Trypanosome Cell Biology - CNRS URA 2581  


  HEADDr. BASTIN Philippe (DR2 INSERM) / philippe.bastin@pasteur.fr
  MEMBERSLISNICK Thierry (experimental officer) BUISSON Johanna (PhD student) COZANET Anne (secretary) HUET Diego (M2 student) Dr. JULKOWSKA Daria (post-doc) Dr. ROTUREAU Brice (post-doc) SUBOTA Ines (PhD student) Dr. VINCENSINI Laetitia (post-doc, collaboration Trousseau hospital)


  Annual Report

Trypanosomes are flagellated parasites responsible for various tropical diseases, including sleeping sickness in Central Africa caused by Trypanosoma brucei and transmitted by the bite of the tsetse fly. During their life cycle, these parasites have to adapt to changing environments (mammalian bloodstream, insect gut and salivary glands) and undergo profound morphological and biochemical modifications. Trypanosomes also represent exciting model organisms as they exhibit unique cellular features and are amenable to modern reverse genetics technology. Our group is interested in two topics: (1) gene expression and RNA interference (RNAi) and (2) flagellum formation and function.

Gene expression and RNAi. RNAi is a sequence-specific RNA destruction process mediated by short or long double-stranded RNA that was discovered in 1998 by C. Mello and A. Fire (Nobel 2006). We have identified a protein that could work downstream of Argonaute cleavage and have shown that it is found in cytoplasmic foci that show co-localisation with mRNA. The protein is only expressed at certain stages of the life cycle where it is essential for parasite survival.

Flagellum formation and functions. The flagellum is a cylindrical organelle made of microtubules and composed of more than 200 proteins. We previously demonstrated that the flagellum is critical for the trypanosome cell cycle and that it is constructed by a specific process called intraflagellar transport (IFT). We have set up optimal conditions to visualise and quantify the movement of fluorescent proteins in the flagellum and are correlating the parameters with the biology of flagellum. We also produced polyclonal and monoclonal antibodies to several IFT motors and partners, revealing the organisation of the IFT process. Some of these antibodies cross-react with the related parasite Leishmania where drastic changes of flagellum length take place during the life cycle. In addition to its functions in motility and morphogenesis, the flagellum has been proposed to act as a sensory organelle. We have now purified intact flagella and analysed the content of their membrane and matrix fraction, what revealed exciting candidates that are currently under investigation. Using our recently set up experimental infections of tsetse flies in the laboratory, we have characterised spectacular changes in the flagellum and in the trypanosome cytoskeleton during the course of differentiation and migration. Importantly, we successfully propagated parasites expressing a GFP fusion protein in the flagellum in tsetse flies, including salivary gland infections, which will allow monitoring live trypanosomes in the insect.

Trypanosome as a model for genetic diseases. Several genetic diseases are linked to defects in cilia and flagella function. Trypanosomes are an excellent model to study these diseases as mammalian, insect or nematode ciliated cells do not propagate in vitro and are poorly amenable to transfection. We are working with two clinical groups to understand the role of cilia and flagella in the Bardet-Biedl Syndrome and in Primary Ciliary Diskynesia, where the role of a novel candidate gene has been dissected in trypanosomes.

Keywords: Trypanosome, RNA interference, flagellum, cytoskeleton, tsetse fly, genetic diseases

Bct.jpg

Left, detergent-extracted trypanosome (Ref. 2); Middle, IFT proteins localisation (green) with DAPI staining (blue); Right, tsetse fly



  Publications

1.ABSALON S., BLISNICK T., BONHIVERS, M., KOHL L., CAYET N., TOUTIRAIS G., BUISSON J., ROBINSON D.R., BASTIN P. (2008) Flagellum elongation is required for correct structure, orientation and function of the flagellar pocket in Trypanosoma brucei. J. Cell Sci., 121, 704-3716. Front cover. PMID: 18940910.

2.ABSALON S., BLISNICK T., KOHL L., TOUTIRAIS G., DORE G., JULKOSWSKA D., TAVENET, A., BASTIN P. (2008) Intraflagellar Transport and Functional Analysis of Genes Required for Flagellum Formation in Trypanosomes. Mol. Biol. Cell19, 929-944. Cited on F1000. PMID: 18094047.

3.DURAND-DUBIEF M., ABSALON S., MENZER L., NGWABYT S., ERSFELD K., BASTIN P. (2007) The argonaute TbAGO1 contributes to large and mini-chromosome segregation and is required for control of RIME retroposons and RHS pseudogene-associated transcripts. Mol. Biochem. Parasitol., 156, 144-153. PMID: 17822785.

4.ABSALON S., KOHL L., BRANCHE C., BLISNICK T., TOUTIRAIS G., RUSCONI F., COSSON J., BONHIVERS M., ROBINSON D.R., BASTIN P. (2007) Basal body positioning is controlled by flagellum formation in Trypanosoma brucei. PLoS One 2, e437. PMID: 17487282.

5.BRANCHE C., KOHL L., TOUTIRAIS G., BUISSON J., COSSON J., BASTIN P. (2006) Conserved and specific functions of axoneme components in trypanosome motility. J. Cell Sci.119, 4343-4355. PMID: 16882690.





Activity Reports 2009 - Institut Pasteur
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