<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 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

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

Production of recombinant <i>Ld</i>Flabarin-His6 and localization of <i>La</i>Flabarin in <i>Leishmania</i>.

<p>(A) 5 µg supernatant of non-induced (SNI), IPTG-induced (SI) and purified <i>Ld</i>Flabarin-His6 (F16) were separated by SDS-PAGE (denaturating polyacrylamide gel electrophoresis). (B) The purified fraction (F16) was submitted to a blue-native gel (BN-PAGE) allowing separation of complex. (C) The complex were separated by electrophoresis in first non-denaturing conditions (BN-PAGE), and then the track was subjected to a second electrophoresis under denaturing conditions (SDS-PAGE) to separate the components of the differents complex. The gel was stained with Coomassie (C1) or transferred to membranes and revealed with 1∶10000 anti-His (C2). (D) 3.10⁶<i>L. amazonensis</i> BA125 untransfected control cells (lanes 1) and <i>Ld</i>Flabarin-mRed-expressing cells (lanes 2) were submitted to SDS-PAGE, transferred to membranes and revealed with 1∶2000 anti-<i>Ld</i>Flabarin (left panel) or 1∶10000 anti-mRed (right panel) and 1∶2500 anti-rabbit IgG conjugate as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076380#pone.0076380-Sahin3" target="_blank">[59]</a>. (E) Localization of native <i>La</i>Flabarin in <i>L. amazonensis</i> BA125 by indirect immunofluorescence. Cells were fixed and incubated with anti-<i>Ld</i>Flabarin (1∶1000) and Alexa-labelled anti-rabbit IgG (8 µg/ml). Panel 1, <i>La</i>Flabarin green fluorescence image of two cells in the same field; panel 2, overlay of panel 1 with DAPI staining (blue) and phase contrast.</p

<i>Ld</i>Flabarin binds and tubulates liposomes in vitro.

<p>Electron micrographs of liposomes incubated with purified recombinant <i>Ld</i>Flabarin-His6 (A–C, F) with <i>Ld</i>Flabarin without His-tag (E) or with BSA (D).</p

Localization of <i>Ld</i>Flabarin-mRed deletion mutants and chimeras.

<p>(A). <i>L. amazonensis</i> cells expressing <i>Ld</i>Flabarin-mRed, <i>Ld</i>Flabarin/C4S-mRed, F(1–255)-mRed and F(1–12)-mRed-F(256–339) were fractionated into soluble and membrane fractions by 100 000 g centrifugation in presence and absence of 0.5% NP-40; equivalent of 7.5 10⁶ cells supernatants (S and SN, N for NP-40) and pellets (P and PN) were submitted to SDS-PAGE and western blotting with anti-<i>Ld</i>Flabarin as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076380#pone-0076380-g002" target="_blank">Fig 2D</a>. (B–E) <i>L. amazonensis</i> expressing red fluorescent proteins were fixed, DAPI stained for nuclear (not always visible) and kinetoplast DNAs coloration (blue), and observed under a fluorescence microscope. Constructs are schematically represented by a multicolored bar with the same color codes as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076380#pone.0076380.s002" target="_blank">Figure S2</a>; the BAR domain is schematized by a black bar under the constructs. (B) <i>Ld</i>Flabarin/N267Q-mRed. (C) <i>Ld</i>Flabarin-mRed (1–339). (D) <i>Ld</i>Flabarin/C4S-mRed. (E) F(1–12)-Red-F(256–339). (F) F(1–255)-mRed. Bars correspond to 5 µm.</p

Ultrastructural localization of <i>Ld</i>Flabarin.

<p>(A) Longitudinal section of a flagellum of <i>Ld</i>Flabarin-mRed-expressing <i>L. amazonensis</i> using anti-<i>Ld</i>Flabarin and an anti-rabbit IgG-gold particle conjugate; black dots represent gold particles; f, flagellum. (B) Upper panel: transverse section of a flagellum of <i>Ld</i>Flabarin-mRed-expressing <i>L. amazonensis</i> using the anti-Red antiserum. PFR, paraflagellar rod; Ax, axoneme; black dots, gold particles. Lower panel: localization of 383 gold particles (white dots) cumulated from 16 transverse cross-section images. (C) Longitudinal section of a flagellum of <i>Ld</i>Flabarin-mRed-expressing <i>L. amazonensis</i> (anti-Red antiserum as in B); black dots, gold particles. (D) Same as A and B, except no anti-<i>Ld</i>Flabarin antiserum. (E) Vicinity of the flagellar pocket of a <i>Ld</i>Flabarin-mRed-expressing <i>L. amazonensis</i> cell (anti-Red antiserum as in B); black dots, gold particles; f, flagellum; fp, flagellar pocket; k, kinetoplast. (F) Gold particle frequency along the flagellum as a function of distance from the reference point (first gold particle counted); distance measurements (4546 gold particles from 50 different flagella) were performed using ImageJ software. (G) Schematic representation of <i>Ld</i>Flabarin organization in the flagellum: black circles indicate <i>Ld</i>Flabarin; Ax and PFR are also represented.</p

Intracellular localization of <i>Ld</i>Flabarin.

<p><i>L. amazonensis</i> BA125 cells were co-transfected with pNUS mRednD-<i>Ld</i>Flabarin and pNUS <i>Ld</i>Flabarin-GFPcH. (A–B) Fluorescence images from a cell expressing both mRed-<i>Ld</i>Flabarin (red)(A) and <i>Ld</i>Flabarin-GFP (green) (B). (C) Overlay of A and B. (D) Overlay of DAPI staining (blue) and phase contrast. (E) A cell expressing <i>Ld</i>Centrin-GFP (green) and <i>Ld</i>Flabarin-mRed.</p