Figure 4: Localization of the T. hominis homologue of mitochondrial Atm1.

(a) Fractionation by differential ultracentrifugation was carried out on healthy and T. hominis-infected RK cells and the fractions were immunostained using an antibody to ThAtm1_1. (b) Proteinase K (PK) protection was performed on the mitosome-enriched 25,000g (25 K) fraction of infected cells, with Triton X-100 (TX) used to solubilize the membranes. A Coomassie-stained gel of the fractions after the protection assay is shown on the right. (c–i) Immunofluorescence of fixed RK-13 cells infected with T. hominis using antibodies raised against ThHsp70 (c,e; green; rat) or ThAtm1_1 (d,e; red; rabbit). DAPI was used to label host and parasite nuclear DNA (blue). The ThAtm1_1 antiserum was pre-purified using uninfected RK-13 cell lysate immobilized on nitrocellulose following SDS–PAGE. Colocalized pixels were identified using Zeiss Axiovision software, and are pseudo-coloured pink as shown in f. The DIC image in (g) shows the individual meronts in the infected RK cell. (h,i) A close-up of a different sample of meronts showing the typical distribution of the signals for the two antibodies. (j) Colocalisation scatter plots against the different channels were generated by Axiovision software and the antibody to the protein import receptor ThTom70 was used as a positive control for colocalisation with ThHsp70. The scatter plots for ThAtm1_1 with ThHsp70 and ThTom70 with ThHsp70 are similar, indicating co-localization of both proteins with ThHsp70. (k–m), The colocalized pixel counts were also quantified (n=3, error bars=s.d.) and represented graphically for ThHsp70 and either (k) ThAtm1_1 or (l) ThTom70. (m) The extent of mitosomal labelling by the ThAtm1_1 antibody relative to other cell compartments was quantified based on the proportion of pixel intensity across six fields of view for each of three replicates (error bars=s.d.).