Fig. 4.

TbTOR1 and TbTOR2 independently control two distinct aspects of cell growth. (A, B, D, and E) RNAi knockdown of TbTOR1, TbTOR2, TbRaptor, and TbAVO3 affects cell proliferation. Growth curves of bloodstream trypanosome cell lines containing inducible RNAi constructs (as indicated) were obtained after RNAi induction with doxycycline or uninduced as a control. Both induced and uninduced cultures were diluted daily to 2.5 × 10⁴ cells per ml to maintain cell density between 2.5 × 10⁴ and 10⁶ cells per ml. (C) Western blot analysis of TbTOR1 and TbTOR2 in total cell extracts (5 × 10⁶ cells per lane) upon RNAi induction and during the following 4 days. Tubulin was used as a loading control. (F) Analysis of the cellular morphology in bloodstream trypanosome living cells after knockdown of TbTOR1, TbRaptor, TbTOR2, and TbAVO3. TbTOR1 and TbRaptor (TORC1)-depleted cells do not show major changes in morphology. TbTOR2 and TbAVO3 (TORC2) depleted trypanosomes display cytokinesis defects leading to the formation of large abnormal cells. Cells were examined by using Nomarski optics after 2 days of RNAi induction as indicated. (Scale bar: 2 μm.) (G) TORC1 and TORC2 disruption by RNAi have opposite effects on cell size. FACS analysis showing forward scatter (FSC) of RNAi cell lines targeting components of both TOR-containing complexes 2 days after RNAi induction. Kolmogorov–Smirnov statistical analysis of the FSC data showing that changes in cell volume upon protein depletion are statistically significant (P < 0.001) is labeled by asterisks. (H) TbTOR1 regulates nucleolar structure and RNA pol I localization to the nucleolus. Exponentially growing RNAi cell lines were analyzed 2 days after RNAi induction. Nucleolar structure was visualized by DAPI stain as a spherical dark region in the DAPI-stained nucleus, and RNA pol I localization was analyzed by IF using anti-TbRPA1 antibody. Note the presence of multiple nuclei and kinetoplasts in TbTOR2 KD cells without major changes in nucleolus structure. (I) TbTORC1 (TbTOR1 and TbRaptor) positively regulates protein synthesis in T. brucei. Total protein synthesis was estimated in TbTOR1, TbTOR2, TbAVO3, and TbRaptor RNAi KD cell lines after 24 and 48 h of RNAi induction as described in SI Materials and Methods. A significant reduction in protein synthesis was detected upon TbTOR1 and TbRaptor depletion, but not upon TbTOR2 and TbAVO3 depletion experiments. Plots of the means ± SD of four assays are shown.