Trypanosoma brucei centrin5 is enriched in the flagellum and interacts with other centrins in a calcium‐dependent manner

Figures

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  Figure 1. Characterization of TbCentrin5. (A) Evolutionary tree of centrins. Numbers on branches represent bootstrap support values. Hs, Human sapiens; Mm, Mus musculus; Cr, Chlamydomonas_reinhardtii; Tg, Toxoplasma gondii; Eo, Euplotes octocarinatus; Sd, Scherffelia dubia; Sc, Saccharomyces_cerevisiae; Tt, Tetrahymena thermophila; Ld, Leishmania donovani; Lm, Leishmania major; Tb, Trypanosoma brucei; (B) The expression of TbCentrin5‐EYFP examined by western blot with anti‐GFP probe. The levels of α‐tubulin served as the loading control. (C) The subcellular localization of TbCentrin5. The localization of TbCentrin5‐EYFP (green) was examined in paraformaldehyde‐fixed intact cells. Cells were stained DAPI for DNA (blue), small blue dots are kinetoplasts, and large blue structures are nuclei. 1N1K, 1N2K, and 2N2K cells were tabulated, respectively. Scale bars: 5 μm.

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  Figure 2. Multiple sequence alignments of TbCentrin5 with other centrins. Tb, Trypanosoma brucei; Hs, Homo sapiens; Lm, Leishmania major. Identical residues are shaded in red box, and conserved residues are colored in red.

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  Figure 3. Structure model of TbCentrin5. The model was built by SWISS‐MODEL based on homology modeling techniques.

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  Figure 4. The model of TbCentrin5 was evaluated by Ramachandran plots.

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  Figure 5. The Ca2+ binding of TbCentrin5. (A) Gel filtration of Superdex G‐75 column and SDS/PAGE analysis of TbCentrin5. (B) Saturated titration of TbCentrin5 with Ca2+ ions was measured by ITC. (C) The model of TbCentrin5 bound by Ca2+ ions. Ca2+‐binding site at the loop of EF‐hand III (D) and EF‐hand IV (E). Cyan‐colored sticks represent the residues which interact with Ca2+ ions, red represents oxygen atom, blue represents nitrogen atom. Magenta lines represent coordinated bonds.

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  Figure 6. Interactions between TbCentrin5 and other TbCentrins. (A) GST‐pull down assay. TbCentrin5‐3HA was pulled down by GST‐TbCentrin1‐4 and detected by western blot. ITCs of TbCentrin1‐4 titrated with TbCentrin5 were performed without (B) or with (C) EGTA treatment.

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  Figure 7. Sedimentation assays to monitor the cell motility. (A) Effect of TbCentrin5 RNAi on cell proliferation. Quantitative RT‐PCR was used to monitor the level of TbCentrin5 mRNA in noninduced control cells and RNAi cells (inset). (B) The parental 29‐13 cell line, the noninduced cells, and TbCentrin5 RNAi cells after tetracycline induction for 3 days were incubated in cuvettes. The change of cell density (ΔOD600) was determined and plotted against the time of incubation (hours). Three independent experiments were carried out, and the error bars represent SD.