand to the lysosome. In conclusion, our endocytosis assay quantifies energydependent internalization of transferrin into the endolysosomal system of T. brucei. GPI-PLC activates transferrin endocytosis in T. brucei
The physiological functions of GPI-PLC in rapidly dividing (i.e. non-differentiating) bloodstream T. brucei are not resolved [15]. c The Authors Journal compilation c 2009 Biochemical Society
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Figure 2
GPI-PLC stimulates endocytosis in T. brucei
(A) Endocytosis of transferrin. Bloodstream-form T. brucei 427 (GPI-PLC +/+ ) or T. brucei RUMP528 (GPI-PLC −/− ) was incubated with transferrin–Alexa Fluor® 594. At the times indicated, cells (5 × 106 ) were washed with 1× BBS/G containing 0.03 mM sodium azide. Fluorescence of cells was measured (excitation at 570 nm and emission at 620 nm). Results −/− T. brucei . T. brucei TfR (the are means + − S.D. (n = 6). (B) TfR is not reduced in GPI-PLC ESAG6–ESAG7 complex) was detected by Western blotting with anti-TfR antibody. The typical pattern of heterogeneously glycosylated ESAG6 and ESAG7 is observed in both wild-type and RUMP528 T. brucei . In control experiments, BiP was detected as a protein-loading control. The position of a 70 kDa protein is indicated.
Figure 1 Sodium azide and 2-deoxyglucose inhibit the endocytosis of transferrin (A) Effect of sodium azide on endocytosis of transferrin. Bloodstream-form T. brucei 427 was incubated in PBS containing sodium azide (0.03 mM) for 15 min at 37 ◦C (control cells were treated with 1× PBS). Subsequently, cells were incubated with transferrin–Alexa Fluor® 594 at 37 ◦C for the times indicated. After specified periods, cells (5 × 106 ) were washed with 1× BBS/G containing 0.03 mM sodium azide, and the fluorescence of cells was measured (excitation at 570 nm and emission at 620 nm). Fluorescence of control cells that did not receive transferrin–Alexa Fluor® 594 was subtracted from the fluorescence signal for each set of triplicate data points. Results are means + − S.D. (n = 3). (B) 2-Deoxyglucose inhibits transferrin endocytosis. T. brucei was incubated in PBS containing 50 mM 2-deoxyglucose or 50 mM glucose (control cells). Trypanosomes were then allowed to endocytose fluorescent transferrin, and the amount of ligand uptake was quantified as described in (A). (C) Transferrin is internalized by the trypanosome. T. brucei 427 cells were exposed to transferrin–Alexa Fluor® 594 (red) at 37 ◦C as described in (A). At indicated times, cells were fixed with paraformaldehyde, permeabilized with methanol and incubated with anti-p67 antibody followed by Alexa Fluor® 488-conjugated goat anti-rabbit IgG (green). DNA (nuclear; n) and mitochondrial (kinetoplast; k) were detected by staining with DAPI (blue).
DAG is a product of GPI-PLC cleavage of GPIs or PtdIns. In vertebrate cells, DAG can regulate endocytosis [31,32]. We therefore tested whether GPI-PLC influenced endocytosis in T. brucei. In initial experiments, we used two strains: T. brucei c The Authors Journal compilation c 2009 Biochemical Society
427 that expresses GPI-PLC, and T. brucei RUMP528, which lacks GPI-PLC genes [18,33]. Receptor-mediated endocytosis of transferrin was quantified in the parasites. Parental T. brucei 427 accumulated up to 5-fold more transferrin than T. brucei RUMP528 (GPI-PLC−/− ) (Figure 2A). We tested a hypothesis that decreased endocytosis in T. brucei RUMP528 (GPI-PLC−/− ) cells resulted from a reduction in the amount of TfR, comprising ESAG (expression-site-associated gene) 6 and ESAG7 proteins [6,34,35]. In Western blot assays, no significant difference was observed in the amount of ESAG6 and ESAG7 between wild-type T. brucei and the GPI-PLC-deficient T. brucei RUMP528 cell lines (Figure 2B). This result implies that the amount of TfR in both cell lines is the same. Therefore the deficiency in transferrin endocytosis of T. brucei RUMP528 (Figure 2A) is not explained by the loss of TfR in T. brucei RUMP528. General membrane endocytosis and exocytosis is not affected by GPI-PLC
Decreased accumulation of transferrin in T. brucei RUMP528 (GPI-PLC−/− ) (Figure 2) could be explained in two other ways.
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First, the rate of transferrin uptake in T. brucei RUMP528 may be lower compared with parental T. brucei 427. Alternatively, a higher rate of exocytosis in T. brucei RUMP528, compared with parental T. brucei 427, could decrease the amount of transferrin that accumulates in the mutant T. brucei. To distinguish between these possibilities, we tested whether exocytosis of membranes differed between T. brucei 427 and T. brucei RUMP528. The fluorescent dye FM4-64 has been used to study recycling of the plasma membrane in Saccharomyces cerevisiae and in neurons [26,36]. At low temperatures, the dye adsorbs to the plasma membrane, which is endocytosed when cells are warmed. Exocytosis of membranes can be evaluated by monitoring the release of FM4-64 into the external medium after pre-loading cells with the dye. We first compared the relative extents of plasma membrane internalization in T. brucei 427 and T. brucei RUMP528 by pre-loading the cells with FM4-64, washing the parasites and quantifying cell-associated fluorescence. Microscopic examination showed that the dye was present in intracellular vesicles in both strains of T. brucei (results not shown; see Figure 4A for an example). Quantitatively, accumulation of FM4-64 was not significantly different between T. brucei 427 and T. brucei RUMP528 (Figure 3A). Therefore the rates of bulk plasma membrane internalization cannot explain the different amounts of transferrin that accumulate in the two strains of T. brucei (Figure 2A). To determine whether exocytosis is affected by loss of the GPIPLC gene, T. brucei RUMP528 and 427 were first allowed to endocytose FM4-64. Cells were washed and ‘chased’ in medium lacking FM4-64, after which (i) cell-associated FM4-64, and (ii) dye exocytosed into the extracellular medium was quantified. During the ‘chase’, similar (relative) amounts of FM4-64 were lost from the two strains of T. brucei (Figure 3B). Furthermore, the two strains of T. brucei exocytosed similar amounts of FM464 into the culture medium (Figure 3C). These results suggest that general exocytosis is not affected by GPI-PLC. Therefore the difference in transferrin retention between T. brucei 427 and T. brucei RUMP528 is not the result of aberrant exocytosis by the mutant cells. GPI-PLC is not associated with endosomes, and its enzyme activity is important for stimulating uptake of transferrin
Two general models may be proposed for how GPI-PLC affects endocytosis in T. brucei. First, the polypeptide itself could act stoichiometrically, by analogy to adaptor proteins or clathrin, to activate the activities of endocytic vesicles. Secondly, GPI-PLC might produce a second messenger which modulates endocytosis, since one product of the enzyme’s reaction is DAG, a well-known second messenger. In an initial test of the first hypothesis (above), we determined whether (or not) GPI-PLC was an endosome protein in T. brucei. For this purpose, endosomes were marked with the fluorescent dye FM4-64, while GPI-PLC was detected with anti-GPI-PLC antibody in the same cells. GPI-PLC did not co-localize with FM4-64 vesicles (Figure 4A), indicating that the enzyme was not endosomal. This conclusion was confirmed in studies with dextran–Texas Red, a marker of fluid-phase endocytosis. GPIPLC does not co-localize with endocytosed dextran–Texas Red (Figure 4B). Thus GPI-PLC is not an endosomal protein. In order to test our ‘second-messenger hypothesis’ (see above), it was important to have an experimental setup with which to express mutant forms of GPI-PLC in bloodstream-form T. brucei so that their effects on endocytosis could be measured without interference from endogenous enzyme activity. A GPI-PLC−/− mutant (T. brucei RUMP528) was the ideal genetic background
Figure 3
Exocytosis of FM4-64 in wild-type and GPI-PLC −/− T. brucei
(A) Uptake of FM4-64. T. brucei 427 (GPI-PLC +/+ ) or T. brucei RUMP528 (GPI-PLC −/− ) were incubated with 200 nM FM4-64 at 37 ◦C. At the times indicated, cells (4 × 106 ) were retrieved, washed with 1× BBS/G containing 0.2 % sodium azide, and levels of FM4-64 were quantified (excitation at 570 nm and emission at 620 nm). Results are means + − S.D. of triplicate determinations. (B and C) Retention and release of FM4-64. T. brucei 427 (GPI-PLC +/+ ) and T. brucei RUMP528 (GPI-PLC −/− ) were incubated with excess FM4-64 (10 μM) at 37 ◦C for 30 min. Cells were then resuspended in fresh IMDM without FM4-64 (chase medium) at 37 ◦C. An aliquot of cells (4 × 106 ) was withdrawn every 10 min, pelleted and washed. Levels of intracellular FM4-64 (i.e. retained FM4-64) (B) and FM4-64 in the chase medium (i.e. released FM4-64) (C) were quantified. Results are means + − S.D. of triplicate determinations.
for these studies: A cell line containing an ectopic copy of the GPIPLC-coding sequence in T. brucei RUMP528, named T. brucei 17.19.18, was obtained for these studies. Cells were induced with tetracycline to express GPI-PLC, and endocytosis of transferrin was measured. Without tetracycline induction of GPI-PLC transcription, transferrin endocytosis in T. brucei 17.19.18 was relatively low (Figure 5A). Following synthesis of GPI-PLC, there was a 3fold (maximal) increase in the uptake of transferrin in T. brucei 17.19.18 (Figure 5A) compared with the uninduced cell line. Our data suggest that a deficiency of transferrin endocytosis c The Authors Journal compilation c 2009 Biochemical Society
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Figure 4
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Immunodetection of GPI-PLC in T. brucei
(A) GPI-PLC is not detected on early endosomes labelled with FM4-64. Endosomes in T. brucei were labelled by uptake of FM4-64 for 10 min. Afterwards, cells were fixed and permeabilized, and GPI-PLC was detected with monoclonal antibody 2A6-6 and fluorescent anti-mouse secondary antibody, and visualized by fluorescence microscopy. Cellular [nuclear (n) and kinetoplast (k) (mitochondrial)] DNA was detected with DAPI staining. (B) GPI-PLC does not associate with endocytic vesicles containing dextran–Texas Red (Dextran-TR). Mutant (GPI-PLC−/− ) and wild-type (GPI-PLC+/+ ) T. brucei were allowed to endocytose dextran–Texas Red for 10 min. The cells were fixed and permeabilized, and GPI-PLC was detected with antibody as described in (A). Parasite DNA was detected with DAPI as in (A).
in T. brucei RUMP528 (GPI-PLC−/− ) can be ameliorated by expression of an ectopic copy of the GPI-PLC gene in the GPIPLC−/− cells. (Higher background endocytosis in the uninduced cells is most likely to be the result of leaky transcription of GPIPLC at the ribosomal DNA locus, as has been reported by other groups [37,38].) We checked whether enzyme activity was important for the effect of GPI-PLC on endocytosis. Genes for unmutated GPI-PLC or an enzymatically inactive Q81L_GPIPLC mutant [21] were expressed in T. brucei RUMP528. Transcription of the gene for the normal or Q81L mutant GPI-PLC was induced with tetracycline, and endocytosis of transferrin was quantified. For the ectopic unmutated GPI-PLC, induction of enzyme synthesis enhanced endocytosis (3-fold maximal) (Figure 5B) compared with cells lacking the protein (i.e. T. brucei RUMP528). The Q81L_GPIPLC mutant failed to activate endocytosis after induction of its synthesis with tetracycline (Figure 5B), suggesting that GPI-PLC activity was important for stimulation of transferrin endocytosis. However, a trivial explanation for these data would be that the mutant protein was unstable in vivo. To test this theory, immunoblotting assays were performed, and the data showed that the Q81L_GPIPLC polypeptide was present in T. brucei RUMP528 cells engineered to express the protein (Figure 5C), as was a (positive) control protein VSG221. However, no GPI-PLC enzyme activity was detected with Q81L_GPIPLC (Figure 5D) [21]. These data indicate that enzyme activity is required for GPIPLC to stimulate endocytosis of transferrin. c The Authors Journal compilation c 2009 Biochemical Society
Phorbol ester and DAGs enhance endocytosis in T . brucei
Since enzyme activity of GPI-PLC is required for its stimulation of transferrin endocytosis (Figures 5A and 5B), we hypothesized that a product of GPI (or PtdIns) cleavage might be a second messenger for GPI-PLC control of endocytosis (Figure 2). DAG is a product of GPI-PLC cleavage of GPIs [39,40], and so we considered the lipid as a possible second messenger produced by GPI-PLC. PMA, a phorbol ester, mimics the biological actions of DAG and is frequently used in place of DAG for physiological studies. We therefore tested whether exogenous PMA or DAG could stimulate endocytosis of transferrin in T. brucei RUMP528 (GPI-PLC−/− ). PMA enhanced transferrin uptake up to 4-fold in T. brucei RUMP528 (Figure 6A), in comparison with control cells that were treated with DMSO, the solvent for PMA. In a timecourse study of T. brucei response to PMA, a 30 min exposure caused a maximal 4-fold increase in endocytosis (Figure 6B). However, PMA treatment of T. brucei for 45 min (or more) inhibited endocytosis (Figure 6B). Dampening of endocytosis after extended PMA exposure, as has been reported in vertebrate cells [41,42], may be caused by desensitization of DAG receptors. In a control experiment, the α-isomer of PMA (i.e. 4α-PMA) failed to stimulate endocytosis (results not shown). This last result indicates that, in T. brucei, the effects of PMA are limited to the physiologically active 4β-PMA isomer, as reported in other biological systems [43]. We also tested whether PMA could
Glycosylphosphatidylinositol-specific phospholipase C
Figure 5
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Enzyme activity of GPI-PLC is required to stimulate endocytosis
(A) Endocytosis can be activated in T. brucei by induction of GPI-PLC expression. T. brucei 17.19.18 expressing an ectopic copy of the GPI-PLC gene was induced with tetracycline (200 ng/ml) for 16 h. Uptake of transferrin–Alexa Fluor® 594 was measured at 37 ◦C (see the legend to Figure 2 for details). Results are means + − S.D. of triplicate determinations, representative of results obtained from four independent experiments. (B) An enzymatically compromised Q81L_GPIPLC mutant cannot stimulate endocytosis in T. brucei . pLewGPIPLC/T. brucei RUMP528 and pLewQ81L_GPIPLC/T. brucei RUMP528 were induced with tetracycline (1 μg/ml) for 16 h. The cells were allowed to endocytose transferrin–Alexa Fluor® 594 at 37 ◦C, and cell-associated fluorescence was obtained in triplicate. Results are means + − S.D. representative of four independent trials. (C) Detection of Q81L_GPI-PLC protein in pLewQ81L_GPIPLC/T. brucei RUMP528. Detergent-solubilized cell extract was resolved by SDS/PAGE (12 % minigel), and Western blotting with anti-GPI-PLC (108 equivalents of cell extract per lane) or anti-VSG antibody (5 × 104 equivalents of cell extract per lane) was performed. (D) Q81L_GPI-PLC lacks enzyme activity in T. brucei . Bloodstream-form T. brucei was lysed hypotonically. Detergent-solubilized cell extract (from 2.5 × 107 cells) was analysed for GPI-PLC enzyme activity. Results are means + − S.D. of duplicate determinations.
enhance endocytosis in parental T. brucei 427 that contains the genes for GPI-PLC. The phorbol ester promoted endocytosis in T. brucei 427 (GPI-PLC+/+ ) to levels similar to that observed in T. brucei RUMP528 (Figure 6B). Furthermore, prolonged exposure of parasites to PMA reduced the uptake of transferrin (Figure 6B), as noted for cells deficient for GPI-PLC activity (GPI-PLC−/− ). DAG, the natural lipid whose actions are mimicked by PMA, is present in T. brucei. It was therefore important to test whether DAG also stimulated endocytosis. Towards this goal, the effects of OAG (1-oleyl-2-acetyl-sn-glycerol) and DMG (dimyristoylsn-glycerol) on transferrin endocytosis were studied. OAG is membrane-permeable DAG used for studies on vertebrate cells [44]. DMG is a component of GPIs in bloodstream T. brucei [45], and it can be produced by GPI-PLC cleavage of GPIs [39,46]. OAG (500 nM) stimulated transferrin uptake 4-fold (Figure 6C), compared with cells that did not receive the lipid. Similarly, pre-treatment of T. brucei with DMG (500 nM) led to 3-fold higher transferrin accumulation, compared with cells exposed to the solvent (DMSO) alone (Figure 6D). These data confirm that a DAG signalling pathway exists in T. brucei. Furthermore, these observations prove that GPI-PLC itself is not needed for the activation of transferrin endocytosis, because either OAG or DMG facilitate endocytosis in cells that lack GPI-PLC genes. We surmise that, in wild-type T. brucei, DAG is a second messenger for the biological effects of GPI-PLC.
DISCUSSION Regulation of transferrin endocytosis: a novel function for GPI-PLC in T . brucei
The biological function of GPI-PLC in non-differentiating bloodstream-form T. brucei has been elusive. The gene for GPI-PLC is not essential for viability of T. brucei [16,18]. However, replication of the parasite in a vertebrate host is compromised in a GPIPLC−/− pleomorphic strain [16]. The molecular basis for the importance of GPI-PLC in the virulence of T. brucei is not resolved. Endocytosis is essential in T. brucei [8,9], probably because some nutrients, e.g. iron and lipoproteins, are obtained by that route [47,48]. Furthermore, endocytosis is important for parasite evasion of host defence, in clearing anti-VSG antibody from the plasma membrane [7]. Parasite-absorbed anti-VSG antibody enables host Kupffer cells to phagocytose and kill T. brucei [49,50]. From this perspective, enhancement of endocytosis in bloodstream-form T. brucei may stimulate the virulence of the parasite. GPI-PLC stimulation of endocytosis (Figures 2, 3 and 5) will lead to more efficient transferrin uptake (i.e. better nutrition; transferrin is a growth factor for T. brucei [4]) and more effective evasion of (host) innate defence pathways (see the previous paragraph). These effects of the enzyme will promote the viability of the parasite in an infected vertebrate, and account, at least in c The Authors Journal compilation c 2009 Biochemical Society
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PMA and DAGs enhance endocytosis
(A) Dose–response of PMA. T. brucei RUMP 528 (5 × 106 /ml) was incubated with different concentrations of PMA for 10 min (or with DMSO, the solvent for PMA). Subsequently, cells were washed and resuspended in serum-free IMDM containing transferrin–Alexa Fluor® 594 at 37 ◦C for 15 min. Cells were washed, and the relative fluorescence was measured (excitation at 570 nm −/− +/+ and emission at 620 nm). Results means + − S.D. of triplicate determinations. (B) Time course of PMA stimulation of endocytosis. T. brucei RUMP 528 (GPI-PLC ) or T. brucei 427 (GPI-PLC ) (5 × 106 /ml) were incubated with PMA (500 nM) for the times indicated before endocytosis of transferrin was measured as described in (A). (C and D) DAGs stimulate endocytosis. T. brucei RUMP528 (5 × 106 /ml) were incubated with various concentrations of OAG (C) or DMG (D) for 30 min, and endocytosis of transferrin was assayed at 37 ◦C for 10 min. After washing in buffer containing 0.2 % sodium azide [30], cells were pelleted and the relative fluorescence was measured (excitation at 570 nm and emission at 620 nm). Background signal from control cells that did not receive the fluorescent cargo was subtracted. Results are means + − S.D. of triplicate determinations.
part, for the decrease in parasitaemia of pleomorphic T. brucei that lacks GPI-PLC genes in a mouse model of human African trypanosomiasis [16]. DAG as a second messenger in T . brucei
Our data indicate that DAG is a second messenger in T. brucei. Exogenous DAGs, as well as PMA, a phorbol ester that mimics the biological activities of DAGs, increased transferrin endocytosis (Figures 5 and 6). These data establish DAG as a regulator of the endocytic system, and provide evidence for a DAG signalling pathway in T. brucei. How might DAG influence the endocytic machinery in T. brucei? In eukaryotes, the effects of DAG on cell physiology depend on proteins with C1-domains that bind to the lipid [51]. Curiously, the classic C1-domain is not present in T. brucei, despite the cellular response of the parasite to the lipid (Figures 2A, 5 and 6). To resolve this perplexing situation, we hypothesized that the parasite may have a C1-related domain that is highly diverged from the mammalian version, because trypanosomatids are ‘deeply diverged’ eukaryotes. To evaluate our theory, we performed, in collaboration with Ga¨elle Blandin (Institute for Genomic Research, Rockville, MD, U.S.A.), a genome-wide bioinformatic search for C1-related domains in T. brucei. That effort produced 21 hits (results not shown). Further analysis of the protein sequences using SMART (http://smart.embl-heidelberg. de/) and PRATT (http://www.ebi.ac.uk/pratt/) revealed that each of the T. brucei C1-related domains (which we term C1_5) c The Authors Journal compilation c 2009 Biochemical Society
contained the motif L-x(11)-C-x(2,4)-C-x(3,9)-E-x(2,9)-F-x-Cx(2)-C-x(4)-C-x(2)-C (PROSITE nomenclature [52]). We hypothesize that C1_5 is a receptor of DAG in T. brucei. [C1_5 is also found in Leishmania sp. and in Trypanosoma cruzi (results not shown).] How might proteins with C1_5 domains activate the endocytic machinery after they bind to DAG? We hypothesize that a C1_5 protein might have a second domain that acts as the ‘effector’ for endocytosis. Consistent with our hypothesis, three C1_5 proteins have other domains with enzyme activity, namely a protein tyrosine kinase (accession number Tb11.01.2290) and ubiquitin ligases (accession numbers Tb09.211.4210 and Tb927.8.1950) (http://www.genedb.org/genedb/tryp/). In vertebrates, tyrosine phosphorylation of some receptors modulates endocytosis [53]. Ubiquitination of membrane proteins in yeasts and vertebrates accelerates their recruitment into endosomes [54,55]. Therefore we predict that DAG regulates transferrin endocytosis by activating a protein tyrosine kinase and/or ubiquitin ligases that can post-translationally activate components (e.g. clathrin or adaptins) of the endocytic pathway. It is unlikely that DAG activates a protein kinase C in the parasite because the genome of T. brucei does not encode a serine/threonine kinase with a C1 or C1_5 domain, implying that T. brucei lacks a classic protein kinase C. FUNDING This work is supported by the National Institutes of Health [grant number AI33383 (to K. M.-W.)].
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