Journal: bioRxiv
Article Title: ABCE1-dependent translational control links Fe-S cluster biogenesis to parasite growth and lipid homeostasis in Toxoplasma gondii
doi: 10.64898/2026.07.01.735774
Figure Lengend Snippet: A. Electron micrograph of intracellular parasites treated with cycloheximide (CHX) for 6 h. Asterisks and square signs denote parasite and host lipid droplets (LDs), respectively. Pixel size: 10 nm. B. Immunofluorescence microscopy imaging of parasites untreated or treated for 6 h and 16 h with translation inhibitors cycloheximide and puromycin. LDs accumulate in both parasites and host cells. LDs were stained with Nile Red (orange), parasites were labeled with an anti-IMC3 antibody (green), and DNA was stained with DAPI. Scale bar = 5 μm. For this experiment, puromycin and CHX were used at 100 µg/ml. C, D. Quantification of lipid droplet number and area in cKD HA-TgABCE1 parasites grown in the absence or in the presence of CHX and puromycin for 6 h or 16 h. A total of 100 parasites were analyzed per condition. Values are represented as the mean ±standard deviation of n = 3 independent biological replicates (different symbols represent different series); ns: not significant ( p -value >0.05), **: p -value ≤0.01, ****: p -value ≤0.0001. p -value s from one-way ANOVA with Dunnett’s multiple comparison test. CHX: cycloheximide, puro: puromycin, SD: standard deviation. E. Immunofluorescence imaging of parasites from the cKD HA-TgZFP2 cell cycle mutant grown in the absence or presence of ATc 48 h, in which the ZFP2 depletion induces an accumulation of LDs (left). Immunofluorescence imaging of parasites pre-cultured for 24 h and then incubated for 48 h with the cell cycle inhibitor oryzalin (ory, 2.5 µM) results in the accumulation of LDs (middle). Ory was then washed out and parasites were left to recover for an extra 48 h : while some vacuoles remained blocked (arrowhead), parasites resuming cell division displayed less LDs (right). LDs were stained with BODIPY 493/503 (green), parasites are outlined, and DNA was stained with DAPI (blue). Scale bar = 5 μm. F, G. Quantification of LD number and area in parasites depleted of TgZFP2, or treated with ory, with or without washout. 100 parasites were analyzed per condition. Values are represented as the mean ± SD of n = 3 independent biological replicates (different symbols represent different replicates); **: p -value ≤0.01, ***: p -value ≤0.001, ****: p -value ≤0.0001, from Student’s t-test. H. Outline of the host cell lipid scavenging protocol: before infection with the parasites, HFF cells were treated for 18h with BODIPY 493/503 and oleic acid (OA, 0.4 mM) to stimulate the accumulation of host LDs. After washing, the HFFs were infected with cKD HA-TgABCE1 in the absence or presence of ATc for 48 h or treated with 2.5 µM ory. OA concentration was maintained at 0.2 mM to sustain host lipid droplets production. Immunofluorescence imaging shows, in both cases, the BODIPY 493/503 signal incorporated in the parasites’ LDs, illustrating scavenging from the host.
Article Snippet: HFF cells were pretreated with oleic acid (HY-N1446, MedChemExpress) at 400 μM and BODIPY 493/503 at 10 μM for 18h, washed five times with PBS and then incubated in complete DMEM medium for 30 min. Then, the cells were incubated with oleic acid at 200 μM and infected with cKD HA-ABCE1 tachyzoites either in the presence or absence of ATc for 48 h or with microtubule-disrupting agent oryzalin (HY-147092, MedChemExpress) at 2.5 μM for 48 h. Upon treatment with 2.5 μM of oryzalin for 48 h, the compound was removed by washes in HBSS and the parasites were then maintained in complete medium for 48 h. The parasites were fixed and permeabilized according to the IFA protocol, after which the LDs were stained with BODIPY493/503 for 30 min.
Techniques: Immunofluorescence, Microscopy, Imaging, Staining, Labeling, Standard Deviation, Comparison, Mutagenesis, Cell Culture, Incubation, Infection, Concentration Assay
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