The Leishmania ARL-1 and Golgi Traffic

We present here the characterisation of the Leishmania small G protein ADP-Ribosylation Factor-Like protein 1 (ARL-1). The ARL-1 gene is present in one copy per haploid genome and conserved among trypanosomatids. It encodes a protein of 20 kDa, which is equally expressed in the insect promastigote and mammalian amastigote forms of the parasite. ARL-1 localises to the Trans-Golgi Network (TGN); N-terminal myristoylation is essential for TGN localisation. In vivo expression of the LdARL-1/Q74L and LdARL-1/T51N mutants (GTP- and GDP-bound blocked forms respectively) shows that GDP/GTP cycling occurs entirely within the TGN. This is contrary to previous reports in yeast and mammals, where the mutant empty form devoid of nucleotide has been considered as the GDP-blocked form. The dominant-negative empty form mutant LdARL-1/T34N inhibits endocytosis and intracellular trafficking from the TGN to the Lysosome/Multivesicular Tubule and to the acidocalcisomes; these defects are probably related to a mislocalisation of the GRIP domain-containing vesicle tethering factors which cannot be recruited to the TGN by the cytoplasmic LdARL-1/T34N. Thus, besides the functional characterization of a new mutant and a better understanding of ARL-1 GDP/GTP cycling, this work shows that Leishmania ARL-1 is a key component of an essential pathway worth future study

Caractérisation des populations hémocytaires de l'huître plate Ostrea edulis à l'aide de lectines

Chez Ostrea edulis, les hémocytes sont actuellement classés en granulocytes, agranulocytes et petits hyalinocytes (Cochennec, 1997). Or, chez cette huître, comme chez Crassostrea gigas et chez Crassostrea virginica, il est possible que l'utilisation de lectines, marqueurs hétérologues, permette de différencier des sous-populations hémocytaires sur un critère autre que morphologique. En effet, les lectines sont des glycoprotéines qui reconnaissent des oses ou polyosides présents à la surface membranaire des cellules et/ou à l'intérieur des cellules. Ainsi, la différenciation des cellules peut-être basée sur la composition en oses ou polyosides de leur membrane plasmique. De plus, d'après certains auteurs, les lectines pourraient jouer un rôle dans la défense immunitaire de certains organismes. En effet, elles ont été décrites comme pouvant expliquer des phénomènes d'agglutination. Lors de ce stage, mon sujet, qui s'intègre dans le programme de pathologie associé à celui de sélection vis-à-vis du parasite Bonamia ostreae, a été de réaliser des marquages d'hémocytes d'Ostrea edulis par des lectines marqués (enzyme ou fluorochrome) afin de voir si ces dernières pourraient servir de marqueur pour différencier des sous-populations hémocytaires. De plus, leur distribution cellulaire a été étudiée chez des populations d'huîtres saines et d'huîtres parasitée

Detection of Freezing of Gait in Parkinson Disease: Preliminary Results

Freezing of gait (FOG) is a common symptom in Parkinsonism, which affects the gait pattern and is associated to a fall risk. Automatized FOG episode detection would allow systematic assessment of patient state and objective evaluation of the clinical effects of treatments. Techniques have been proposed in the literature to identify FOG episodes based on the frequency properties of inertial sensor signals. Our objective here is to adapt and extend these FOG detectors in order to include other associated gait pattern changes, like festination. The proposed approach is based on a single wireless inertial sensor placed on the patient’s lower limbs. The preliminary experimental results show that existing frequency-based freezing detectors are not sufficient to detect all FOG and festination episodes and that the observation of some gait parameters such as stride length and cadence are valuable inputs to anticipate the occurrence of upcoming FOG events

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

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

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

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