Calcium transfer from the ER to other organelles for optimal signaling in Toxoplasma gondii
- Center for Tropical and Emerging Global Diseases, University of Georgia and Department of Cellular Biology, University of Georgia, United States
- Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, United States
Figures
Figure 1 with 2 supplements
The role of extracellular calcium in the filling of intracellular stores.
(A) Scheme depicting the Fura-2-AM loading and the experimental setup. (B) T. gondii tachyzoites loaded with Fura-2 were in suspension in Ringer buffer with 100 µM EGTA. Thapsigargin (TG) was added at 100 s, at two different concentrations (1 and 2 µM). (C) Same conditions as in B. 1 µM Thapsigargin (TG) was added at 100 s for T1 and 400 s for T2. 1 µM ionomycin (IO) was added at 400 sec for trace 1 (T1) and at 100 sec for trace 2 (T2). Bar graph shows Ca2+ increase after adding TG before (T1) and after IO (T2). (D) Same conditions as in B. 1.8 mM CaCl2 was added at 100 s, followed by 2 µM TG at 300 s (dark blue trace). The light blue trace shows the same experiment without the addition of CaCl2. (E) Similar to D but using 40 µM glycyl-L-phenylalanine-naphthylamide (GPN) instead of TG. (F) Same experimental setup to the one shown in D but using the mitochondrial uncoupler CCCP. (G) Same experimental setup to the one shown in D but adding the potassium ionophore nigericin (Nig), 10 µM. The quantification for D, E, F, and G shows the % increase of cytoplasmic calcium compared with the same condition without previous addition of calcium. (H) 1.8 mM CaCl2 was added at 100 s, 1 µM TG was added at 300 sec followed by 40 µM GPN at 500 s. The light blue trace shows the same experiment without the addition of TG. The quantification shows the Ca2+ increase after adding GPN ± previous addition of TG. (I) Identical experiment to H but instead using 10 µM Nig at 500 sec. The quantification shows the Ca2+ increase after adding Nig ± previous addition of TG. (J) 1 µM TG was added at 100 s followed by 40 µM GPN at 300 s. The light blue trace shows the same experiment without the addition of TG. The buffer contains 100 µM EGTA. Quantification shows Ca2+ increase after adding GPN ± previous addition of TG. (K) Similar conditions to J but with 10 µM Nig at 300 s instead. Data are presented as mean ± SD for all comparisons. p-value: unpaired two-tailed t-test performed in all comparisons.
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Figure 1—source data 1
Source data for Figure 1 showing Fura2 calcium measurements.
- https://cdn.elifesciences.org/articles/101894/elife-101894-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Intracellular calcium pools.
T. gondii tachyzoites loaded with Fura-2 were used for these measurements. The suspension was in Ringer buffer with 100 µM EGTA. (A) Cyclopiazonic acid (CPA), 10 µM was added at 100 s (CPA-1) followed by Ionomycin 1 µM at 400 s (purple trace). CPA-2, pink trace: 1 µM ionomycin (IO) was added at 100 s followed by CPA 10 µM at 400 s. The bar graph shows the analysis of cytosolic Δ[Ca²+] measured after the addition of CPA first (C1), compared with the response to CPA after addition of IO (C2), based on data from more than three independent biological experiments. (B) Thapsigargin (TG) 1 µM was added at 100 s followed by CPA at 300 s. (C) CPA 10 µM was added at 100 sec followed by TG 1 µM at 300 s. The bar graph shows the analysis of the cytosolic Δ[Ca2+] after the addition of TG (T1 and T2) or CPA (C1 and C2) from more than three biological experiments. Data are presented as mean ± SD. p-value: unpaired two-tailed t-test performed in all comparisons. *, p≤0.05. **, p≤0.01. ***, p≤0.001. ****, p≤0.0001.
Figure 1—figure supplement 2
Intracellular calcium pools.
T. gondii tachyzoites loaded with Fura-2 were used for these measurements. The suspension buffer was Ringer with 100 µM EGTA. (A) 1 µM Thapsigargin (TG) was added at 100 s followed by 1.8 mM Ca2+ at 300 s and 40 µM GPN at 500 s (orange trace). The peach trace shows the same additions of Ca2+ and GPN without TG addition. The bar graph shows the analysis of the rate of Δ[Ca2+]cyt per second obtained after the addition of GPN with and without the previous addition of TG from more than three biological experiments. (B) Similar experimental setup: 1 µM TG was added at 100 s, followed by 40 µM GPN (dark blue trace) and 100 µM Zaprinast at 500 s. The bar graph shows the analysis of the rate of the cytosolic Δ[Ca2+] change after the addition of Zaprinast from more than three biological experiments. Data are presented as mean ± SD. p-value: unpaired two-tailed t-test performed in all comparisons.
Figure 2 with 1 supplement
Ca2+ uptake by intracellular stores.
(A) Scheme showing the loading with Mag-Fluo-4 AM followed by permeabilization with digitonin of a T. gondii tachyzoite (RH parental strain) suspension (IS, intracellular store). (B) Fluorescence measurements (see Materials and methods for specifics) of the suspension of parasites loaded with Mag-Fluo-4. MgATP (500 µM), the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) substrate was added at 50 s. The bar graph shows the quantification of the slope of the increase in fluorescence after adding MgATP. The concentration of free Ca2+ was varied, and it is indicated. The calculation of free Ca2+ was done using MaxChelator. (C) A similar experimental setup to the one shown in B with 220 nM free Ca2+, with varied concentrations of MgATP as indicated in the bar graph, which shows the quantification of the slope of fluorescence increase after adding MgATP. (D) Experiment was done with 500 µM MgATP and 220 nM free Ca2+. Thapsigargin (TG) was added to inhibit SERCA causing calcium to be released from the store. The concentrations used are indicated. The bar graph shows the negative slope after the addition of TG. (E) Similar to D, but adding various concentrations of ionomycin (IO). The concentrations used are indicated and the slopes were measured after the addition of IO. Data are presented as mean ± SD for B-E. p-value: unpaired two-tailed t-test performed for all comparisons. ns, not significant, p>0.05. *, p≤0.05. **, p≤0.01. ***, p≤0.001. ****, p≤0.0001.
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Figure 2—source data 1
Source data for Figure 2 showing MagFluo4 calcium measurements.
- https://cdn.elifesciences.org/articles/101894/elife-101894-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Inhibition of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity by thapsigargin (TG) and cyclopiazonic acid (CPA).
T. gondii tachyzoites loaded with Mag-Fluo4 and permeabilized with digitonin were used for these measurements. (A) 125 µM MgATP was added at 100 s, followed by the addition of DMSO (control) or 1 µM TG or 10 µM CPA at 350 s. The slope of the fluorescence decrease after CPA addition was normalized to the slope of the fluorescence decrease after TG addition, which was set to 100% in the bar graph. (B) MgATP was added at 100 sec followed by TG at 350 s for both runs, CPA at 500 s was added to the experiment represented by the green trace. (C) MgATP was added at 100 s followed by CPA at 350 s for both runs, TG was added at 500 s to the experiment represented by the blue trace.
Figure 3 with 1 supplement
The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is essential for the T. gondii lytic cycle.
(A) Scheme showing the strategy used for generating conditional knockouts of TgSERCA by promoter insertion and regulation by 0.5 µg/ml Anhydrotetracyclin (ATc). The resulting mutants were named iΔTgSERCA or iΔTgSERCA-3HA (C-terminally HA-tagged). DHFR, dihydrofolate reductase gene (pyrimethamine selection); CAT, chloramphenicol acetyltransferase gene (chloramphenicol selection). (B) Western blots of iΔTgSERCA-3HA parasites grown ± ATc. TgSERCA expression was detected using an anti-HA antibody, showing reduced levels with ATc treatment. (C) Plaque assays comparing the growth of iΔTgSERCA tachyzoites (150 parasites/well) cultured ± 0.5 µg/ml ATc for 8 days. Plaques formed by the parental TatiΔku80 strain are shown for comparison. (D) Quantification of the size of the plaques presented in C. (E) Replication assay using the iΔTgSERCA-RFP mutant. The number of parasites per parasitophorous vacuole (PV) was quantified 24 hr post-infection of fibroblast cells and compared between parasites grown ± 0.5 µg/ml ATc. (F) Average number of parasites per PV counted at 24 hr after the initial infection. The graph to the right shows the number of parasites per PV of the iΔTgSERCA (+ATc) for 24 or 48 hr after the initial infection. (G) Invasion assay of the iΔTgSERCA mutant following 24 hr of ATc treatment, performed using the red-green assay described in the Methods section. (H) Egress assays with fibroblast monolayers infected with iΔTgSERCA-RFP parasites for 24 or 48 hr. Egress was triggered with ionomycin (IO; 100 nM or 50 nM) or saponin (0.01%). Natural egress was monitored following treatment with 1 μM compound 1 as described in the Methods section. % Vacuoles: 100 X Number of vacuoles egressed/total vacuoles. Data (D, E, F, G, H) are presented as mean from at least three biological replicates ± SD. Statistical significance was assessed using an unpaired two-tailed t-test.
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Figure 3—source data 1
Source data for Figure 3 data of growth, replication, invasion, and egress of the mutant T. gondii compared to control.
- https://cdn.elifesciences.org/articles/101894/elife-101894-fig3-data1-v1.xlsx
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Figure 3—source data 2
Original files for western blot analysis displayed in Figure 3B.
- https://cdn.elifesciences.org/articles/101894/elife-101894-fig3-data2-v1.zip
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Figure 3—source data 3
PDF file containing original western blots for Figure 3B, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/101894/elife-101894-fig3-data3-v1.zip
Figure 3—figure supplement 1
Regulation of the expression of T. gondii sarco/endoplasmic reticulum Ca2+-ATPase (TgSERCA).
(A) Western blots of total lysates of T. gondii tachyzoites probed with mouse anti-TgSERCA (left panel) (gift from David Sibley) or guinea pig anti-TgSERCA generated in this work (right panel) (1:1,000). (B) IFAs of TatiΔku80 or iΔTgSERCA-3HA (±ATc). The mouse monoclonal antibody αHA was used at 1:200 dilution (green signal). The guinea pig antibody against TgSERCA was used at 1:500 dilution (red signal). The HA signal partially co-localizes with the TgSERCA signal. Both signals disappear in the iΔTgSERCA-3HA mutant when cultured with ATc for 24 hr. Scale bars are 5 µm.
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Figure 3—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 3—figure supplement 1A.
- https://cdn.elifesciences.org/articles/101894/elife-101894-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
PDF file containing original western blots for Figure 3B, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/101894/elife-101894-fig3-figsupp1-data2-v1.zip
Figure 4
Organellar calcium pools in the iΔTgSERCA mutant.
(A) The iΔTgSERCA mutant was grown ±ATc and was loaded with Fura-2 for cytosolic Ca2+ measurements. 1 µM thapsigargin (TG) was added at 200 s to a suspension of tachyzoites. The purple trace shows the response of the parental cell line grown without anhydrotetracycline (ATc) and the pink trace shows the response of the same mutant grown with ATc for 24 hr. The bar graph shows the analysis of the Δ[Ca2+]cyt from three biological experiments. (B) Same experimental setup as the one in A but adding 1.8 mM extracellular Ca2+ at 200 s. (C) Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity measured in Mag-Fluo-4 loaded iΔTgSERCA tachyzoites grown ±ATc. Parasites were collected, loaded with Mag-Fluo-4AM, and permeabilized with digitonin as described in the Methods section. Free Ca²+ in the buffer was set at 220 nM, and MgATP (0.125 mM) was added at 100 s. The purple trace represents the control (no ATc), while the other traces correspond to parasites treated with ATc for 24 or 48 hr. TG (1 µM) was added as indicated. The bar graph shows the quantification of the initial slope after adding MgATP. (D) Ca²+ entry measured in Fura-2–loaded iΔTgSERCA parasites grown ±ATc. Extracellular Ca²+ (1.8 mM) was added at 200 s. The inset shows ΔF values from three independent experiments, indicating no significant differences. (E) Similar conditions to the ones used in A but adding 100 µM Zaprinast. The bar graph shows the quantification of the Δ[Ca2+] from three biological experiments. (F) Similar conditions to the ones used in B but adding 1.8 mM extracellular calcium at 200 s and 100 µM Zaprinast at 400 s. The bar graph shows the quantification of the Δ[Ca2+] from three biological experiments. (G) Same as A but adding 40 µM glycyl-L-phenylalanine-naphthylamide (GPN). The bar graph shows the analysis of the Δ[Ca2+] from three biological replicates. (H) Same setup as in F but adding 1.8 mM Ca2+ at 200 s followed by 40 µM GPN at 400 s. The bar graph shows the quantification of the Δ[Ca2+] from three biological replicates. Data are presented as mean ± SD. p-value: unpaired two-tailed t-test performed in all comparisons.
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Figure 4—source data 1
Source data for Figure 4 showing calcium measurements with Fura2 and MagFluo4.
- https://cdn.elifesciences.org/articles/101894/elife-101894-fig4-data1-v1.xlsx
Figure 5 with 1 supplement
Mitochondrial calcium uptake.
(A) Fluorescence image of T. gondii tachyzoites of the RH strain expressing SOD2-GCaMP6f (pDT7S4H3-SOD2-GCaMP6f). The generation of this cell line is described in the Methods section. Scale bar is 10 µm. (B) Ca²+ uptake in digitonin-permeabilized T. gondii tachyzoites expressing SOD2-GCaMP6f. Parasites (5×10⁷) were permeabilized as described in the Methods section and suspended in buffer containing 100 µM EGTA. Ca²+ was added at 100 s to reach final free concentrations of 0.25, 0.5, 1, 10, 50, 100, and 200 µM, calculated using Maxchelator. (C), ΔF was measured as the change in fluorescence between the baseline and the maximum value obtained 20 s after Ca²+ addition. Data represent the average of three independent biological experiments. (D) Fura-2-loaded T. gondii tachyzoites expressing SOD2-GCaMP6f in suspension. The experimental setup was identical to that described in Figure 1A–B. CaCl₂ (1.8 mM) was added at 400 s, and fluorescence measurements were performed under Fura-2 conditions. (E) GCaMP6f fluorescence measurements of the same parasites from D but the fluorescence was recorded using optimized settings for GCaMP6 detection. (F) Tachyzoites expressing SOD2-GCaMP6f loaded with Fura-2 in suspension. 1 µM thapsigargin (TG) was added at 100 s followed by 1.8 mM CaCl2 at 400 s. Fura-2 conditions were used. (G) Same additions and same parasites as in F but measuring fluorescence of GCaMP6f. (H) Response to 1 µM TG of iΔTgSERCA-SOD2-GCaMP6f parasites (transfected with the pCTH3-SOD2-GCaMP6f plasmid), grown with (pink trace) or without (blue trace) anhydrotetracycline (ATc). Fluorescence measurements were performed under the same conditions as in panel G using intact parasites. The bar graph shows ΔF values from three independent biological replicates. (I) Same as H but using 40 μM glycyl-L-phenylalanine-naphthylamide (GPN). (J) Same as H but using 100 μM Zaprinast. (K) Same as H but using 1 μM Ionomycin (IO). Data are presented as mean ± SD from three independent biological experiments. p-value: unpaired two-tailed t-test performed in all comparisons.
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Figure 5—source data 1
Source data for Figure 5 showing Fura 2 and GCaMP6 calcium measurements.
- https://cdn.elifesciences.org/articles/101894/elife-101894-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Mitochondrial localization of the GCaMP6f.
(A) T. gondii tachyzoites of the iΔTgSERCA-SOD2-GCaMP6f clonal mutant live show localization of the fluorescence signal in the mitochondrion. Scale bars, Top row: 10 µm, middle and bottom rows: 5 µm. (B) Live intact iΔTgSERCA-SOD2-GCaMP6f mutant parasites in suspension show the lack of response to the addition of extracellular Ca2+. The lower trace shows a similar experiment with the addition of TG at 300 s. Parasites were in suspension in Ringer buffer.
Figure 6 with 4 supplements
Endoplasmic reticulum (ER)-mitochondria-Plant-Like Vacuolar Compartment (PLVAC) associations revealed by immunofluorescence and electron microscopy.
(A) Super-resolution IFAs of intracellular parasites with the mitochondrion labeled with the αTom40 (green, 1:20,000) antibody and the ER labeled with the αTgcalumenin antibody (an ER calcium binding protein) (red, 1:1,000) or the αTgSERCA (red 1:1,000). (B) IFAs of extracellular tachyzoites with the same antibodies used for part A. Close associations between the mitochondrial and ER membranes are observed at several regions. (C) The PLVAC was labeled with the αTgVP1 antibody (green, 1:200) or the αTgCPL antibody (green, 1:500). The ER was labeled with the αTgcalumenin antibody (red). The points of contact between the ER and the PLVAC are yellow. Scale bars in A-C are 5 µm. (D) Transmission Electron Microscopy imaging of the contact sites formed between ER and PLVAC, ER and Apicoplast, ER and mitochondria. Scale bars are 100 nm.
Figure 6—figure supplement 1
Electron microscopy images of the iΔTgSERCA mutant treated with anhydrotetracycline (ATc) for 24 hr.
(A) Representative images of the iΔTgSERCA mutant highlighting a large empty parasitophorous vacuole (PV). Scale bar represents 500 nm. (B) Representative image of the iΔTgSERCA mutant with a large residual body inside the PV. Scale bar represents 500 nm. (C) The iΔTgSERCA mutant treated with ATc still showed contacts between the endoplasmic reticulum (ER) and mitochondria. Scale bar represents 100 nm. (D) The iΔTgSERCA mutant treated with ATc still showed contacts between the ER and the PLVAC. Scale bar represents 100 nm. (E–G) Quantitative assessment of the contact area between ER and organelles (mitochondrion, apicoplast, or PLVAC), length of the limiting membrane of the organelle in contact with ER tubules at a distance less than 30 nm. This was measured and divided by the total length of the limiting membrane of the organelle. A total of 47–85 sections was analyzed for each population of organelles. (E) Comparison of contacts measurements for iΔTgSERCA ±ATc for ER-mitochondria. (F) Comparison of contacts measurements for iΔTgSERCA ±ATc for ER-apicoplast. (G) Comparison of contacts measurements for iΔTgSERCA ±ATc for ER-PLVAC. All p-values were calculated by two-tail t-test comparing iΔTgSERCA ±ATc. p-values ER-mito: 0.403; ER-Api: 0.492; ER-PLVAC: 0.244.
Figure 6—video 1
IFA of intracellular parasites labeled with the mitochondria marker αTom40 (green) antibody, and the ER labeled with the αTgERC antibody (red).
Image acquisition using the Zeiss Elyra super-resolution microscope and 3D visualizations using Imaris version 10.1.
Figure 6—video 2
Imaris 3D optimal visualization of extracellular parasites labeled with the αTom40 (green) antibody and the endoplasmic reticulum (ER) labeled with the αTgERC antibody (red).
Figure 6—video 3
Endoplasmic reticulum (ER) membrane contacts sites with the Plant-Like Vacuolar Compartment (PLVAC).
Imaris 3D visualization of immunofluorescence of extracellular tachyzoites. The PLVAC was labeled with αVP1 antibody (green) and the ER was labeled with αTgERC antibody (red).
Figure 7
Hypothetical model showing Ca2+ entry through two different types of Ca2+ channels, uptake by T. gondii sarco/endoplasmic reticulum Ca2+- ATPase (TgSERCA) into the endoplasmic reticulum (ER) and distribution to the other organelles via transfer from the ER to the mitochondria, Plant-Like Vacuolar Compartment (PLVAC), and apicoplast.
The mitochondrion is shown in close contact to the ER which constitutively leaks Ca2+ into the cytosol. Ca2+ could leak from the ER through the TgTRPPL-2 channel previously described (Márquez-Nogueras et al., 2021). The mitochondria take up Ca2+ from the ER through an unknown mechanism. Voltage-dependent anion channel (VDAC) could be involved in the transfer through the outer mitochondrial membrane (Mallo et al., 2021). The PLVAC interacts with the ER and may also interact with the mitochondrion and the apicoplast. TgA1, a calcium ATPase previously characterized may be the pump involved in Ca2+ uptake (Luo et al., 2001; Luo et al., 2005). The mechanism of release is unknown. The Two Pore Channel (TgTPC) was shown to be involved in the mechanism of transfer of Ca²+ between the ER and the apicoplast (Li et al., 2021). Question marks point to molecules or mechanisms partially or not yet identified.
Author response image 1
T. gondii tachyzoites in suspension exposed to Thapsigargin Calcium and GPN.
The dashed lines shows and experiments using the same conditions but parasites were permeabilized with digitonin shows a similar experiment with parasites exposed to MgATP.to release the cytosolic Fura. Part B
Additional files
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MDAR checklist
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Supplementary file 1
Primers used in this study.
- https://cdn.elifesciences.org/articles/101894/elife-101894-supp1-v1.pdf
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Calcium transfer from the ER to other organelles for optimal signaling in Toxoplasma gondii
eLife 13:RP101894.
https://doi.org/10.7554/eLife.101894.3
