Checkpoints of apicomplexan cell division identified in Toxoplasma gondii
Fig 5
Loss of TgCrk1 that forms a stable sub-nuclear complex with TgCycL leads to abnormal assembly of the daughter cytoskeleton.
(A) Cytological defects of TgCrk1 tet-OFF mutant were analyzed and quantified (B) using antibodies against the basal complex marker MORN1 (red), cytoskeletal marker IMC1 (green) and nuclear staining DAPI (blue). The guide panel shows schematics of the parasites captured in IFA images (#) and parasite features are summarized in the description panel. During normal mitosis depicted in the –ATc images, membrane protein MORN1 localizes to the mother basal complex, intranuclear spindle compartment centrocone, and to the attached MORN rings (image a). Downregulation of TgCrk1 with 1 μg/ml ATc for 16 h resulted in incomplete assembly (image c) or fragmentation (image e) of the MORN rings and associated accumulation of IMC sacs (image f). Fully arrested vacuoles shown in the bottom panel (24 h +ATc) exhibit a catastrophic phenotype; the chaotic daughter cytoskeletal mass (image h) disconnected from parasite cytoplasm. (B) Vacuoles undergoing proper budding (normal buds) or containing parasites displaying cytological defects depicted in the images c-f (piled IMC1/MORN1), or in the images g and h (cytological defects) were quantified in the TgCrk1 tet-OFF populations grown with or without 1 μg/ml ATc for 24 h. Results are plotted on the graph. Statistically different values are indicated with asterisks (** ≤ 0.01; *** ≤ 0.001). (C) TgCrk1 tet-OFF mutant was stained with α-ISP1 antibodies to monitor changes in the apical complex. Image on the left illustrates proper expression and localization of the marker in S/M tachyzoites (-ATc). Downregulation of TgCrk1 (+ATc) caused severe morphological changes in the apical cone and reduced expression of ISP1 protein. Dotted line indicates boundary of the mis-shaped parasite. Major abnormalities are labeled and indicated by double-headed arrows. (D) TgCrk1-TgCyclin complexes were immunoisolated from parasites co-expressing TgCrk1HA and myc-tagged TgPHO80, TgCycL, TgCycY and TgCycH (used transgenic strains are listed in S1 Table). The soluble fraction before [In] and after immunoprecipitation [DF], and protein complexes on the beads [IP] were probed with α-myc antibody to detect TgCyclins and with α-HA antibody to verify TgCrk1 pulldown (top panel). The results revealed a dominant TgCrk1-TgCycL complex. (E) Dual-tagged parasites expressing TgCrk1HA and TgCycLmyc were stained with a α-HA (green) and α-myc (red) antibodies. Proteins display similar localization patterns with particular accumulation in the nuclear sub-compartment (insert, arrow).