<i>Tb</i>SAXO RNAi knockdowns exhibit impaired flagellar motility.

<p>Inducible RNAi<i>^TbSAXO</i> in PCF (<b>a, b, c, d</b>) and BSF (<b>e, f, g, h</b>) cells. Growth curves of PCF (<b>a</b>) and BSF (<b>e</b>) RNAi<i>^TbSAXO</i> cell lines. Corresponding WBs (PCF in <b>b</b>, BSF in <b>f</b>) of WT (parental), RNAi non-induced (-), and 24 h and 96 h induced cells probed with mAb25 and L8C4 (anti-PFR2). For PCF 5.10⁶ cells were used and 1.25×10⁵ cells for BSF. <b>c</b>. Sedimentation assay of PCF RNAi. WT (closed squares). RNAi non-induced (−TET) (closed triangles) and induced (+TET) (open circles). <b>d</b>. Mobility graph obtained from Movie S1. The positions of individual cells are plotted at 2.5 s intervals. Open circle: starting position of each cell. Arrowhead: ending position. Number in parentheses: time in seconds of a given cell was within the field of view. Bar, 10 µm. <b>g</b>. Graph of cell populations with orthodox and unorthodox kinetoplast number in BSF RNAi cultures (72 h of induction). K: kinetoplast. N: nucleus. Asterisks indicate statistical significance compared with the WT population, and −TET <i>versus</i> +TET condition (*<i>P<0.1</i>; ** <i>P<0.05</i>; ***<i>P<0.01</i>). <b>h</b> Electron-micrograph of a thin section of an aberrant BSF RNAi induced cell (72 h). (*) indicates a flagellum. Scale bar, 2 µm. Error bars in a, c, e, and g represent the standard error from 3 independent experiments.</p

<i>Tb</i>SAXO is an axoneme-associated protein in <i>T. brucei</i>.

<p><b>A</b>. Immuno-labeling and localization of <i>Tb</i>SAXO on PCF cytoskeletons. Left panel: <i>Tb</i>SAXO localization in the flagellum was identified by the mAb25 antibody (green). Labeling extends along the entire length of the flagellum from the flagellar transition zone (*, labeled with the FTZC antibody) to the distal tip. The PFR is labeled red and begins where the flagellum exits the cell (antibody L8C4). Right panel: a merge of IF and phase contrast. N: nucleus. K: kinetoplast. F: flagellum. Bar, 5 µm. <b>B</b>. Images of the proximal flagellar regions of PCF cytoskeletons from cells through mitosis and cytokinesis. In each row, the left panel shows the PFR and FTZC (*) (red), the center panel <i>Tb</i>SAXO (green), and the right panel shows merged images. The cell cycle stages are defined as 1K1N1F (1 Kinetoplast, 1 Nucleus, 1 Flagellum), 1K1N2F, 2K1N2F and 2K2N2F in rows a–d respectively. <i>Tb</i>SAXO labeling is present immediately distal to the FTZC and is clearly distinct from PFR staining. Bar, 1 µm. <b>C</b>. Immuno-gold electron microscopy reveals that <i>Tb</i>SAXO is localized in the axoneme. Mab25 immuno-gold particles can be seen mainly on the axoneme and not on the PFR of flagella of PCF WT cells. Bars, 100 nm.</p

<i>Tb</i>SAXO is a microtubule-associated protein and a microtubule-stabilizing protein.

<p>Mammalian cells (U-2 OS) expressing either MAP6-1-GFP (row a), <i>Tb</i>SAXO-Myc (row b), or various truncated versions of <i>Tb</i>SAXO-Myc (rows c–j) (constructs are represented on the schemes on the right panel). In each case, the transfected cells were incubated at 37°C or 4°C to test for MTs cold stability. Anti-tubulin (TAT1) and anti-Myc antibodies provided the images in left and centre columns respectively at each temperature. The right columns for each temperature set are merged images. The cells were subjected to short extraction before fixation and immuno-labeling. <i>Tb</i>SAXO MT stabilization is seen in images b, c, e, h and j. MT stabilization is also observed in the positive control MAP6-1-GFP expressing cells (a). Nuclei were labeled with DAPI. Bar, 20 µm.</p

Identification of SAXO proteins, a MAP6-related protein family.

<p><b>A</b>. Motif 1. The N-terminal domain and its cysteine consensus sequence. Left panel: alignment of the N-terminal sequences of the proteins used for the MEME analysis in C. The boxed sequences correspond to motif 1. Amino acids corresponding to the regular expression of motif 1 are shown in blue. Right panel: motif 1 is represented as a position-specific probability matrice derived from the MEME analysis in C. <b>B</b>. Motif 2. Mn domains in mouse Map6-1, Map6d1, and Mn-like domains and inter-repeats in mouse Saxo1, <i>Plasmodium</i> SAXO and <i>Trypanosoma</i> SAXO. Left panel: characters in blue correspond to the regular expression of the Mn and Mn-like domains identified by the MEME analysis in C; the Mn-like domains identified manually are in italics. The underlined sequences in Map6-1 and Map6d1 correspond to the experimentally identified Mn domains in mouse <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031344#pone.0031344-GoryFaure1" target="_blank">[4]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031344#pone.0031344-Bosc2" target="_blank">[6]</a>. CP motifs are boxed. IR: inter-repeat regions. Right panel: the Mn-like regular expression is represented as position-specific probability matrix derived from MEME analysis in C. <b>C</b>. Identification of a family of proteins containing Mn-Like domains. MEME analysis using mouse Map6s, mouse Saxo1, and only protozoan SAXO sequences identified a characteristic N-terminal motif (motif 1, dark blue boxes) in SAXO proteins and Mn-like domains (motif 2, light blue boxes) common to the SAXO and MAP6 proteins. Manually identified supplementary motifs 2 are in grey boxes (Motif 2 manual). The asterisk indicates a conserved CP sequence in the last Mn-like domain.</p

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 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

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

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