Fig. 4

Mitochondrial morphology and structure are disrupted by the absence of UBLCP1 in Toxoplasma gondii. A, Indirect immunofluorescence of HSP60 (marker protein of mitochondrial matrix), TOM40 (marker protein of mitochondrial outer membrane), and F1β ATPase (marker protein of mitochondrial inner membrane) in RHΔku80 and ΔUBLCP1 tachyzoites. Red indicates IMC1. Scale bar, 2 µm. B, Statistical analysis of collapsed mitochondrial morphology in RHΔku80 and ΔUBLCP1 tachyzoites detecting by polyclonal rabbit anti-HSP60 sera. At least ten images for each sample were acquired stochastically with a Zeiss LSM880 confocal laser scanning microscope to analyze. C, Transmission electron micrographs of RHΔku80 and ΔUBLCP1 tachyzoites. Insets show representative mitochondrion using Mi as an indication. The mitochondrion is outlined with black box lines and enlarged to show significantly reduced mitochondrial density and cristae number in ΔUBLCP1 tachyzoites. D, The number of mitochondrial cristae per μm² of RHΔku80 and ΔUBLCP1 tachyzoites. E, Protein expressions of HSP60, TOM40, and F1β ATPase in RHΔku80 and ΔUBLCP1 tachyzoites, which are analyzed on the basis of the grey intensity of protein bands using ImageJ software. SAG1 is employed as the internal control. F, ATP content in RHΔku80 and ΔUBLCP1 tachyzoites. G, Mitochondrial membrane potential (ΔΨm) of RHΔku80 and ΔUBLCP1 tachyzoites, as assessed by JC-1 probe. Representative histograms of red fluorescence are recorded by flow cytometry. H, The ratio of red to green fluorescence intensity between RHΔku80 and ΔUBLCP1 tachyzoites. Over 10,000 JC-1 positive tachyzoites of each strain are analyzed by flow cytometry. The values are the means ± standard deviation (SD). *P < 0.1; **P < 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, by unpaired t test. I, The schematic diagram illustrates the mitochondrion abnormalities in terms of morphology, ultrastructure, and function in the UBLCP1 deletion strain