Intraflagellar transport during the assembly of flagella of different length in Trypanosoma brucei isolated from tsetse flies

Eloïse Bertiaux, Adeline Mallet, Brice Rotureau, Philippe Bastin

Posted on: 4 June 2020

Preprint posted on 15 May 2020

Article now published in Journal of Cell Science at http://dx.doi.org/10.1242/jcs.248989

Peeking into the flagella of the successful swimmer and invading parasite, Trypanosoma brucei.

Selected by Mariana De Niz

Categories: cell biology, developmental biology

Background

Various organisms, including the parasite Trypanosoma brucei, assemble flagella of different lengths according to the stage of their life cycle. Within the tsetse fly, the length of T. brucei flagella ranges between 3 and 30µm during parasite development. Cilia and flagella are found in various organisms, and share a similar architecture, namely a cylinder of 9 doublet microtubules, with varying length within and across organisms. Some organisms even possess several types of flagella in the same cell, which makes studying the mechanisms regulating flagellar length, a complex task, while they provide an opportunity to compare different flagella in the same cell.

Flagella are constructed by addition of new blocks at their distal end, via intraflagellar transport (IFT); moreover, the total amount of IFT proteins correlates with the length of the flagellum. Altogether, IFT is considered an important candidate for flagellum length regulation. All IFT genes are conserved in trypanosomes, and functional investigation by RNAi knockdown has revealed that they are essential for flagellum construction. In their work here, Bertiaux and colleagues investigated IFT in several life cycle stages of T. brucei that exhibit flagella of different lengths (1) (Figure 1).

Figure 1.Evolution of flagellum length during the parasite cycle. The main morphological stages of the *T. brucei* parasite cycle encountered in the tsetse fly cardia are presented. (From Fig. 1, ref 1).

Key findings and developments

Findings using electron microscopy and FIB-SEM

In their work, Bertiaux et al used various approaches to study the IFT in T. brucei parasites, including imaging platforms such as electron microscopy, FIB-SEM, immunofluorescence in fixed cells, and fluorescence-based live imaging. To investigate IFT trains of T. brucei parasites infecting tsetse flies, the authors dissected the tsetse fly cardia, where parasites display the longest and shortest flagella. Areas with high parasite density were selected for semi-thin sectioning, to perform electron microscopy. IFT trains were observed between the membrane and the microtubules, and were mostly encountered in the proximity of doublets 3-4 or 7-8. A limitation of this method is that it does not allow identification of parasite stages. To overcome this, the authors performed FIB-SEM, which also allowed confirmation of the IFT train and observation of its location. Despite the possibility to observe IFT in great detail, the authors acknowledge the limitation of FIB-SEM in being extremely laborious to find all stages of interest using this technique. To overcome this hurdle, the authors went on to use fluorescence-based imaging in live and fixed cells to study IFT proteins in various parasite life stages.

Findings using fluorescence microscopy in fixed and live cells

To study the link between IFT amounts and flagellum length, the authors studied the distribution of IFT proteins IFT172, and IFT22 as well as an axonemal marker. IFT proteins were present as a succession of diffuse spots all along the length of the flagellum, with a brighter signal at the base present in all stages. For both IFT proteins, a direct correlation between the total amount of IFT proteins and the length of the corresponding flagellum was found, independent of the parasite’s life cycle stage. To analyse the IFT proteins in a dynamic manner, live trypanosomes expressing a IFT81-TdTomatofusion protein were used. In all parasite stages studied, a higher concentration of IFT proteins was detected at the base of the flagellum, while IFT trafficking was clearly visible. A limitation found with this approach was the speed of flagellar beating, which complicates quantitative analysis of some of the T. brucei stages. In stages that could be analysed, analysis of IFT protein speed and frequency showed similar results in short and long flagella, suggesting that modulation of these parameters does not explain the changes in flagellum length observed during T. brucei development. Again, IFT proteins were found in discrete spots along the flagellum and concentrated at the base of the flagellum in all trypanosome cell types. Moreover, a large IFT pool was detected at the distal end of the short flagella in epimastigotes. Fluorescence recovery after photobleaching (FRAP) was used to evaluate if this pool was dynamic. Fresh anterograde trains were then detected, emerging from the basal pool, and traveling towards the tip of the flagellum. Within seconds, these trains reached the tip and progressively replenished the distal pool.

Exploring the role of cell division on defining flagellar length

To explain the mechanisms regulating flagellar length, in previous work, the authors had proposed a grow-and-lock model whereby the mature flagellum would be locked, preventing further elongation (2,3). This model has been validated on procyclic cells in culture, but had not been tested in parasites coming from the fly. In this model, two parameters regulating flagellar length are a) the rate of elongation and b) the timing of the locking event – linked to cell division. To explore whether a modification of the timing of cell division could explain the production of flagella with different length in the context of the natural cyclical development of trypanosomes, cell division was chemically inhibited in parasites isolated from infected tsetse cardia. They found that the flagellum of short epimastigote cells does not increase after inhibition of cell division, suggesting that length is restricted.

What I like about this preprint

I chose this preprint because it explores an interesting aspect of cell biology relevant to parasitology and other organisms. The Bastin lab has explored multiple aspects of T. brucei flagellar biology, and the question addressed here in my opinion is very relevant to understand other questions beyond cell biology, such as T. brucei pathology, and T. brucei heterogeneity during an in vivo infection either in the fly or the mammalian host. In this work, the authors also generated methods and tools useful to the community.

Open questions

Beyond the life cycle stage, does flagellar length vary across body locations in vectors and mammals? If so, what do you think is the biological reason/advantage of having flagella of different lengths?
In your discussion, you mention that the assembly of flagella of different lengths is likely achieved by different mechanisms in procyclic trypanosomes as opposed to epimastigotes. In general, evolutionarily and biologically, do you have a hypothesis of why a different mechanism might exist for both? Why is it not conserved?
In different organs of the hosts (mammalian or vector), is there a specific flagellar length that would advantageous for survival given the specific composition of such organ? If you were able to interfere with the ‘lock’ mechanism, what would be the advantage/disadvantage of a much more enlarged flagellum?
How does IFT and flagellar length relate to other parameters of the cell, suggesting a potential biophysical equilibrium optimal for motility? For instance, are all cells with a flagellum of a specific length equal in width and length? Again, would this not potentially aid in adaptation to specific tissues?
In inducible KDs, what would be your expectation if the flagella can reach a certain length, and then transport of specific nutrients/proteins (systematically explored) is altered?
Given previous description (4) of different parasite behaviours in vitro, in your work, does a specific flagellar length influence the type of motion that the parasites have?
You mentioned within the text, that a limitation you faced was the difficulty in quantifying flagellar speed. If it is a relevant parameter, is this tool suitable (Ref 6)?

References

Bertiaux E et al, Intraflagellar transport during the assembly of flagella of different length in brucei isolated from tsetse flies, bioRxiv, 2020.
Bertiaux E and Bastin P, Dealing with several flagella in the same cell, Microbiol. e13162, 2020
Bertiaux E et al, A grow-and-lock model for the control of flagellum length in Trypanosomes, Biol. 28, 3802-3814 e3, 2018.
Bargul JL, et al, Species-specific adaptations of trypanosome morphology and motility to the mammalian host, Plos Pathogens, 12(2), 2016.
Kohl L, et al, Novel roles for the flagellum in cell morphogenesis and cytokinesis of Trypanosomes, EMBO J, 22(20), 2003.
Walker BJ, and Wheeler RJ, High-speed multifocal plane fluorescence microscopy for three-dimensional visualization of beating flagella, Cell Sci. 132(16), 2019.

doi: https://doi.org/10.1242/prelights.21668

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Author's response

Philippe Bastin shared

Open questions

1.Beyond the life cycle stage, does flagellar length vary across body locations in vectors and mammals? If so, what do you think is the biological reason/advantage of having flagella of different lengths?

Flagellum length increases when parasites migrate from the posterior midgut to the anterior midgut. Since parasites have to swim against the flow, it is tempting to say that they use forward motility for that and that a longer flagellum facilitates migration. Brice Rotureau showed a few years ago that parasites that cannot swim forward because of an absence of outer dyne arms failed to reach the foregut. In the mammalian stage, stumpy cells have a shorter flagellum but I’m not sure it has been investigated why.

A shorter flagellum in SE could make sense because it is thought that this is that stage that reaches the salivary glands. It needs to attach via the flagellum and so it can elongate the short flagellum to stick to microvillae of the epithelium.

2.In your discussion, you mention that the assembly of flagella of different lengths is likely achieved by different mechanisms in procyclic trypanosomes as opposed to epimastigotes. In general, evolutionarily and biologically, do you have a hypothesis of why a different mechanism might exist for both? Why is it not conserved?

Different mechanisms are observed in animal cells, for example for the formation of the spermatozoa flagellum, especially in insects, but also in mammals, or between primary cilia and cilia of photoreceptors in the retina. Another example is found in cilia of neurons in C. elegans that even harbour different IFT machineries. So, it can happen in other species. The reasons for that are more difficult to explain but could be related to the history of each cell type. After all, protists with various life cycle stages behave a bit like multicellular organisms: they do different cells with the same genome but different genetic programmes. Possibly the grow-and-lock model applies to other cell types but it has not yet been investigated (to our knowledge).

3. In different organs of the hosts (mammalian or vector), is there a specific flagellar length that would advantageous for survival given the specific composition of such organ?

Indeed, parasites display flagella of different length according to the tissues where they are encountered, suggesting it is the optimal length for that environment. This is genetically controlled since the cell types that can be maintained in culture retain that flagellum length.

If you were able to interfere with the ‘lock’ mechanism, what would be the advantage/disadvantage of a much more enlarged flagellum?

It depends on the environment really, a longer flagellum is often thought to be more efficient for motility but it also requires synthesis of more proteins and theoretically more energy. Extremely long flagella encountered in insect sperm for example are barely motile. So it must be a balance. A longer flagellum could be advantageous to have more sensors given the proposed sensing functions.

4. How does IFT and flagellar length relate to other parameters of the cell, suggesting a potential biophysical equilibrium optimal for motility? For instance, are all cells with a flagellum of a specific length equal in width and length? Again, would this not potentially aid in adaptation to specific tissues?

Actually, there is a direct link between flagellum length and cell body length in trypanosomes. Many years ago, we showed that reducing flagellum length by reducing the amount of IFT proteins led to the formation of shorter cell bodies (Kohl et al, (Ref 5)). The flagellum is attached to the cell body in trypanosomes and people also noticed that cells with longer flagella were more elongated, possibly to reduce friction during movement.

5. In inducible KDs, what would be your expectation if the flagella can reach a certain length, and then transport of specific nutrients/proteins (systematically explored) is altered?

We might have a partial answer to this question in a project we did in collaboration with the lab of Sue Vaughan in Oxford. She identified a protein called CEP164C that is localised at the base of the flagellum and that is a very interesting candidate for controlling the locking mechanism. This protein is only present at the base of the mature flagellum when the cell grows a new flagellum. When it is removed by RNAi KD, the mature flagellum becomes too long and yet it still beats and cells grow normally. However, the new flagellum is too short, possibly because it does not receive enough tubulin from the rather limited soluble pool.
The paper is on BioRxiv: doi: https://doi.org/10.1101/872952 However, we have not tried to use these cells to infect flies so we don’t know how they would behave in a real environment.

6.Given previous description (4) of different parasite behaviours in vitro, in your work, does a specific flagellar length influence the type of motion that the parasites have?

In the cardia, we noticed that cells with a longer flagellum were more motile. It is more difficult to evaluate in the salivary gland since the cells are attached via their flagellum, so they beat their flagellum but they do not swim. In culture, we noticed that cells with shorter flagellum (for example in the kinesis KD cell line (Bertiaux et al 2018) were swimming less well.

7. You mentioned within the text, that a limitation you faced was the difficulty in quantifying flagellar speed. If it is a relevant parameter, is this tool suitable (Ref 5)?

Thanks for the information! Our aim here was not to quantify parasite motility but to monitor IFT in flagella of different lengths.

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Intraflagellar transport during the assembly of flagella of different length in Trypanosoma brucei isolated from tsetse flies

Background

Key findings and developments

What I like about this preprint

Open questions

References

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