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