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First published online 3 June 2003
doi: 10.1242/jcs.00596

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Intracellular calcium stores in Toxoplasma gondii govern invasion of host cells

Jennie L. Lovett and L. David Sibley*

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, USA



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  		Fig. 1. Effect of extracellular calcium on attachment and invasion. Host cells were challenged with parasites in the presence of 1 mM BAPTA, a diluent control (ctl; refers to water or DMSO), 5 mM CaCl2, or 5 mM EGTA in pH 6.0 or pH 7.2 low calcium Ringer's. To examine the role of intracellular calcium, parasites were pretreated with 50 µM BAPTA-AM (T. gondii pretreatment). In parallel, host cells were loaded with 50 µM BAPTA-AM (host cell pretreatment) or a diluent control (ctl) prior to contact with parasites. Invasion (solid bars) and attachment (open bars) were monitored as described in the methods and average percentages calculated from the results of three independent experiments (mean±s.e.).

 


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  		Fig. 2. Effect of calcium on trail formation by gliding parasites. Formation of gliding trails by parasites treated with (A) BAPTA-AM, (B) BAPTA, (C) a diluent control, (D) CaCl2, (E) EGTA in pH 6.0 low calcium Ringer's or (F) EGTA in pH 7.2 low calcium Ringer's. Coverslips were stained with a fluorescently conjugated antibody against the plasma membrane marker SAG1 to visualize trails. Images shown are representative of three independent experiments showing similar results. Bar, 10 µm.

 


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  		Fig. 3. Effect of calcium on microneme secretion by T. gondii. Western blot analysis of secretion by parasites treated with agents to chelate extracellular or intracellular calcium. Chelation of intracellular calcium with BAPTA-AM completely blocked secretion. Cellular MIC2 (cMIC2) forms present in dilutions of parasite pellets (cell stds) migrate at a larger size compared to the secreted form of MIC2 (sMIC2) found in supernatants. Ethanol combined with incubation at 37°C was used to stimulate secretion in all the samples. Unstimulated cells that were not treated with ethanol or raised to 37°C were used as a negative control. Unstimulated actin provided a control for inadvertent cell lysis in the experiment. Example shown is a representative blot from three independent experiments with similar results.

 


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  		Fig. 4. Host calcium response during parasite invasion. (A) Fluo-4 calcium flux in untreated host cells. Fluo-4 AM loaded fibroblasts were observed by time-lapse fluorescent video microscopy with frames captured every 0.5 seconds. The bright object in the center is the nucleus of the cell. Shown are selected frames with the time elapsed between frames in seconds indicated. Bar, 10 µm. (B) Phase images of T. gondii parasites invading fluo-4 AM loaded fibroblasts and recorded by time-lapse video. Phase and fluorescent images were acquired with 0.5 seconds between each pair. Bar, 5 µm. (C) Fluorescent images corresponding to the panels in (B) are shown, with the elapsed time between frames indicated. Also see Movie 2 at jcs.biologists.org/supplemental.

 


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  		Fig. 5. Calcium responses in T. gondii during gliding and cell invasion. (A) Calcium fluxes in gliding parasites. Fluo-4 AM loaded parasites were observed by time-lapse fluorescent video microscopy, with both phase and fluorescent images acquired with 0.5 seconds between each pair. Shown are selected images that were merged, with the elapsed time between frames indicated. Scale bar, 5 µm. (B,C) Merged images of fluo-4-labeled parasites during invasion into fibroblasts. Shown are selected merged pairs of images, with the elapsed time between subsequent frames indicated. Scale bar, 5 µm. Also see Movie 4 at jcs.biologists.org/supplemental.

 


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  		Fig. 6. Parasite calcium responses during gliding and invasion. (A) Frame-by-frame quantification of calcium changes in a parasite gliding on serum-coated glass. The parameters monitored for data in Table 1 are indicated. (B) Frame-by-frame analysis of calcium changes in a parasite during host cell invasion. Arrows indicate first contact with the host cell (contact), as well as when the parasite is fully inside the host cell (intracellular). Background is indicated by the dashed line.

 

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© The Company of Biologists Ltd 2003