Biology subjects

BONNEFOY, S.

Publications and source records attributed to BONNEFOY, S..

5 recordsLinked to original sources

bioRxiv2026

Life without heterotrimeric kinesins: trypanosomatids use a combination of homodimeric kinesin-2 motors to drive intraflagellar transport

Heterotrimeric kinesin 2 is the canonical motor protein for anterograde intraflagellar transport (IFT), driving movement of protein complexes towards the tip of cilia and flagella. Here, we show that all members of the Euglenozoa group lack genes for heterotrimeric kinesins and instead possess a variable number of genes for two homodimeric kinesins termed KIN2A and KIN2B. When expressed in vitro, both Trypanosoma brucei kinesins form homodimers and move processively along brain microtubules, KIN2A being faster than KIN2B. Studies in T. brucei and Leishmania mexicana show anterograde and retrograde IFT of both kinesins, with KIN2A travelling throughout the whole length of the flagellum, while KIN2B is concentrated at its base. In the proximal portion of the flagellum, most KIN2B molecules travel without IFT proteins, except for a few particles that are associated with IFT proteins and reach the tip. Surprisingly, the absence of KIN2A has mild effects on IFT and flagellum assembly, whereas KIN2B is essential for both. Investigation of trypanosome flagella deprived of KIN2B revealed that IFT proteins do not access these flagella but that KIN2A can still circulate. We propose a division-of-labour model where KIN2B would be responsible for the import of IFT proteins while KIN2A would ensure most of the anterograde transport.

cell biology↗

bioRxiv2025

Mass spectrometry reveals novel features of tubulin polyglutamylation in the flagellum of Trypanosoma brucei

Tubulin polyglutamylation is a prominent feature of eukaryotic cilia and flagella. In the protist parasite Trypanosoma brucei, nine different tubulin tyrosine ligase-like (TTLL) enzymes are potentially involved in tubulin glutamylation. Given this enzymatic diversity, it is important to qualitatively and quantitatively characterize tubulin polyglutamylation, in order to understand the trypanosome tubulin code and pave the way for functional assignments of different TTLLs. Robust mass spectrometry (MS) based proteomics approaches for analysis of posttranslational modifications have been developed in recent years, but none to study polyglutamylation. We therefore optimized a nanoLC-MS/MS pipeline, from sample preparation to data analysis, using synthetic polyglutamylated peptides for quantification. Applied to the flagellum of T. brucei, our approach enabled the detection and quantification of C-terminal tubulin peptides with up to eleven supplementary glutamates on -, and five on {beta}-tubulin. In addition to the known E445 on - and E435 on {beta}-, a novel glutamylation site of {beta}-tubulin was discovered at residue E438. Furthermore, our data revealed an increase in enzymatic detyrosination with increasing length of the glutamate chains, especially for -tubulin. This indicates crosstalk between the modifications and different rates of detyrosination of the two tubulin types. Our results complement the existing knowledge of the tubulin code in T. brucei and introduce an efficient analytical pipeline for the characterization of polyglutamylated proteins.

biochemistry↗

bioRxiv2025

A novel approach to tagging tubulin reveals microtubule assembly dynamics of the axoneme in Trypanosoma brucei

The protozoan parasite Trypanosoma brucei is a mono-flagellated cell during the G1-phase of its cell cycle. In order to duplicate, it assembles a new flagellum alongside the mature one, in which further elongation is prevented. Our group proposed a model where the mature flagellum is locked after construction to full length (Bertiaux et al. 2018) and access of new building blocks for elongation is exclusive to the new flagellum. To test this hypothesis directly, we developed a tool for the inducible expression of tagged tubulin. Alpha-tubulin that was tagged with an intragenic Ty-1-epitope behaved indistinguishable from untagged tubulin. Its incorporation was monitored after inducible expression, to follow the assembly dynamics of microtubules in the cell body, the mitotic spindle and the flagellum. In this study we observed that integration of tubulin occurs at the distal flagellum tip at a linear rate and is indeed restricted to the new flagellum in bi-flagellated cells. This is direct evidence that trypanosomes avoid competition between the two flagella by allowing tubulin incorporation only in the new organelle. However, by tracing flagella over several cell cycles we could also show that mature flagella do not remain locked indefinitely. The restriction is lifted briefly after the bi-flagellated cell has divided and the daughter cell inheriting the old flagellum shows incorporation of newly synthesized building blocks again. It then has to lock again before the cell can assemble a new flagellum. Our findings suggest regular incorporation of tubulin at the tip of previously locked flagella. This evidence was supported with an orthogonal approach, with which we monitored the incorporation of HaloTag-tagged radial spoke protein 4/6. Since flagellum length in trypanosomes is stable, this indicates that the entire axoneme is subject to regular events of transient disassembly followed by assembly at its distal tip.

cell biology↗

bioRxiv2023

Novel roles for the LRRC56 protein, an IFT cargo protein, in docking of dynein arms in Trypanosoma brucei

Outer dynein arms (ODAs) are responsible for ciliary beating in eukaryotes. They are assembled in the cytoplasm and shipped by intraflagellar transport (IFT) before attachment to microtubule doublets via the docking complex. The LRRC56 protein has been proposed to contribute to ODAs maturation. Mutations or deletion of the LRRC56 gene lead to reduced ciliary motility in all species investigated so far, but with variable impact on dynein arm presence. Here, we investigated the role of LRRC56 in the protist Trypanosoma brucei, where its absence results in distal loss of ODAs, mostly in growing flagella. We show that LRRC56 is a transient cargo of IFT trains during flagellum construction and surprisingly, is required for efficient attachment of a subset of docking complex proteins present in the distal portion of the organelle. This relation is interdependent since the knockdown of the distal docking complex prevents LRRC56s association with the flagellum. Intriguingly, lrrc56-/- cells display shorter flagella whose maturation is delayed. Inhibition of cell division compensates for the distal ODAs absence thanks to the redistribution of the proximal docking complex, restoring ODAs attachment but not the flagellum length phenotype. This work reveals an unexpected connection between LRRC56 and the docking complex.

cell biology↗

bioRxiv2020

Redistribution of FLAgellar Member 8 during the trypanosome life cycle: consequences for cell fate prediction.

The single flagellum of African trypanosomes is essential in multiple aspects of the parasite development. The FLAgellar Member 8 protein (FLAM8), localised to the tip of the flagellum in cultured insect forms, was identified as a marker of the locking event that controls flagellum length. Here, we investigated whether FLAM8 could also reflect the flagellum maturation state in other stages. We observed that FLAM8 distribution extended along the entire flagellar cytoskeleton in mammalian infective forms. Then, a rapid FLAM8 concentration to the distal tip occurs during differentiation into early insect forms, illustrating for the first time the remodeling of an existing flagellum in trypanosomes. In the tsetse cardia, FLAM8 further localizes to the entire length of the new flagellum during an asymmetric division. Strikingly, in parasites dividing in the tsetse midgut and in the salivary glands, the amount and distribution of FLAM8 in the new flagellum was seen to predict the daughter cell fate. We propose and discuss how FLAM8 could be considered as a meta-marker of the flagellum stage and maturation state in trypanosomes. Summary statementThe trypanosome protein FLAM8 displays a dynamic and stage-specific distribution during the entire parasite cycle, representing a novel marker of the flagellum stage and maturation state.

microbiology↗