Checkpoints of apicomplexan cell division identified in Toxoplasma gondii
Fig 6
Nuclear TgCrk6 regulates spindle biology in tachyzoites.
(A) TgCrk6 tet-OFF mutant parasites were grown in the absence (upper row) or presence of 1 μg/ml ATc for 16 h (bottom row) and analyzed by IFA using α-MORN1 (red, centrocone and basal complex) and α-IMC1 (green, parasite shape and internal buds) antibody. Chromosome dynamics was detected by DAPI staining (blue). Downregulation of TgCrk6 (+ATc) led to an inability of the centrocone compartment to duplicate during mitosis. The guide panel includes a cartoon and a description of the analyzed structures and observed deficiencies. Single (red) and duplicated (blue) centrocones (α-MORN1) were quantified in 25–50 randomly selected budding vacuoles (α-IMC1) (for raw data, see S1 Table) revealing a significantly larger number of the vacuoles with a single centrocone when TgCrk6 tet-OFF mutant was treated with ATc for 24 h (+ATc). P-value ≤ 0.0001 (***) was calculated using unpaired two-tail t-test. (B) Kinetochore dynamics were analyzed in the TgCrk6 tet-OFF mutant parasites co-expressing TgNdc80myc using α-myc antibody. To identify vacuoles in cytokinesis, microtubules of the growing internal buds were stained with antibody against acetylated Tubulin A (green). Kinetochore dynamics in TgCrk6 tet-OFF mutant after 24 h treatment with or without 1μg/ml ATc is summarized in the guide panel on the right. TgCrk6-deficient parasites (+ATc) retained a single assembled kinetochore (image g) positioned between two internal daughters (image h).