LdFlabarin, a new BAR domain membrane protein of Leishmania flagellum.

International audienceDuring the Leishmania life cycle, the flagellum undergoes successive assembly and disassembly of hundreds of proteins. Understanding these processes necessitates the study of individual components. Here, we investigated LdFlabarin, an uncharacterized L. donovani flagellar protein. The gene is conserved within the Leishmania genus and orthologous genes only exist in the Trypanosoma genus. LdFlabarin associates with the flagellar plasma membrane, extending from the base to the tip of the flagellum as a helicoidal structure. Site-directed mutagenesis, deletions and chimera constructs showed that LdFlabarin flagellar addressing necessitates three determinants: an N-terminal potential acylation site and a central BAR domain for membrane targeting and the C-terminal domain for flagellar specificity. In vitro, the protein spontaneously associates with liposomes, triggering tubule formation, which suggests a structural/morphogenetic function. LdFlabarin is the first characterized Leishmania BAR domain protein, and the first flagellum-specific BAR domain protein

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

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

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

<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

SPINK2 deficiency causes infertility by inducing sperm defects in heterozygotes and azoospermia inhomozygotes

International audienceAzoospermia, characterized by the absence of spermatozoa in the ejaculate, is a common cause of male infertility with a poorly characterized etiology. Exome sequencing analysis of two azoospermic brothers allowed the identification of a homozygous splice mutation in SPINK2, encoding a serine protease inhibitor believed to target acrosin, the main sperm acrosomal protease. In accord with these findings, we observed that homozygous Spink2 KO male mice had azoospermia. Moreover, despite normal fertility, heterozygous male mice had a high rate of morphologically abnormal spermatozoa and a reduced sperm motility. Further analysis demonstrated that in the absence of Spink2, protease-induced stress initiates Golgi fragmentation and prevents acrosome biogenesis leading to spermatid differentiation arrest. We also observed a deleterious effect of acrosin overexpression in HEK cells, effect that was alleviated by SPINK2 coexpression confirming its role as acrosin inhibitor. These results demonstrate that SPINK2 is necessary to neutralize proteases during their cellular transit toward the acrosome and that its deficiency induces a pathological continuum ranging from oligoasthenoteratozoospermia in heterozygotes to azoospermia in homozygotes