|Trypanosoma Cell Biology|
|Director : BASTIN Philippe (email@example.com)|
Trypanosomes are flagellated parasites responsible for sleeping sickness in Central Africa. Trypanosomes also represent exciting organisms to study as they exhibit unique cellular features. Our lab is interested in two topics. First, we have developed several tools for functional analysis by RNAi and have demonstrated the involvement of this process in mitosis and in the control of transposon RNA abundance. More recently, we have demonstrated the importance of RNAi activity in the control of expression of some pseudogenes. Second, we have shown that the flagellum is critical for the trypanosome cell cycle and have characterised its involvement in cell motility. We have identified more than 50 genes coding for putative flagellar proteins and defined their function after individual silencing by inducible RNAi. Recent evidence suggest that trypanosomes could be an excellent model to study genetic diseases related to cilia and flagella defects.
1. RNA interference in trypanosomes
(M. Durand-Dubief, S. Ngwabyt, L. Menzer & P. Bastin)
RNAi is now the prime-choice system for functional studies in trypanosomes as in other model organisms. We determined specificity and efficiency of various RNAi methods and have developed two new systems for functional complementation of RNAi mutants. Complementation is performed after expression, in the first case, of the target gene but with a different 3'UTR and, in the second case, of a heterologous gene from a different species. This one is sufficiently different at the nucleotide level to escape RNAi whereas protein is conserved enough to complement the phenotype. This strategy can be applied to any organism providing expression systems are available. This allows demonstration that phenotype observed upon RNAi silencing are indeed due to silencing of the targeted gene. This offers interesting perspectives for screening of human gene functions in trypanosomes (see Part 2).
We have generated a cell line where both copies of the TbAGO1 gene (one of the Argonaute genes in T. brucei) have been deleted by gene knockout. These cells cannot do RNAi no matter the way of dsRNA delivery. However, these trypanosomes are viable although they exhibit a slower growth rate. RNAi activity is restored upon inducible expression of a GFP::TbAGO1 fusion protein that demonstrated cytoplasmic and perinuclear localisation. Two other phenotypes were noted: mitotic defects, due to problems in spindle formation and chromosome anchoring and (2) over-expression of ingi and SLACS retrotransposon RNA.
Mitotic defects are progressively compensated during continuous cell culture, indicating that cells can compensate for the absence of RNAi in vitro. In contrast, over-expression of retroposon transcripts is even more pronounced, although transposition was not observed. These results indicate that RNAi would act on mitosis and retroposon control by two separate means, possibly via the action of specific effectors. We then analysed transcription from peripheral regions of the transposons, called RIME (that can be compared to SIINE elements of the human genome). Short RNAs are detected only in the presence of RNAi whereas longer transcripts are only detected in the presence of RNAi, indicating that they might be targets of natural RNAi. RT-PCR analysis, followed by sequencing of the products, demonstrated that these transcripts correspond to pseudogenes made of transposable element(s) inserted in genes belonging to the RHS family. These genes of unknown function are present as multi-copies in the genome and are frequently found close to telomeres and contain a sequence likely to correspond to the transposon endonuclease recognition site. These data show that RNAi is required for the control of pseudogene expression.
2. Assembly and functions of flagella
(C. Adhiambo, C. Branche, S. Absalon & P. Bastin)
We have demonstrated that molecular components of the trypanosome flagellum function in a similar way as in other eukaryotes regarding flagellum construction and motility. These experiments validate the trypanosome as model to study genetic defects in cilia and flagella. In collaboration with the team of S. Amselem and E. Escudier (Créteil-La Pitié), we have investigated the DNAI1 gene, the first one whose involvement in human primary ciliary dyskinesia was demonstrated. Inhibition of flagellum movement has unexpected consequences on the trypanosome cell cycle, that could be significant both for basic and therapeutic research. First, motility is required to complete the last stage of cell division. Second, motility is essential for basal body positioning. These centriole equivalents are key partners in cytoskeleton organisation and their movement at mitosis and cytokinesis has been extensively analysed. We have shown for the first time that basal body positioning is defined by the flagellum. In the absence of flagellum or in the absence of discrete cytoskeletal components associated to the flagellum, basal body migration and positioning is severely affected.
Flagellum construction is controlled by intraflagellar transport (IFT). First, we have shown that reducing IFT protein abundance leads to the assembly of shorter flagella. It is striking that in these conditions, cell size is concomitantly reduced, demonstrating a direct link between flagellum length and cell size. Morphometric analysis showed that the flagellum acts at this level by defining the point of initiation of cell cleavage. When flagellum formation is completely blocked, trypanosomes are very short, lose their typical shape and polarity and fail to divide. The elongation of the flagellum controls the formation of associated cytoskeletal structures that act as molecular organiser of the cell.
By comparing fully sequenced genomes, several teams identified genes present only in ciliated species (human, insects) and missing from non-ciliated species (yeast, high plants). Several of these genes are of unknown function and some of them are associated to human genetic diseases (Bardet-Biedl, Alström). We have identified more than 50 genes of interest conserved in trypanosomes, 42 have been cloned and 34 have been successfully silenced by RNAi. Spectacular phenotypes were observed at the flagellar and cellular level and are currently under investigation. Examples include new G proteins required for flagellum construction and BBS proteins whose silencing produces complex phenotypes. Given the complexity of this disease in humans, function studies of the genes involved in a model organism is a key step in comprehension of the human disease.
Photo 1. Wild-type trypanosomes or form the TbAGO1-/- mutant in which chromosome do not migrate properly at mitosis. DNA is shown in blue.
Photo 2. Trypanosomes where flagellum (green) assembly was perturbed. DNA is hsonw in red, basal bodies and adhesion filament are shown in red.
Keywords: trypanosome, RNA interference, flagellum, cytoskeleton, genetic diseases
|Publications 2005 of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
|BRANDT Maryse||BASTIN Philippe (head of unit) firstname.lastname@example.org||ADHIAMBO Christine (postdoctoral fellow)
BRANCHE Carole (postdoctoral fellow)
ABSALON Sabrina (doctoral fellow)
NGWABYT Sandra (M2 student)
|BLISNICK Thierry (engineer)|