<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

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

<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 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

The EF-hand domains modulate the morphology of polymers formed by BILBO1.

<p>(A) Immunofluorescence labelling of cytoskeletons from cells expressing mEFH1:myc, mEFH2:myc, mEFH1+2:myc using anti-NTD (green) and anti-myc (red) after six hours of induction. Scale bar in the magnified inset represent 1 μm. Scale bar for all other images represents 5μm. (B) Immunofluorescence labelling of cytoskeleton extracted cells expressing mEFH1:myc, mEFH2:myc, mEFH1+2:myc using anti-NTD (green) and anti-myc (red) after 24 hours of induction. The asterisks indicate a dot in the new detached flagellum. (C) Western-blot of corresponding cells. 2.10⁶ cells (WC) or cytoskeletons (CK) non-induced (-) and induced (+) for six hours, were loaded on a 10% SDS-PAGE, transferred and immuno-probed with anti-myc and anti-tubulin as loading control. WT is the non-transfected parental cell line. All expressed proteins except mEFH1+2:myc are insoluble. (D) Growth curves of the parental cell line (WT, black) compared to the cell lines non-induced (NI, red open squares) or induced (I, red closed square) for the expression of mEFH1, mEFH2, mEFH1+2 myc-tagged proteins. (E) Overexpression of BILBO1 coiled-coil truncations or EF-hand domain mutations induces detached flagellum phenotypes. The percentage of cells with a detached flagellum was determined using phase contrast microscopy after six or 24 hours of protein expression. (F) WT cells or cells expressing recombinant proteins (induced for six hours), were fixed and immunolabelled with anti-myc and anti-NTD. The graph represents the measurements of the FPC diameter in WT cells, in BILBO1:myc, mEFH1:myc, mEFH2:myc and mEFH1+2:myc expressing cell lines. The graph also shows the diameter of the helix and comma/annulus shaped polymers formed by mEFH1:myc.</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

BILBO1 forms polymers in a heterologous system and the coiled-coil domain is required for polymerization.

<p>Heterologous expression of BILBO1:GFP (A), or un-tagged BILBO1 (B) in mammalian U-2 OS cells demonstrate that BILBO1 has self-polymerizing properties. After 6 hours post-transfection of BILBO1:GFP the polymers formed were observed by direct GFP fluorescence. After 6 hours post-transfection of un-tagged BILBO1, extracted cells were immuno-labelled with anti-BILBO1 monoclonal antibody. (C-F) Electron micrograph of extracted and negative stained U-2 OS cells after six hours of BILBO1 expression. (G) Electron micrograph of extracted and negative stained U-2 OS cells after six hours of BILBO1:GFP expression. (H) Measurements of fibre width and diameter of termini in U-2 OS cells expressing BILBO1 after six or 24 hours post-transfection and immunolabelled with anti-NTD. (I) Measurements of total fluorescence emitted per cell, as arbitrary units, after a fixed time of acquisition on anti-NTD immunolabelled BILBO1 expressing U-2 OS cells, and number of complex or simple termini in each corresponding cell. The red line indicates the 50% of maximum fluorescence intensity limit. (J) Determination of the percentage of complex polymers with termini at one end (left) or at both ends (right) in BILBO1 U-2 OS expressing cells after six or 24 hours transfection. Scale bars represent 10 μm in A and B, 1 μm in insets, 500 nm in C, E, F, and 250nm in D, G.</p

The coiled-domain is required for polymer formation in <i>T. brucei</i>.

<p>(A) Immunofluorescence labelling of cytoskeletons from cells expressing BILBO1:myc, T1:myc, T2:myc, T3:myc and T4:myc using anti-NTD (green) and anti-myc (red) after six hours of induction. (B) Immunofluorescence labelling of cytoskeletons expressing BILBO1:myc, T1:myc, T2:myc, T3:myc and T4:myc using anti-NTD (green) and anti-myc (red) after 24 hours of induction. (C) Western-blot of corresponding cells. 2.10⁶ non-induced (-) or induced (+) for six hours from whole cells (WC) or cytoskeletons (CK) were loaded on a 12% SDS-PAGE, transferred and immuno-probed with anti-myc, or anti-tubulin (tubulin is a loading control). WT is the non-transfected parental cell line. (D) Growth curves of the parental cell line (WT, black circles) compared to the cell lines non-induced (NI, red open squares) or induced (I, red closed squares) for the expression of BILBO1, T1, T2, T3, T4 myc-tagged truncations. Scale bars in A and B represent 5μm.</p

The conserved residues in the loops of the EF-hand domains are required for calcium binding.

<p>(A) Schematic depicting the <i>Tb</i>BILBO1-EFh (residues 177–250) expression construct used in the <i>in vitro</i> calcium-binding experiments. The protein was expressed with an N-terminal tag containing maltose-binding protein (MBP) together with a 10 × histine linker. (B) Purified proteins of wild-type (WT) and three mutants of MBP-His₁₀-TbBILBO1-EFh were analyzed by SDS-PAGE and stained using Coomassie blue. The minor bands are degradation products. The last lane shows purified MBP-His₁₀ alone. (C) Superimposition of the <i>Tb</i>BILBO1-EFh model onto the modeling template 1ggz.pdb, (a human epithelial cell calmodulin-like protein). The two proteins share 30% identities and 47% similarities in their primary sequences. (D) Ribbon diagram of the <i>Tb</i>BILBO1-EFh derived from homology-based modeling shown in (C). The structure is color-ramped from blue at the N-terminus to red at the C-terminus. Conserved D/E/N residues in the loops, which are predicted to coordinate calcium binding, are shown as sticks. The two calcium sites from the modeling template (1ggz.pdb) are shown as semi-transparent magenta spheres. (E) ITC titration and fit curve for the wild-type <i>Tb</i>BILBO1-EF-hand domains. N, which represents the molar ratio between Ca²⁺ and the protein, was determined to be approximately 2, suggesting that both EF-hand domains of <i>Tb</i>BILBO1 bind calcium. (F-H) ITC titration results for mEFH1, mEFH2, and mEFH1+2. None of the three mutants were able to bind calcium. (I) ITC titration result for the MBP-His₁₀ tag. The fusion tag by itself did not bind calcium.</p

Mean T3 and T4 width after expression in U-2 OS cells.

<p>Measurements of the mean T3 and T4 width was done at six and 24 hours of expression in U-2 OS cells. Mean T3 width increased over time whereas T4 did not.</p><p>Mean T3 and T4 width after expression in U-2 OS cells.</p