Checkpoints of apicomplexan cell division identified in Toxoplasma gondii
Fig 2
Conditional expression of T. gondii Crks.
The upper schematic illustrates anhydrotetracycline (ATc) mediated control of gene expression in the tet-OFF system. In the absence of ATc the tetracycline transactivator (tTA) binds to the tet-operator (tetO) maintaining the active transcription of the gene of interest (GOI). Downregulation of the GOI product is achieved by transcriptional repression with ATc that prevents tTA binding to tet-operators incorporated into the tet-OFF promoter of the GOI. IFA images on the left show predominant localization of the epitope-tagged kinases (α-HA, green) relative to nuclear staining (DAPI, blue) and IMC compartment (IMC1, red). Downregulation of TgCrk expression after 24 h treatment with 1μg/ml ATc was verified for seven kinases by IFA and Western Blot analysis (α-Tubulin A staining was used as a loading control). The essentiality of each kinase was tested by the ability of TgCrk tet-OFF mutant parasites to form plaques after 6 days with 1μg/ml ATc (representative DIC images on the right). Parent Tati-RHΔKu80 strain was included as positive control (top DIC image). Percentage number indicated below each DIC image represents the number of plaques relative to the -ATc condition for each tet-OFF mutant. All analyzed kinases were either essential or required for tachyzoite growth, with the exception of TgCrk8. Downregulation of TgCrk2, resulted in growth arrest of morphologically normal looking parasites, while knockdown of TgCrk1, TgCrk4 and TgCrk6 led to major morphological abnormalities that are indicated in the IFA images and quantified in the S2 Fig.