Figure 3. CPL deficient bradyzoites develop Atg8-positive autophagosomes associated with the VAC.
a, Phase contrast microscopy of in vitro encysted bradyzoites differentiated for 7 days. Arrows indicate cytosolic inclusions in CPL deficient strains. Scale bar, 5 μm. b, Quantification of dark inclusion size (area of individual puncta, left panel) and number (per 100 μm2 of cyst area, right panel) shown as violin plots. Data are mean ± SEM of 3 biological replicates. The following number of total cysts and puncta, respectively, were quantified: Pru 74, 409; PΔcpl 102, 3812; PΔcpl:c/CPL 120, 743; PΔcpl:s/CPL 86, 1324; PΔcpl:b/CPL 101, 464; Pru-DMSO 75, 237; and Pru-LHVS 110, 3024. Shape indicates distribution of the pooled data. ****, p<0.0001 Mann Whitney test. c, Fluorescence microscopy of autophagosomes in bradyzoites expressing tdTomato-Atg8. Bradyzoites were differentiated for 7 days, fixed, and viewed for bradyzoite specific expression of GFP and tdTomato-Atg8. Examples in the top 3 rows are of parasites expressing WT tdTomato-Atg8, whereas the bottom row shows parasites expressing a tdTomato-Atg8 bearing a glycine to alanine mutation rendering it refractory to lipidation. Scale bar, 10 μm. The same scale applies to the fluorescence images, which were captured and processed identically. d, Western blots of bradyzoite lysates probed with α-Atg8 or α-BAG1. e, Fluorescence microscopy of autophagosomes in Pru bradyzoites treated with solvent (DMSO) or 1 μM LHVS. Bradyzoites were differentiated for 7 days, treated for 4 days, stained with cytoID (green), fixed, and stained with α-CPL (red). An arrow indicates an example of CPL at the periphery of an autophagosome shown as an inset of the boxed area. Scale bar, 5 μm. The same scale applies to the fluorescence images. f, Transmission electron microscopy of bradyzoites differentiated for 1 week. Abbreviations used: c, conoid; m, microneme; rh, rhoptry; dg, dense granule; mt, mitochondrion. Scale bars, 500 nm.