Description

Context. Cilia and flagella are essential organelles for the development and the functioning of animals. Their common architecture relies on a cylinder of 9 doublet microtubules called the axoneme. Despite their conservation, they display amazing variations, with either complexification or simplification of their structure, in relation with their function. In spermatozoa, extra-axonemal elements are present while photoreceptors or olfactory neurons exhibit spectacular membrane extensions. By contrast, primary cilia have a simplified structure and yet perform key sensory function. It is often said that development recapitulates evolution and indeed these variations are encountered across ciliated species, especially in protists that represent the largest biodiversity on earth.
Question. What drives the evolution of cilia and flagella?
Hypothesis. Cilia and flagella are made of highly conserved proteins such as alpha- and beta-tubulins. These undergo important post-translational modifications (PTM) that tune their functions by regulating their interactions with molecular motors or microtubule-associated proteins. It has been proposed that these modifications are involved in the diversification of flagellar structure and function.
Objective. To figure out the contribution of tubulin PTM to the formation of different types of flagella, the student will investigate their distribution and their function in several protists that display different structural configurations, such as the parasites Trypanosoma brucei (responsible for sleeping sickness) or Crithidia fasciculata (infects mosquitoes) and the free-living protists Micromonas pusillatum or Salpingoeca rosetta.
Two main aims.
1. Localisation of tubulin PTM within the flagellum. The extreme density of microtubules (9 doublets within 180 nm) so far hindered subcellular analysis, restricting the ability to discriminate them. This is now overcome by a combination of ultrastructure expansion microscopy and super-resolution microscopy, allowing individual resolution of microtubule doublets. The host team has optimised these procedures and discovered that for example polyglutamylation (the enzymatic addition of supplementary glutamates forming a side chain) was enriched in a limited set of microtubules the trypanosome flagellum, exactly where a supplementary structure is attached, suggesting possible function in linking skeletal elements. Such an analysis will be performed with the collection of organisms mentioned above and available in the host laboratory.

2. Functional analysis of the significance of tubulin PTM. The presence of multiple tubulin alleles and large families of enzymes responsible for PTM has complicated the generation of mutants and limited functional studies. Recent progress of CRISPR-Cas9 approaches, yielding high efficiency of transformation without the need of selection markers, has opened new horizons. It is now possible to selectively mutate the tubulin residues targeted for modifications or to inhibit multiple enzymes simultaneously and to monitor the impact on flagellum architecture and on cellular function.

Expected insights. The team gathered a collection of unique protists (parasitic or free-living) with different flagellar anatomy necessary to evaluate the breadth of diversity. These species are amenable to genetic manipulation (which is not accessible for most animal ciliated cells) and so perfectly suited to relate structure with function. Recent technological progress have broken several deadlocks, making the project exciting, feasible and timely. The student will benefit from an experienced team and from national and international collaborations already in place, especially for imaging, genetics or proteomics.

Compétences requises

Cellular and molecular biology, microscopy

Bibliographie

A novel approach to tagging tubulin reveals microtubule assembly dynamics of the axoneme in Trypanosoma brucei
Daniel Abbühl, Martina Pružincová, Luděk Štěpánek, Eleonore Bouscasse, Rita Azevedo, Mariette Matondo, Vladimir Varga, Serge Bonnefoy, Philippe Bastin
bioRxiv 2025.01.27.634986; doi: https://doi.org/10.1101/2025.01.27.634986

Intraflagellar Transport Selectivity Occurs with the Proximal Portion of the Trypanosome Flagellum
Aline Araujo Alves, Jamin Jung, Gaël Moneron, Humbeline Vaucelle, Cécile Fort, Johanna Buisson, Cataldo Schietroma, Philippe Bastin
bioRxiv 2024.12.13.628094; doi: https://doi.org/10.1101/2024.12.13.628094

The tubulin code and its role in controlling microtubule properties and functions.
Janke C, Magiera MM. Nat Rev Mol Cell Biol. 2020 Jun;21(6):307-326. doi: 10.1038/s41580-020-0214-3. Epub 2020 Feb 27.

Mallet, A., and Bastin, P. (2022). Restriction of intraflagellar transport to some microtubule doublets: An opportunity for cilia diversification? Bioessays 44, e2200031.

Mass spectrometry reveals novel features of tubulin polyglutamylation in the flagellum of Trypanosoma brucei
Marija Nisavic, Thibault Chaze, Serge Bonnefoy, Carsten Janke, Philippe Bastin, Mariette Matondo, Julia Chamot-Rooke
bioRxiv 2025.02.06.636787; doi: https://doi.org/10.1101/2025.02.06.636787

Mots clés

Cils et flagelles, Biodiversité, Microscopie, CRISPR-Cas9