Lab topics

Trypanosomes

 

The trypanosome cell

African trypanosomes are protozoa (unicellular organisms) belonging to the Kinetoplastida order. They are elongated cells (20-25 µm long, 3-5 µm wide), although certain variations are observed during the life cycle. They are eukaryotic organisms and possess a nucleus surrounded by a nuclear membrane with classic nucleopores. The genome of Trypanosoma brucei contains 35 Mb, with 11 “large” chromosomes and about 100 “mini-chromosomes”.  Its full sequence was published in July 2005 and data can be accessed at GeneDb



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The 10 main morphological stages of the T. brucei parasite cycle, found in the mammalian bloodstream and in the tsetse fly vector. The scale bar represents 5 mm and the old (arrow) and new (arrowhead) basal body positions are indicated. All stages are presented in a chronological order and the following name code has been used. SL: slender trypomastigote; ST: stumpy trypomastigote; PC: procyclic trypomastigote; MS: mesocyclic trypomastigote; E: proventricular epimastigote; DE:asymmetrically dividing epimastigote; LE: long epimastigote; SE: short epimastigote; AE: attached epimastigote; MT: metacyclic trypomastigote. Rotureau et al. 2011


The cell is covered by a dense surface coat that is different according to stages of the life cycle. When trypanosomes infect the bloodstream, the surface coat made of Variant Surface Glycoprotein (VSG) is regularly replaced, ensuring immune evasion.  In the insect midgut stages, a different coat, made of procyclins is present. Both families of proteins are anchored to the membrane by a modified phospholipid, the GPI anchor (first discovered in trypanosomes). Once in the salivary glands, trypanosomes express yet a different surface molecule called BARP.
 

Trypanosomes possess a single flagellum that is always motile and that exhibits several features, including the fact of being attached to the cell body (see Trypanosome Flagellum section).  Underneath the plasma membrane is found a dense network of microtubules that defines cell shape. It is so stable that it survives detergent extraction. The presence of this corset restricts endocytosis that can only occur at the level of the flagellar pocket, a surface invagination from which the flagellum emerges from the cell body.  Its collar is maintained by a cytoskeletal structure essential for the maintenance and/or formation of the flagellar pocket.

 

The cellular organelles of trypanosomes exhibit amazing features. They possess a single mitochondrion that is very large and spreads throughout the cell body. Its mitochondrial DNA is made of a mass of large and small circles that are tightly packed and is called the kinetoplast. It is the only mitochondrial DNA that can be readily detected by light microscopy without staining procedure. It is physically linked by fibres to the basal body of the flagellum. It is the site of RNA editing-the phenomenon of addition and deletion of U in mitochondrial transcripts- that was actually first discovered in trypanosomes! In staed of classic peroxisomes, trypanosomes possess "glycosomes" that contain the first 7 enzymes of glycolysis and different enzymes according to the life cycle stage. When present in the bloodstream, trypanosomes exclusively rely on glycolysis as energy source.

 

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Transmission electron microscopy section of a procyclic trypanosome showing the axoneme (red), the flagellar membrane (purple), the golgi apparatus and associated membranes (green) and the kinetoplast (yellow). From Buisson et al. 2010

 

The trypanosome flagellum

 

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Transmission electron micrograph showing a cross-section of the flagellum. The outer doublet microtubules, the central pair and the PFR are visible. Kohl et al. 2005

Trypanosomes possess a single flagellum that emerges from the flagellar pocket and that is attached for most of its length at the surface of the cell body with the exception of its distal tip. The region of the cell body found underneath the flagellum is specialised and defined as the flagellum attachment zone or FAZ. The length and positioning of the flagellum varies extensively during the life cycle and was used as a central criterion to define the different development stages, especially when parasites infect the tsetse fl

Sections through the flagellum reveal a different organisation according to the position. When the flagellum is found at the surface of the cell, it contains two cytoskeletal elements: the axoneme, made of nine doublets of microtubles surrounding a central pair (as in most ciliated or flagellated eukaryotes), and the paraflagellar rod (PFR), wrapped by the flagellar membrane that is in tight contact with the plasma membrane. The PFR is unique to the Euglenoids and not encountered in other groups of eukaryotes, although morphologically related structures have been described in dinoflagellates. The PFR is not present in the portion of the flagellum found in the flagellar pocket. At the distal end, the flagellum is free and not anchored to the cell body.

 

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Longitudinal section through the flagellum attachment zone in a procyclic trypanosome. The repetitive nature of FAZ electron-dense structures found immediately underneath the plasma membrane is easily recognised. From Buisson et al. 2010

 

 

 

The Paraflagellar Rod
 

This is a lattice-like structure that is unique to Euglena and Kinetoplastida protozoa (see picture above). It is tightly attached to the axoneme as only trypsin treatment can separate the two structures. It is composed of two main coiled-coil proteins termed PFR1 and PFR2 (formerly known as PFRC and PFRA in T. brucei and PAR3 and PAR2 in T. cruzi). Ablation of any of these two proteins leads to formation of a rudimentary PFR restricted to the proximal domain and to severe defects in flagellum beating (Bastin et al, 1998, 1999; Durand-Dubief et al, 2003). Cells are almost paralysed, suggesting that the PFR is contributing to cell motility. Although several hypotheses have been raised to explain this result, the actual way by which PFR contributes to cell motility remains a mystery. As PFR proteins are unique to the parasites and essential for parasite development in the bloodstream, they represent attractive drug targets.

An exhaustive proteomic study of the T. brucei flagellar skeleton containing the axoneme and the PFR has been published by the group of Keith Gull (Oxford) and has revealed the presence of at least 331 proteins and functional analysis implied several of them in flagellum beating (Broadhead et al, Nature 2006). 

Flagellum construction

The flagellum is constructed by addition of axoneme and PFR proteins at the distal tip of the elongating structure, a bit like a tower is built by addition of new bricks at its top. This process involves intraflagellar transport (IFT), a dynamic process by which 'trains' made of protein complexes travel between the flagellar membrane and the axoneme doublets, from base to tip (anterograde) and then from tip to base (retrograde). Our group was the first one to demonstrate the importance of IFT for flagellum formation (Kohl et al. 2003). The data also demonstrated formally that the length of the flagellum controls the length of the cell body. More recently, we were the first ones to successfully visualise IFT in live trypanosomes (Absalon et al. 2008). This was quite challenging as trains can travel at speed of up to 7 µm per second! The current model proposes that tubulin and other flagellum components are transported by the IFT system, although direct evidence for such a transport are still missing.

 

 

Updated on 27/01/2014

News

18-07-2014 Brice is awarded an ANR "Jeunes Chercheurs" grant (Young Scientists)! Only 8% of the applications were funded.

 

17-07-2014 Eloïse is awarded a doctoral fellowship to study flagellum length regulation and IFT loading

 

 

All news

We and 3 other European groups are organising the 2nd European Cilia meeting in Paris.

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We are coordinators of the French network GDR CILS that groups 30 teams throughout France working on cilia and flagella in diverse species.

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The third Trypanosomatid meeting will be held at the Muséum d'Histoire Naturelle.

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