A Novel Bipartite Centrosome Coordinates the Apicomplexan Cell Cycle
Fig 5
The outer centrosome core protein TgSfi1 has an essential role in the parasite cell cycle.
(A) Growth of the chemical mutant 9–86E4 is inhibited by high temperature, leading to lethal arrest (40°C). Cultures were pre-synchronized by limited invasion, and the average number of parasites per vacuole was calculated after 0, 8, 16, and 24 h at indicated temperature. Bar graph shows mean values and standard deviations for three growth experiments with a minimum of 50 vacuoles per time point (for all raw data, see S1 Data). (B) Duplication of the outer core detected by anti-Centrin staining is severely affected in the 9–86E4 mutant grown at 40°C for 20 hours but not when the mutant is grown at the permissive temperature (34°C). Culture temperatures are indicated in the upper left of each image panel. The included guide panel is a non-colored inverted image of the merged red (anti-IMC1) and green (anti-Centrin) stains. Red asterisks indicate the position of the Centrin-positive structures. Note the parasite at 40°C with duplicated nuclei with a single TgCentrin1-associated core (circled) where, normally, there should be two cores. (C) Genetic complementation of mutant 9–86E4 with cosmid genomic libraries identified the defective locus on chromosome VIII (see top diagram) and this was further resolved to gene 2 (TGME49_274000) with individual cosmids that span the locus; gene 2 encodes centrin co-factor, TgSfi1 (see S1B Fig. and S2 Fig.) [38]. (D) Whole genome sequencing of mutant 9–86E4 independently confirmed the ts-TgSfi1 protein was mutated with a E1759K change shown by a red bar in the TgSfi1 protein diagram. Putative centrin binding sites (black box) are also indicated (see also S2 Fig.). (E) Western blot analysis of the mutant 9–86E4 parasites expressing ts-TgSfi1HA protein after 24 h growth at 34°C or 40°C. Total lysate of 20 x 106 parasites were probed with anti-HA-epitope or anti-α-Tubulin antibodies. (F) The average of TgCentrin1 containing outer cores (anti-Centrin staining) per parasite, determined by anti-IMC1 staining of the mother parasite, was quantified in ten microscopic fields with an average of 3–10 vacuoles (for all raw data, see S1 Data) revealing a significant reduction of the outer cores when mutant 9–86E4 was shifted to 40°C (red dots). P-value ≤ 0.0005 (***) was calculated using paired two-tail t test. The loss of the outer core at high temperature in the ts-TgSfi1 mutant does not prevent replication of the inner core. To monitor inner core, TgCEP250-L1HA was introduced in the mutant 9–86E4 (see Generation of transgenic tachyzoite strains in Materials and Methods). Co-staining of TgCEP250-L1HA (inner core) and anti-Centrin (outer core) showed that amplification of the inner core occurred in parasites in which duplication of the outer core was inhibited. (G) Multiple inner cores (TgCEP250-L1HA, red) in mutant 9–86E4 parasites at 40°C showed tight alignment and matched duplication of the nuclear centrocone (anti-TgMORN1stain, green).