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Role for the flagellum attachment zone in the resolution of cell membrane morphogenesis during Leishmania cell division

Clare Halliday, Ryuji Yanase, Carolina Moura Costa Catta-Preta, Flavia Moreira-Leite, Jitka Myskova, Katerina Pruzinova, Petr Volf, Jeremy C. Mottram, Jack D. Sunter

Preprint posted on 26 March 2020 https://www.biorxiv.org/content/10.1101/2020.03.26.009928v1

Article now published in PLOS Pathogens at http://dx.doi.org/10.1371/journal.ppat.1008494

Untangling the flagellum attachment zone and its function in Leishmania parasites.

Selected by Mariana De Niz

Background

Leishmania are parasites that cause leishmaniasis in humans, resulting in pathology ranging from mild cutaneous lesions to severe visceral disease. The shape of the parasites is adapted to their environment in mammalian hosts, and in sandflies – the vectors responsible for transmission. The shape of Leishmania parasites is defined by the sub-pellicular microtubule array and the positioning of the nucleus, kinetoplast and the flagellum within this array. The flagellum emerges from the anterior end of the cell body through an invagination called the flagellar pocket. The flagellar pocket is a critical site, as it is where all endo- and exocytosis in Leishmania parasites occurs.

The flagellum is attached to the side of the flagellar pocket by a cytoskeletal structure called the flagellum attachment zone (FAZ). The FAZ and flagellar pocket have been the subject of great interest both in Leishmania parasites and in Trypanosoma brucei parasites. Despite the conservation in protein content, there are important differences in cytoskeletal elements of the Leishmania and T. brucei FAZ.

In their work, Halliday et al investigated the role of the FAZ in the morphogenetic resolution of the anterior cell tip during cell division (1). They focused on investigating the function of FAZ2, as in T. brucei parasites, this protein is an essential FAZ filament component (2).

 

Key findings and developments

The authors studied the effects of deletion of the FAZ filament protein FAZ2 on cell morphology, survival, and pathology. Although the deletion did not have a lethal phenotype on the parasites, nor did it affect the overall parasite morphology, the authors found that FAZ2 plays an important role in cell segregation after cytokinesis: a) in vitro, the authors detected cell rosettes with cells connected to each other via a membranous bridge between their flagella; b) evaluation of the cell cycle stage of parasites suggested a problem in late stage resolution of cell division. (Figure 1, obtained from Ref. 1).

Figure 1. FAZ2 plays an important role in cell segregation after cytokinesis.

 

Although the overall organization of the flagellar pocket was maintained, deletion of FAZ2 resulted in a shorter flagellar pocket and a very short FAZ filament. Unlike T. brucei, where FAZ2 plays a role in maintaining the attachment of the flagellum to the cell body, this is not a prominent phenotype in FAZ2 Leishmania mutants. Besides the structural changes described above, FAZ2 loss results in errors in morphogenesis that disrupts the overall organization of the FAZ. Analysis at the molecular level showed that FAZ2 deletion resulted in mislocalization of various FAZ proteins. This mislocalization ultimately affects late stage anterior cell morphogenesis. The flagellum-to-flagellum connections observed in FAZ2 mutants were mediated by FAZ proteins from the intermembrane and flagellum FAZ domains.

Altogether, the disruption of cell shape caused by FAZ2 deletion resulted in detrimental effects for infections of vectors and rodent hosts in vivo, as mutant parasites were unable to develop and proliferate in sandflies, and had a significantly reduced burden in mice.

 

What I like about this preprint

I like this preprint because the authors addressed a knowledge gap on Leishmania structural components, and defined important differences between this parasite and T. brucei. Moreover, they found a link allowing a better understanding of membrane-cytoskeletal components that define the shape and form of an individual parasite, and the interconnection with cell division. I like that they used a wide range of tools to characterize the FAZ2 mutants. Together, they have contributed another piece on a larger picture aiming to understand the relevance of the FAZ and flagellar pocket to parasite shape, function, and virulence.

Acknowledgement

I thank Jack Sunter for engaging on this highlight, and his helpful answers.

References

  1. Halliday C, Yanase R, Moura Costa Catta-Preta C, Moreira-Leite F, Myskova J, Pruzinova K, Volf P, Mottram JC, Sunter JD, Role for the flagellum attachment zone in the resolution of cell membrane morphogenesis during Leishmania cell division, bioRxiv, 2020.
  2. Zhou Q, Hu H, He CY, Li Z, Assembly and maintenance of the flagellum attachment zone filament in Trypanosoma brucei, J Cell Sci, 2015.

 

Posted on: 20 April 2020

doi: https://doi.org/10.1242/prelights.18967

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Author's response

Jack Sunter shared

Open questions 

1. You found in your work that FAZ2 deletion leads to the mislocalization of other FAZ proteins. Is it a next step for you to dissect how/if each of those affected proteins is important for membrane segregation during the late stages of cell division, rather than only a direct effect of FAZ2 loss?

Essentially, yes. A major focus of the lab at the moment is to systematically interrogate all the FAZ proteins in Leishmania to find out their function – are they important for flagellum attachment or cell morphogenesis or flagellar pocket morphology or other functions or all of these? This will give us an integrated understanding of FAZ function as I think it will have multiple roles.

2. If you could do a conditional knock out of FAZ2, and induce FAZ2 loss at different points of the late stages of cell division, how do you think this would affect parasite morphology and virulence? That is, is there a specific timing when separation of the flagella is crucial?

The connected flagella are the most noticeable aspect of the FAZ2 phenotype and are the result of errors in late stage cell division and cell morphogenesis, but I think they are the manifestations of defects in FAZ assembly that occur early in the cell cycle. Therefore, if you specifically lost FAZ2 from late stage cells they would not have this connected flagella phenotype.

3.Although they are different parasites, and therefore full conservation of phenotypes is not expected, why do you think the phenotype is so different between brucei and Leishmania, regarding FAZ2 deletion?

I think the difference in phenotype between Leishmania and T. brucei reflects the different layout of the flagellum attachment zone between the two organisms. In Leishmania the FAZ filament in which FAZ2 is found, is separated from the main attachment region, whereas in T. brucei the FAZ filament is adjacent to this attachment region with distinct connections to it. Loss of FAZ2 in both organisms disrupts the FAZ filament but due to their differing positions this results in different effects on flagellum attachment. Moreover, these parasites have different overall cell morphologies, requiring different cell morphogenesis solutions, which are reflected in the different phenotypes we see.

4.Is there any suggestion of compensation by other proteins upon the loss of FAZ2?

At the moment, we haven’t found any evidence for compensation from other FAZ proteins on loss of FAZ2; however, this does lead to an interesting point about experimental approaches. Here, we used a gene deletion approach to get rid of the FAZ2 and this requires selection of cells able to grow without FAZ2. Therefore, it is possible that there are compensatory mechanisms occurring in these cells that we have yet to find. Furthermore, if we were to do perform this experiment with an inducible approach gene deletion approach we might see a more severe phenotype.

5.One of your conclusions is that flagellar pocket architecture is important for parasite pathogenicity in the mammalian hosts. Can you expand further on the findings that your group and others have found regarding this point – is it well characterized which flagellar pocket components affect different functions in the parasite -for instance are there specific groups affecting endo- and exo-cytosis, and others affecting motility?

As you point the flagellar pocket does not have a single function and actually has a role in many different processes in the cell including endo/exocytosis, motility, and morphogenesis. Despite the central role of the flagellar pocket, its protein components are not well characterised. Until recently we only knew of a handful of proteins that localised to the flagellar pocket; however, that is starting to change with the TrypTag project I am involved with, which is tagging and localising every protein coding gene in the T. brucei genome and has found a lot of new flagellar pocket proteins.

Other groups have identified proteins that are important for endocytosis from the flagellar pocket or are critical for its morphogenesis; however, disruption of these proteins leads to cell death. This clearly shows the importance of the flagellar pocket but makes it impossible to look at what is going on in the mammalian host. In Leishmania, we have found that deletion of FAZ2 (and FAZ5 – in one of other recent publications) causes defects in the flagellar pocket but importantly these are non-lethal so we can investigate their effect in vivo and show that small changes to the flagellar pocket architecture and hence function can have massive effects on the ability of the parasite to cause disease in animals and develop in the sand fly vector.

6. Are other proteins known to be involved in parasite segregation, and are they also related to the flagellar pocket and the FAZ?

In the related parasite T. brucei there is a group of proteins – Cytokinesis Initiation Factors (CIFs) that are important for cytokinesis. In T. brucei the distal end of the new FAZ is the point at which cytokinesis begins and these CIF proteins appear to hitch a ride on the tip of the FAZ. Depletion of the CIF proteins causes severe defects in parasite segregation in T. brucei. Many of the CIFs are conserved in Leishmania but their function and whether they localise to the FAZ has not been investigated in this parasite.

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