Checkpoints of apicomplexan cell division identified in Toxoplasma gondii
Fig 3
Conditional expression of T. gondii cyclins in the tet-OFF model.
(A) IFA analysis of TgCyclins localization. Tagged at the genomic locus TgCycHHA, TgCycLHA and TgCycYHA were visualized using α-HA antibody (green). Due to low native expression level, localization of ectopically expressed DDmycTgPHO80 is shown after a 3 h induction with 100nM Shield1. (B) TgCycYHA is dynamically expressed in G1 stage. Yellow dotted line indicates individual vacuoles. The cell cycle stage was determined based on intensity of the nuclear staining (DAPI, blue). (C) Transgenic tet-OFF clones of TgCyclins were established with alternative 3xHA or myc tags as described in Material and Methods and in the S1D Fig. IFA images show a representative vacuole after 24 h growth with 1μg/ml ATc. Parasite shape and nucleus were visualized with α-IMC1 (red) and DAPI (blue) staining, respectively. ATc-induced downregulation of the TgCyclins was confirmed by Western blot analysis using α-Tubulin A staining as a loading control. Results of the plaque assays are shown after 6 days growth with 1μg/ml ATc (DIC panel on the right). The percentage is the number of plaques formed relative to no ATc condition. The phenotype of TgPHO80 and TgCycL deficient parasites suggested the factors' involvement in the cell cycle regulation.