Biogenèse de la poche flagellaire , caractérisation de l’organelle d’endo-exocytose chez Trypanosoma brucei. – Bio-Pocket

Trypanosoma brucei is a flagellated protozoan parasite that is used as a model organism to study biological phenomena such as gene expression, protein trafficking and cytoskeleton biogenesis. In T. brucei and related parasites endo- and exocytosis occurs exclusively through a specialized, sequestered organelle called the flagellar pocket (FP), an invagination of the pellicular surface membrane. In order to obtain a better understanding of flagella and cytoskeleton biogenesis in general, our studies have focused on immunological and proteomic analysis of minor flagellar proteins of T. brucei. We made a series of monoclonal antibodies against minor cytoskeleton proteins, the most interesting of which recognizes an unknown collar-like structure localized at the point of flagellum emergence from the flagellar pocket (the flagellar pocket collar FPC). Using this antibody, Western blots from two-dimensional gels and mass spectrometry we recently identified an novel and unusual protein that we named BILBO1. BILBO1 is the first cytoskeleton flagellar pocket and FPC protein to be identified in T. brucei and related parasites The organisation of some mammalian cilia is very similar to that of the flagellum in the trypanosome flagellar pocket. Primary cilia have pocket-like structures where it is believed that endocytosis takes place. However, the molecular functions of the vesicle of primary cilia are not known. We have carried out in vivo RNAi knockdown of BILBO1 RNA transcripts using an inducible RNAi system in the insect form and the bloodstream form. BILBO1 RNAi caused failure to complete cytokinesis after 24hrs. The dominant phenotype appears after 36 hours of induction. Cells accumulate in pre-cytokinesis stage but more surprisingly however is that knockdown of BILBO1 prevents new flagellar pocket biogenesis. We have made and tested a second antibody that recognizes a second flagellar pocket structural protein we have named BILBO2. BILBO2 may also be essential in the biogenesis of the pocket and is part of the proposed analysis of this project. We are the first and only laboratory to do identify structural proteins in the FP. In this proposal I now wish to gain a thorough understanding of FP function/biogenesis by asking the following questions: a). Why is the flagellar pocket collar essential' b). What are the partners of BILBO1/2 ' can their interactions be blocked in vitro or in vivo' c). Is BILBO1/2 or their partners exposed to the FP lumen' The search for BILBO1/2 partners is important because it is not unlikely that we will find proteins that have homologues in the genomes of a wider distribution of organisms including man. Thus from this viewpoint our work and results may attract specialists from non-parasite domains, who may wish to search for structural homologues of BILBO1/2 partner proteins to understand the positioning of flagella and cilia (i.e. in primary cilia and cilia flagella related diseases). Thus we will gain significant insights into the organisation and localisation of mammalian cilia. In order to address the questions raised above we will; 1). Characterise the inter-molecular binding potential of BILBO1 with itself and with BILBO2 via AlphaScreen® analysis. 2). Identify and /characterise BILBO1/2 partner proteins and their function. This will be done using cross-linking protocols and two-hybrid screening. 3). Analyse the biogenesis of the FP using BILBO1/2 using tagged versions of BILBO1/2 and visualise their role in flagellar pocket biogenesis, division and turnover. 4). Analyse BILBO2 function in detail by; RNAi knockdown, GFP and histidine tagging, domain functional analysis and over-expression studies in insect and bloodstream forms. 5). Identify exposed domains of the flagellar pocket collar by the exposing BILBO1/2 or partners proteins or domains in the lumen of the pocket by probing live cells with purified FAB fragments against these proteins. The analysis of flagellar pocket biogenesis has far reaching applications for medical and veterinary parasitology because it identifies a single protein that is responsible for a biogenesis role of the flagellar pocket. This is important because three major functions of the pocket are eliminated in the absence of this organelle rendering the parasite essentially visible and exposed to the host immune system, unable to remove host antigens and unable to endo/exocytose. This makes BILBO1 and 2 highly patentable dug target candidates. Furthermore, we will be able to define how the pocket is made and so translate this information to mammalian systems such as the primary cilia and test whether the pocket of the cilia is formed in a similar manner to that of trypanosomes and identify potential roles in cilia related diseases due to disruption of cilia formation and function.