Laboratoire de Microbiologie Fondamentale et Pathogénicité

banniere

 

bouton3

Cytoskeleton biogenesis of Trypanosomes.

 

Responsible :

Derrick ROBINSON, Mélanie BONHIVERS.

 

Team members :

Researcher, professor, assistant professor :

ITA :

Student :

 

 

Research project :

Tbb BILBO1

 

 

 

Figure 4 : Bilbo1 protein is a component of the flagella pocket collar strucure

Fig4

Axoneme = Red, BILBO1 = Green.

 

We also focus on novel proteins and structures including uncharacterised basal body and spindle proteins. Currently, our main focus has centred on a component of a previously unknown collar-like structure localised at the point of emergence the flagellum from the cell. Using western blots from two-dimensional gels and mass spectrometry we identified a 67kDa protein. The gene encoding the 67kDa protein was cloned, histidine-tagged and the protein affinity purified. Finally mouse polyclonals/monoclonal were raised against this protein.

 

Using GFP and Immunofluorescence studies show that this protein is a component of a collar-like structure located at the site where the flagellum exits the cell. This collar is also a component of the flagellar pocket (FP). The FP is the sole organelle responsible for endo and exocytosis in this these cells (See figures 4 and 5). We have named the 67kDa protein BILBO1.

 

BLAST analysis in the trypanosome database GeneDB identified 3 orthologues of BILBO1; Two in T. cruzi (South American trypanosome) and one in Leishmania major.

Interestingly, the FP is a sequestered site but paradoxically, is in constant contact with components of the hosts innate immune system. Furthermore, during cell differentiation between the insect form (procyclic form) and the host mammalian form (bloodstream form), the cytoskeleton and FP undergo numerous structural changes that facilitate the expression (on the cell surface and the FP) of Variable Surface Glycoproteins (VSG) in bloodstream forms and the expression of procyclin in procyclic forms. Procyclin aids cell survival in the insect host whilst, VSG’s are a major proportion of the bloodstream form protein traffic in the cell. VSG’s are essential and traffic through the pocket en route to the cell surface.

Fig5

Figure 5 : Knockdown of BILBO1 blocks Flagella pocket collar formation

 

The pocket maintains a fixed position in G1 cells and is always maintained at the site of flagellum exit. Surprisingly, the proteins involved in FP biogenesis are not known. However, some cytoskeletal proteins close to the neck of the pocket remain intact even after extensive detergent extraction Therefore, the pocket is not only multi-functional, it is vital for cell survival.

 

 

BILBO1 is essential for parasite survival. We have carried out RNAi knockdown of BILBO1 RNA transcripts using an inducible RNAi system. BILBO1 RNAi stimulated failure to complete cytokinesis after 24hrs. The dominant phenotype detectable appears after 36 hours of induction as observed by growth of a new flagellum that is motile and detached from the cell body. This flagella growth occurred at the most posterior end of the cell rather than the normal case of 4-6µm from the posterior end. Cell death occurred within 36-48 hours. Surprisingly, although new flagella grew, no new FP was formed.

 

 

 

This phenotype is also characterised by a change in cell shape and the posterior flagellum positioning. Because BILBO1 is essential for cell survival we have an excellent subject for further study of flagellar pocket biogenesis.

 

The SAXO proteins, from protozoa to mammals: a role in cilia function?

The dynamics of assembly and disassembly of tubulin polymers (MTs) is tightly regulated, in part by post-translational modifications (PTMs), or due to their association with proteins such as Microtubule-Associated Proteins (MAPs), tektins and ribbon filaments.
In vertebrates, most MTs will disassemble at low temperature, however the resistance of some MTs to cold-induced depolymerization is largely due to their association with the class of MAPs known as MAP6. MAP6 proteins stabilize MTs via two types of MT-binding modules: a central Mc module repeat that conveys resistance to cold-induced disassembly, and a Mn module repeat that convey resistance to both cold- and nocodazole-induced disassembly.
Centrioles, cilia and flagella have cold-stable microtubules, but MAP6 proteins are most likely not involved in their stabilization, as they have not been localized to these structures. The molecular component responsible for the cold and nocodazole resistance of these MTs has thus not been identified yet.

We have recently identified a MAP6-related family of proteins that we called the SAXO proteins (Dacheux et al., 2012). Contrary to the MAP6 genes, which are present only in vertebrates, SAXO genes are present in eukaryotes from protozoa to mammals, but only in ciliated or flagellated organisms. These proteins (MAP6 and SAXO) share a cysteine-rich N-terminal domain and Mn modules.

The first member of the family to be characterized was TbSAXO, a protein of the parasitic flagellated pathogen Trypanosoma brucei. TbSAXO stabilized MTs upon cold or nocodazole treatments and stabilization is mediated through the Mn modules.

The first member of the family to be characterized was TbSAXO, a protein of the parasitic flagellated pathogen Trypanosoma brucei. TbSAXO stabilized MTs upon cold or nocodazole treatments and stabilization is mediated through the Mn modules.

More work is needed to understand the physiopathological roles of SAXO1 and SAXO2 in mammals, in particular in regards of the dysfunction of cilia in ciliopathies.

 

Funding :

Work in my lab has been and is supported by:

- LabEx ParaFrap (2012-2016)
- ANR Blanc MAS Flagella (partenaire) (2014-2018)
- ANR Blanc Mucofertil (partenaire) (2012-2014)
- Appel Interne SFR TRansBioMed (2012)
- Preciput ANR Bordeaux (2011)
- ANR Blanc Bio-Pocket (coordination) (2009-2012)
- CNRS PEPS (2009)
- Protéomique et Génie des Protéines (2004-2006)
- ACI Dynamique et réactivité des assemblages biologiques (2003)
- FRM Accueil Jeune Equipe (2001-2002)
- CNRS ATIPE (2001-2003)

 

Past members :

 

Name

Position in the group

Current position

Florian LAURUOL
( 2015 - 2016 )
Master 2 Microbiologie - Immunologie Maîtrise à l’Université de Laval, Canada.
Alexis Carreaux
( 2014 )
BTS Biotechnologie L3 student Bordeaux University / Sherbrooke University, Canada
Damien GELIN
( 2013 )
Licence 3 Bordeaux University R&D Engineer Intern – Biotechnology Applications at Procelys - Lesaffre Fermentation Nutrients
Marie EGGENSPIELER
( 2012 - 2013 )
Master 1, 2 Microbiologie - Immunologie Clinical Research Associate at J&J (Parexel Consultant)
Emmanuel ROCHE
( 2011 )
Master 2 Genetics PhD student, VINCO, Bordeaux
Benoit ROGER
( 2011 - 2014 )
Assistant Professor (MC) Bordeaux University Assistant Professor (MC) Bordeaux University, UMR5234, Wodrich group.
Karen EGUIENTA
( 2011 )
Master 2 Microbiologie - Immunologie PhD Student, IBGC, Bordeaux
Elodie Berdance
( 2011 - 2014 )
Phd student Seeking for engineer / Post Doc position
Lucie CHANSEL-DEBORDEAUX
( 2011 - 2012 )
Master Pro Assistante, CHU Pellegrin, Biologie de la reproduction – CECOS, Bordeaux.
Elodie DAVID
( 2010 )
Master 2 Microbiologie - Immunologie Professeur de Sciences (Biochimie - Biologie - Chimie) Athénée Royal de Namur, Belgium
Christophe CHOPARD
( 2010 )
Master 2 Microbiologie - Immunologie PhD student, UMR5236, Montpellier
Guillaume GILBERT
( 2010 )
summer rotation, Master 1 Biologie Cellulaire ATER, U1045, Bordeaux
Célia FLORIMOND
( 2009 - 2013 )
Master 1, 2 Microbiologie - Immunologie ; PhD student Bordeaux University Postdoc in the Blader lab - Department of Microbiology and Immunology, University at Buffalo, N.Y. USA.
Quentin OSSMAN
( 2009 )
Master 1 Microbiologie - Immunologie PhD student, UMR5234, Bordeaux
Annelise SAHIN
( 2009 - 2011 )
Ingénieure CDD ANR PostDoc, UMR-5248 CBMN, Bordeaux
Christine KIEFER
( 2006 - 2007 )
Erasmus Master 2 Microbiologie - Immunologie Technical University of Darmstadt, Germany
Nathalie BOUCHER
( 2006 - 2007 )
Postdoctoral fellow Translation and Interpretation (NBtraduction)
Céline DAGREOU
( 2005 - 2007 )
License, Master 2 Microbiologie - Immunologie -
Dr. S. KRISHNASWAMI
( 2004 - 2005 )
Postdoctoral fellow Postdoc, U. California
Josiane TACHOW
( 2004 )
License -
Magali THONNUS
( 2004 )
License professionnelle AI, CNRS at UMR5234, Bordeaux
Sophie NOWACKI
( 2004 - 2005 )
Master 1, 2 Microbiologie - Immunologie Internal Auditor / Risk Manager at Daiichi Sankyo Europe, Germany
Alizée GARCIA
( 2004 )
BTS 1st year -
Dr. A. C. Cabrera
( 2002 - 2003 )
Postdoctoral fellow Investigator at CEMITEC, Pamplona Spain
Carol JANIS
( 2002 )
Master 1 Scientific Product Manager, Biomerieux
Lydie PRADEL
( 2002 - 2005 )
PhD student Bordeaux University Associate Professor at TAGC/INSERM U 1090, Marseille.
Dr. N. DOWNEY
( 1991 - 2001 )
Postdoctoral fellow Faculty, University of Wisconsin - La Crosse

 

Publications :

Dauchy FA, Bonhivers M, Landrein N, Dacheux D, Courtois P, Lauruol F, Daulouède S, Vincendeau P, Robinson DR. Trypanosoma brucei CYP51: Essentiality and Targeting Therapy in an Experimental Model.PLoS Negl Trop Dis. 2016 Nov;10(11):e0005125

Perdomo D, Bonhivers M, Robinson DR. The Trypanosome Flagellar Pocket Collar and Its Ring Forming Protein-TbBILBO1.Cells. 2016 Mar 2;5(1)

Komatsu T, Dacheux D, Kreppel F, Nagata K, Wodrich H. A Method for Visualization of Incoming Adenovirus Chromatin Complexes in Fixed and Living Cells.PLoS One. 2015;10(9):e0137102

Austen JM, Reid SA, Robinson DR, Friend JA, Ditcham WG, Irwin PJ, Ryan U. Investigation of the morphological diversity of the potentially zoonotic Trypanosoma copemani in quokkas and Gilbert's potoroos.Parasitology. 2015 Sep;142(11):1443-52

Florimond C, Sahin A, Vidilaseris K, Dong G, Landrein N, Dacheux D, Albisetti A, Byard EH, Bonhivers M, Robinson DR. BILBO1 is a scaffold protein of the flagellar pocket collar in the pathogen Trypanosoma brucei.PLoS Pathog. 2015 Mar;11(3):e1004654

Dacheux D, Roger B, Bosc C, Landrein N, Roche E, Chansel L, Trian T, Andrieux A, Papaxanthos-Roche A, Marthan R, Robinson DR, Bonhivers M. Human FAM154A (SAXO1) is a microtubule-stabilizing protein specific to cilia and related structures.J Cell Sci. 2015 Apr 1;128(7):1294-307

Dacheux D, Landrein N, Thonnus M, Gilbert G, Sahin A, Wodrich H, Robinson DR, Bonhivers M. A MAP6-related protein is present in protozoa and is involved in flagellum motility.PLoS One. 2012;7(2):e31344

May SF, Peacock L, Almeida Costa CI, Gibson WC, Tetley L, Robinson DR, Hammarton TC. The Trypanosoma brucei AIR9-like protein is cytoskeleton-associated and is required for nucleus positioning and accurate cleavage furrow placement.Mol Microbiol. 2012 Apr;84(1):77-92

Giroud C, Ottones F, Coustou V, Dacheux D, Biteau N, Miezan B, Van Reet N, Carrington M, Doua F, Baltz T. Murine Models for Trypanosoma brucei gambiense disease progression--from silent to chronic infections and early brain tropism.PLoS Negl Trop Dis. 2009 Sep 1;3(9):e509

Absalon S, Blisnick T, Bonhivers M, Kohl L, Cayet N, Toutirais G, Buisson J, Robinson D, Bastin P. Flagellum elongation is required for correct structure, orientation and function of the flagellar pocket in Trypanosoma brucei.J Cell Sci. 2008 Nov 15;121(Pt 22):3704-16

Bonhivers M, Landrein N, Decossas M, Robinson DR. A monoclonal antibody marker for the exclusion-zone filaments of Trypanosoma brucei.Parasit Vectors. 2008 Jul 10;1(1):21

Bonhivers M, Nowacki S, Landrein N, Robinson DR. Biogenesis of the trypanosome endo-exocytotic organelle is cytoskeleton mediated.PLoS Biol. 2008 May 6;6(5):e105

Sahin A, Espiau B, Tetaud E, Cuvillier A, Lartigue L, Ambit A, Robinson DR, Merlin G. The leishmania ARL-1 and Golgi traffic.PLoS One. 2008 Feb 20;3(2):e1620

Zhao Z, Lindsay ME, Roy Chowdhury A, Robinson DR, Englund PT. p166, a link between the trypanosome mitochondrial DNA and flagellum, mediates genome segregation.EMBO J. 2008 Jan 9;27(1):143-54

Milman N, Motyka SA, Englund PT, Robinson D, Shlomai J. Mitochondrial origin-binding protein UMSBP mediates DNA replication and segregation in trypanosomes.Proc Natl Acad Sci U S A. 2007 Dec 4;104(49):19250-5

Absalon S, Kohl L, Branche C, Blisnick T, Toutirais G, Rusconi F, Cosson J, Bonhivers M, Robinson D, Bastin P. Basal body positioning is controlled by flagellum formation in Trypanosoma brucei.PLoS One. 2007 May 9;2(5):e437

Si-Tayeb K, Monvoisin A, Mazzocco C, Lepreux S, Decossas M, Cubel G, Taras D, Blanc JF, Robinson DR, Rosenbaum J. Matrix metalloproteinase 3 is present in the cell nucleus and is involved in apoptosis.Am J Pathol. 2006 Oct;169(4):1390-401

Pradel LC, Bonhivers M, Landrein N, Robinson DR. NIMA-related kinase TbNRKC is involved in basal body separation in Trypanosoma brucei.J Cell Sci. 2006 May 1;119(Pt 9):1852-63

 

 

 

Top

Accueil Imprimer Contact Plan du site Credits
cnrs ub2 sfr chu