Checkpoints of apicomplexan cell division identified in Toxoplasma gondii
Fig 7
Loss of cytoplasmic TgCrk4 leads to abnormal centrosome duplication.
(A) TgCrk4 deficiency affected duplication of the structures localized in the cytoplasm. TgCrk4 tet-OFF mutant parasites were treated with 1μg/ml ATc for 24 h and stained with α-IMC1 (surface), DAPI (DNA) and the markers of the following structures: centromere (α-TgCenH3), outer core of the centrosome (α-Centrin1) and apicoplast (α-TgAtrx1). To visualize inner core of centrosome, we introduced recombinant TgCEP250myc protein on the fosmid in TgCrk4 tet-OFF mutant and stained parasites with α-myc antibody. Dynamics of organelles and structures are indicated with red arrows and summarized in the Guide panel. General deficiencies caused by the loss of TgCrk4 are indicated with white arrows. (B) Quantification of the morphologically abnormal vacuoles (blue, determined by α-IMC1 and DAPI co-staining) containing under-duplicated (green) or over-duplicated (red) centrosomes (visualized by α-Centrin1 staining) in the TgCrk4 tet-OFF mutant grown without or with ATc for 20 h is shown on the bar graph (for raw data see S1 Table). Significant difference between two indicated conditions was verified by unpaired t-test that returned the p-value 0.02 (*). Note that centrosome re-duplication is a predominant defect and together with centrosome under-duplication affected about 30% of the TgCrk4 deficient population. (C) The reduplicated centrosome of TgCrk4-deficient parasites preserved internal integrity. TgCrk4 tet-OFF mutant parasites co-expressing TgCEP250myc were stained with α-Centrin1 (outer centrosomal core) and α-myc (TgCEP250myc, inner centrosomal core) after 16 h growth with or without 1μg/ml ATc. White arrows indicate re-duplication of both centrosome cores in TgCrk4 deficient parasite. The enlarged merged image shows that only one of the centrosomes remains connected to the nucleus. (D) Reduplicated centrosomes are associated with assembled kinetochores in TgCrk4 deficient parasites. Transgenic TgCrk4 tet-OFF mutant expressing kinetochore marker TgNdc80myc was grown with or without 1μg/ml ATc for 16 h and co-stained with α-Centrin1 (centrosome), α-myc (TgNdc80myc, kinetochore), and DAPI. In mitotic cells expressing TgCrk4 (-ATc), centrosome duplication and kinetochore assembly occurs once per chromosome cycle. Kinetochores are assembled after centrosome segregation (2 centrosomes:1 kinetochore, images a, b), duplicated after the spindle break (2 centrosomes: 2 kinetochores, images c, d) and segregated/disassembled early in the budding (2 centrosomes: 0 kinetochores, images e, f). Lower panel (+ATc) shows vacuole of four TgCrk4-deficient parasites with three cells (white arrows) containing reduplicated centrosomes associated with assembled kinetochores (4 centrosomes: 2 kinetochores, images g, h). Number of structures per parasite is shown in each insert. Parasite shapes are outlined in the merge panel.