TbKINX1B: a novel BILBO1 partner and an essential protein in bloodstream form Trypanosoma brucei

Figure 1. TbKINX1B is an N-kinesin with a classical motor domain. A. Predicted secondary structure representation of TbKINX1B protein. The kinesin motor domain (TbKINX1BMD aa. 4-496 with P-loop aa. GQTGSGKT, Switch I aa. NEHSSRSH and Switch II aa. DLAGSE), TbKINX1B leucine zipper (LZ, aa. 756–784), the coiled-coil (aa. 1082–1297), and the TbBILBO1-binding domain (B1BD aa. 517–715) are shown. B. Alignment-based homology model of TbKINX1B motor domain to Mus musculus KIF1A motor domain (PDB 1I5S) by Phyre2 software. The predicted tertiary structure is represented as a superposition of both kinesin motor domains, TbKINX1B (colored: yellow for β-sheet and red for α-helix) and KIF1A (white), corresponding to 100% match. C. Western blot analysis of TbKINX1B in PCF and BSF cells. TbKINX1B, TbBILBO1 and Tubulin were probed on whole cells (WC), detergent-extracted cytoskeleton (CSK) and soluble fraction (S) of lysed cells.

Figure 2. TbKINX1B displays a dual localization. A. Co-immunolabeling of TbKINX1B and TbBILBO1 on PCF detergent-extracted cells using anti-BILBO1 (red) and anti-TbKINX1B (green). B. Co-immunolabeling of TbKINX1B and BILBO1 on BSF detergent-extracted cells using anti-BILBO1 (red) and anti-TbKINX1B (green). C. Quantification of TbKINX1B BB and/BB+FPC localization in PCF and BSF from immunofluorescence experiments shown in A and B. BB localization (white bars) or BB and FPC localization (grey bars) were quantified at different cell cycle stages (200 cells, n = 3). D. Co-immunolabeling of TbKINX1B (green) and the basal bodies marker YL1/2 (red) and the transition zone marker FTZC (cyan) on detergent-extracted PCF cells cytoskeleton and flagellum. E. Immuno-TEM using PCF isolated flagella that were labelled with anti-TbKINX1B (15 nm gold particles), and co-immunolabeled with anti-TbKINX1B (15 nm gold) and anti-TbBILBO1 (6 nm gold). The images show positive labelling of the protein at the basal bodies (pBB, mBB), Flagellar Pocket Collar (FPC) and at the MTQ. The zoom image shows the TbBILBO1 6 nm gold beads on the MTQ and the colocalization of TbKINX1B and TbBILBO1. Scale bars in A, B and C…

Figure 3. Microtubule and FPC localization of TbKINX1B depends on its motor domain and BILBO1-binding domain respectively. A. Immuno-precipitation of TbKINX1B and immuno-detection by western blotting of TbKINX1B and TbBILBO1 on input (I), flow-through (FT), wash (W), and elution (E) fractions. B. Induction of the expression of TbKINX1Bmyc, TbKINX1BmycMD or TbKINX1BmycB1BD does not affect PCF cell growth, as compared to WT (n = 3). C. Western blot of PCF WT cells and cells non-induced (–) or tetracycline-induced 24 h, 48 h, 72 h for the expression of TbKINX1Bmyc (151 KDa), TbKINX1BmycMD (62 KDa) or TbKINX1BmycB1BD (28 KDa). Expressed proteins were detected using anti-myc antibody and using PFR2 (L8C4) as a loading control. No alteration on TbBILBO1 protein levels was observed. D. Co-immunolabelling on detergent-extracted cells of TbKINX1Bmyc and TbKINX1BmycB1BD proteins (green) with TbBILBO1 (red), and of TbKINX1BmycMD (green) with Tubulin (red).

Figure 4. TbKINX1B is involved in basal body/FPC segregation in PCF. A. Left panel, Western blot analysis of whole cell protein extracts from PCF TbKINX1BRNAi cell line in the presence (+) or absence (−) of tetracycline after 24 h, 48 h or 72 h probed with anti-TbKINX1B, anti-TbBILBO1, and anti-Tubulin (TAT1) as a loading control (left panel). Right panel, PCF growth curves showing that induction of RNAi down-regulation of TbKINX1B (TbKINX1BRNAi) does not dramatically affect cell growth when compared to WT and non-induced cells (n = 3). B. Down-regulation of TbKINX1B induces misplaced kinetoplasts. Left panel, IFA micrographs from CSK preparation of WT (a) and TbKINX1BRNAi cell lines after 48 h of induction (b, c). Kinetoplasts (K) and nuclei (N) were stained with DAPI. Right panel, quantification of kinetoplast localization in 1K1N WT cells and in TbKINX1BRNAi cells after 24 h, 48 h and 72 h of induction. C. Left panel, immunolocalization of TbKINX1B (green) in TbBILBO1RNAi induced detergent-extracted cells. Right panel, immunolocalization of TbBILBO1 (green) in TbKINX1BRNAi induced detergent-extracted cells. Scale bars 5 μm.

Figure 5. TbKINX1B is essential in bloodstream forms. A. Left panel, western blot of BSF whole cell protein extracts from TbKINX1BRNAi cell line in the absence (−) or presence (+) of tetracycline at 24 h, 48 h and 72 h using anti-TbKINX1B, and anti-tubulin (TAT1) as a loading control. Right panel, down-regulation of TbKINX1B in BSF (+) inhibits cell growth after 48 h of induction, as compared to non-induced cells (−) (n = 3). B. Left panel, IFA micrographs of WT and of 48 h induced TbKINX1BRNAi BSF cell lines. Immunofluorescence shows that down-regulation of TbKINX1B leads to multi-flagellated and multinucleated cells. Flagella were labelled with anti-PFR2 (green), and kinetoplasts and nuclei were DAPI-stained (blue). A large FP or vacuole is marked with an asterisk. Scale bars 5 μm. Right panel, quantification of cell division phenotypes in WT and in TbKINX1BRNAi cell lines after 24 h, 48 h and 72 h of induction. Normal phenotypes are underlined (200 cells, n = 3). C. Transmission Electron Microscopy (TEM) thin-sections of WT BSF and of TbKINX1BRNAi induced for 48 h. The structural organization of WT cells reveal well-defined FP (black asterisk), FPC, Golgi (G), endoplasmic reticulum…

Figure S1:. TbKINX1B can hydrolyse ATP in vitro. A. ATPase activity using the ELIPA in vitro assay. Kinesin activity was measured by the amount of generated phosphate per minute, in the presence of taxol-stabilized microtubules and ATP. The human kinesin heavy chain motor domain (KHCMD) is the positive control kinesin in presence of MTs (+MTs, black line) or negative control in the absence of MT (−MTs, grey line), the TbKINX1BMD (green), TbKINX1BMD deleted for the ATPase domain (ΔP-loopMD, red line), and 6HisTRX purified protein (yellow line), (n = 3). B. Recombinant TbKINX1B proteins purification. Western blot analysis (upper panel) and Coomassie-stained SDS-PAGE (lower panel) of the purification of the Trx-6HisTbKINX1BMD (A), Trx-6HisTbKINX1B-ΔP-loopMD (B), and Trx-6His (C) proteins. The recombinant proteins were immunolabelled using anti-Histidine tag. Abbreviations: Total (T), non-induced (NI), induced (IND), supernatant (S), flow-through (FT), wash (W, different wash fractions numbered), Elution (E, fractions numbered).

Figure S2:. Impact of RNAi knockdown of TbKINX1B or TbBILBO1 in PCF on expression and localization. A. Minor phenotypes were observed in TbKINX1BRNAi cells induced for 24 h to 72 h. (a) The flagella were labelled with anti-PFR antibody (L8C4). (b) The FPC structures were labelled with anti-TbBILBO1 antibody. (c) The axonemes were labelled with anti-TbSAXO antibody (mAb25). (d) The FAZ structure was labelled with the anti-FAZ antibody (L3B2). Scale bars 5 μm. B. Expression level of TbKINX1B upon RNAi down-regulation of TbBILBO1. Immunoblotting of TbKINX1B, TbBILBO1 and Tubulin in whole cell protein extracts (WC), detergent-extracted cytoskeleton fraction (CSK), and soluble fraction (S) of lysed TbBILBO1RNAi non-induced (NInd) or induced for 36H (Ind) cells.