Fig. 3.

Interfering with TgArk1 function results in a nuclear segregation defect. a The overexpression of DD-Myc-TgArk1D/A kinase results in formation of nucleus-deficient parasites. IFA performed on intracellular parasites of the ark1D/A-Δku80 strain shows an IMC marker (TgIMC1, red), a nucleus marker (TgBUB3-HA₃, green, the two upper panels) and nuclei stained by DAPI (lower panel). In the presence of shield-1 for 12 h, nucleus-deficient parasites appear, whereas normal nucleus segregation is observed in the absence of shield-1 for ark1D/A-Δku80 strain, as expected. A white asterisk indicates the parasites lacking nuclei. In the intermediate panel, a nucleus is observed outside the parasites, in the residual body. b Quantification of the number of nucleus-deficient parasites in three different strains (RH-Δku80, ark1WT-Δku80 and ark1D/A-Δku80) in the presence or absence of shield-1 for 12 h. The percentage of parasites displaying nucleus segregation defect was determined for at least 300 parasites. The results shown are from three independent experiments. c Quantification of the number of vacuoles showing nuclei in residual bodies in three different strains (RH-Δku80, ark1WT-Δku80 and ark1D/A-Δku80) ± shield-1 for 12 h. At least 300 vacuoles were examined for each condition. Values are mean ± SD for three independent experiments. d Flow cytometric analysis of ethanol (EtOH)-fixed wild-type and mutant parasites stained with propidium iodide after RNase treatment. Parental (RH-Δku80) and dominant negative mutant (ark1D/A-Δku80) parasites were grown in the presence of shield-1 for 12 h. The 1 N population was considered in the G1 phase, while the 1.8–2 N population is considered in the S/M phase of the cell cycle. ark1D/A-Δku80 dominant negative mutant parasites showed an important increase in their sub-1 N aneuploidy population that is consistent with nuclei loss