BILBO1—FPC4 interaction involves their N-terminal and C-terminal domains respectively.

<p>(A) BILBO1-FPC4 yeast two-hybrid interaction test. Left panel: Schematic overview of BILBO1 and FPC4 domains and of the combinations tested by Y2H. The EF-hand calcium binding sites of BILBO1 are represented in green and the coiled-coil domains in BILBO1 and FPC4 are represented in blue. The FPC4 B1BD is represented in violet. Right panel: The interactions tested were probed on–histidine selective medium (-His) and on growth control medium (control) (n = 3). Positive control involved p53 and T-antigen, whereas negative control involved Lamin and T-antigen. (B) Heterologous expression and co-expression in U-2 OS cells of FPC4 and FPC4 truncations fused to a C-terminal GFP tag, and of BILBO1. BILBO1 (a), FPC4 (b), FPC4-ΔB1BD (c) and FPC4-B1BD (d) were expressed alone and cells were probed with anti-BILBO1 (red) and anti-GFP (green). In e, f, g, cells were expressing BILBO1 + FPC4, BILBO1 + FPC4-ΔB1BD, and BILBO1 + FPC4-B1BD respectively and were also probed with anti-BILBO1 (red) and anti-GFP (green). Cells were extracted before labelling, except in (d) to show the cytoplasmic localisation of FPC4-B1BD, which is not visible on extracted cells. The transfections were performed more than three independent times. Nuclei were DAPI stained (blue). Scale bar represents 10 μm.</p

FPC4 binds to the remnant MtQ complex of isolated flagella.

<p>(A) Co-labelling of myc-tagged proteins (green) and tubulin (red) on isolated flagella from WT cells (a), and cells over-expressing myc-FPC4 (b) or myc-FPC4-ΔB1BD (c). White arrowheads are pointing to the zoom areas. Scale bars 5 μm. (B) Immuno-gold labelling of myc-FPC4-ΔB1BD (anti-myc, 10 nm gold beads) and tubulin (15 nm gold beads) on isolated flagella. In the zoom image (b), the arrows highlight the MtQ decorated with anti-tubulin and anti-myc labelling that appears close to the MtQ. In the zoom image (c), the black arrowheads highlight the FPC structure decorated by the anti-myc antibody. All the experiments were performed more than three independent times.</p

FPC4 is a hook complex and an FPC protein.

<p>(A) (a) Immunolabelling of endogenous FPC4 on cytoskeleton extracted cells from <i>Tb</i>427 29–13 cells using the rat anti-FPC4 antibody. (b) Cytoskeletons from the cell line expressing endogenously myc-tagged FPC4 probed with anti-myc. (c and d) Cytoskeletons from the cell line overexpressing myc-FPC4 using anti-myc. Co-labelling with anti-BILBO1 (a, b, c) or anti-MORN1 (d). (B) Anti-myc immuno-electron microscopy on flagella isolated from myc-FPC4 expressing cells (a), and the respective enlargements (b, c and d). (C) FPC4 immunolabelling using anti-FPC4 on CK extracted <i>Tb</i>427 90–13 BSF cells. (D) Co-labelling on cytoskeleton extracted <i>Tb</i>427 29–13 cells using anti-FPC4 and anti-MORN1 (a-d), and rat anti-FPC4 and anti-BILBO1 (e-h). (E) BILBO1^<i>RNAi</i> was induced for 36 h and cells were detergent extracted and probed with rat anti-FPC4 (green) and anti-MORN1 (red). The white arrowheads indicate the area that is enlarged in the zoom image and highlight the labelling of FPC4 and MORN1 within the new detached flagellum (a) and at the base of the axoneme of the new detached flagellum (b). Kinetoplasts and nuclei were DAPI stained in A, C, D and E. Scale bars in A, C, D, and E represent 5 μm. In A c and d where the cells were induced for 24 h. All the experiments were performed more than three independent times.</p

The overexpression of FPC4-ΔB1BD induced morphological phenotypes.

<p>(A) Co-localisation of GFP-FPC4 (anti-GFP, green) and BILBO1 (anti-BILBO1, red) by immunofluorescence on cytoskeletons. The cells were induced to express GFP-FPC4 for 48 h. (B) Transmission electron micrograph of a thin section of embedded cells that were over-expressing GFP-FPC4 (72 h of induction). Black arrowheads highlight the electron dense structure observed in these cells resembling the filament observed by immunofluorescence. (C) Co-localisation of myc-FPC4-ΔB1BD (anti-myc, green) and BILBO1 (red), MORN1 (red) and FAZ (red) by immunofluorescence on cytoskeletons. The cells were induced for 48 h. Several phenotypes were observed: filament connection between 2 FPCs (a, c, e), epimastigote-like cells (b), and localisation of myc-FPC4-ΔB1BD together with MORN1 at the distal end of the cell body (d). Abnormal cells still exhibit FAZ structures that were positively labelled with L3B2 (f). (D) Immuno-electron microscopy localisation of gold particles of myc-FPC4-ΔB1BD (anti-myc, 15 nm gold) and BILBO1 (anti-BILBO1, 10 nm gold) on isolated flagella from cells expressing myc-FPC4-ΔB1BD. (E) Transmission electron microscopy of thin sections of embedded cells over-expressing myc-FPC4-ΔB1BD showing an epimastigote-like phenotype. (F) Counts of cells showing different phenotypes related to kinetoplast positioning. Normal phenotype (brown - 1K1N, 2K1N, and 2K2N cells where cell morphology was normal), abnormal (yellow—Zoids and multinucleated), and abnormal (green—misplaced kinetoplast) for WT and non-induced and induced (24, 48, and 96 h) myc-FPC4-ΔB1BD over-expressing cells. The misplaced kinetoplast category was then divided into subcategories (48 h induction). Kinetoplasts and nuclei were DAPI stained and scale bars represent 5 μm in A and C. Initial immuno-localisations were performed on two individual clones.</p

Immuno-localisation of endogenously tagged 10xTY1-FPC4, BILBO1 and MORN1.

<p>(A) Wide-field microscopy observation of the triple labelling of BILBO1, MORN1 and FPC4 on cytoskeleton from a SmOxP427 cell line expressing endogenously 10xTY1 tagged FPC4. (B) Immuno-electron microscopy on flagella labelled with anti-TY1 for FPC4 (5 nm gold beads), anti-BILBO1 (15 nm gold beads), and anti-MORN1 (15 nm gold beads). (C) STED confocal microscopy views of the co-labelling of BILBO1, MORN1 and FPC4 on cytoskeletons from a SmOxP427 cell line expressing endogenously 10xTY1 tagged FPC4 (a-c). The asterisks indicate the kinetoplast position relative to the structure. The snapshots of the 3D reconstructions show the close proximity of BILBO1 (green), FPC4 (red) and MORN1 (cyan). (D) Normalised intensities Plot corresponding to the section indicated by the white lines (a, b) indicate the co-localisation of the proteins. In A and B, scale bars represent 1 μm.</p

The formation of a stable complex between the BILBO1-NTD and FPC4-B1BD depends on the residues Y87 and F89 on the BILBO1-NTD.

<p>(A) SEC elution profile (S-200 16/60) of the mixture of purified BILBO1-NTD’ (aa1-120) and FPC4-B1BD (aa357-440). Both proteins were co-eluted in the elution peak of 86.88 ml. The 44.95 peak is the void volume. Inset: Coomassie-stained SDS-PAGE gel showing proteins in the fractions indicated with the coloured bars in the elution profile. Upper arrow indicates the BILBO1-NTD’ and lower arrow indicates FPC4-B1BD. (B) SLS results of the complex of BILBO1-NTD’ and FPC4-B1BD. The apparent molecular mass of 24.9 kDa indicates the formation of a hetero-dimer. (C) ITC analysis showing that interaction with FPC4-B1BD is partially reduced in BILBO1-Edge-Mut but completely abolished in BILBO1-Centre-Mut. (D) Co-expression in U-2 OS cells and immunolabelling on permeabilised cells (no extraction) or extracted cells (extraction) of FPC4-B1BD-GFP (anti-GFP, green) with either BILBO1-Centre-Mut or BILBO1-Edge-Mut (anti-BILBO1, red). Nuclei were DAPI stained. Scale bar represents 10 μm. The experiments were performed more than three independent times. (E) WB and growth curves for the PCF WT and induced cell lines for the expression of Ty1-tagged BILBO1, BILBO1-Centre-Mut, and BILBO1-Edge-Mut. Error bars represent the standard error from 3 independent experiments (and are smaller than the data point mark).</p

FPC4 is a microtubule binding protein.

<p>(A) Anti-acetylated tubulin and anti-GFP immunolabelling on U-2 OS cells expressing GFP tagged FPC4 (a), FPC4-ΔB1BD (aa 1–356) (b), FPC4-B1BD (aa 357–444) (c), FPC4-1-217 (d), and FPC4-shuffled-1-217 (e). Scale bar represents 10 μm. The transfections were performed more than three independent times. (B) Microtubule binding assay (n = 3). Increasing concentrations of purified FPC4-1-260_6His (a) or _6HisFPC4-B1BD were incubated with fixed concentration (7.2 μM) of tubulin (polymerised microtubules). After centrifugation, pellets (P) and supernatants (S) were loaded on SDS-PAGE (12% in a, 15% in b). Proteins were stained using Instant Blue.</p

The B1BD of FPC4 is not involved in the targeting to the FPC/hook complex.

<p>(A) Immunofluorescence on cell lines over-expressing (48h of induction) myc-FPC4 (a), myc-FPC4-ΔB1BD (b), myc-FPC4-B1BD (c, d), and GFP-FPC4 (24 h of induction) (e) using anti-myc or anti-GFP (green) and anti-BILBO1 (red). Immunofluorescence was done on cytoskeletons except in (d) were cells were permeabilised. Kinetoplasts and nuclei were DAPI stained. Scale bars represent 5 μm. Initial immunofluorescence analyses were performed on two individual clones. (B) Growth curves for WT cells (grey diamond), and cells non-induced (black square) or induced (white square) for the expression of myc-FPC4, myc-FPC4-ΔB1BD, myc-FPC4-B1BD, and GFP-FPC4. Error bars represent the standard error from 3 independent experiments (errors bars are smaller than the data point mark). (C) Western blot analysis of the level of expression of myc-FPC4, myc-FPC4-ΔB1BD, and GFP-FPC4 using anti-FPC4, and <i>Tb</i>SAXO (mAb25) as loading control. Whole cell extracts from 5.10⁶ cells were loaded on 12% SDS-PAGE gels and immuno-blotted.</p

mEFH:myc induced detached flagellum phenotypes after expression in <i>T</i>. <i>brucei</i> cells.

<p>Quantification of the percentage of cells exhibiting detached flagella phenotypes after six and 24 h expression of mEFH1:myc, mEFH2:myc and mEFH1+2:myc tagged proteins in procyclic <i>T</i>. <i>brucei</i> cells. Expression of all EF-hand mutations induced detached flagella phenotypes.</p><p>mEFH:myc induced detached flagellum phenotypes after expression in <i>T</i>. <i>brucei</i> cells.</p

Schematic diagram of BILBO1 primary structure and yeast two-hybrid identification of its interaction domains.

<p>(A) BILBO1 has two predicted EF-hand calcium-binding domain EFH1 and EFH2, from amino acids Lysine 185 to Tyrosine 249, and a long domain predicted as coiled-coil. Serine 163 is phosphorylated <i>in vivo</i>. The main truncations used in this study are shown schematically and labelled as T1-T4. The mutations in the EF-hand calcium binding domains are indicated below the wild-type amino acid sequence. (B) Bait (BILBO1 full-length) and prey interactions were tested by drop tests on adenine-free selective medium. Full-length BILBO1 interacts with full-length BILBO1, whilst truncations T1 and T2 do not interact with full-length BILBO1. T3 and T4 truncations interactions were positive, demonstrating that the coiled-coil domain is required for BILBO1-BILBO1 interaction.</p