The single flagellum of Leishmania has a fixed polarisation of its asymmetric beat

Ziyin Wang, Tom Beneke, Eva Gluenz, Richard John Wheeler

Posted on: 6 April 2020

Preprint posted on 21 March 2020

Article now published in Journal of Cell Science at http://dx.doi.org/10.1242/jcs.246637

Deciphering what makes parasites tick: visualising swimming mechanisms of Leishmania parasites.

Selected by Mariana De Niz

Categories: biophysics, cell biology, microbiology

Background

Flagella and cilia are organelles that have essentially indistinguishable ultrastructures, but originally received different names based on their biological function, including their location within organisms, their structure, and their motion. Eukaryotic flagella undergo a beat that is symmetric planar near-sinusoidal, and there are few per cell. Conversely, eukaryotic cilia undergo an asymmetric planar wafting beat, and there are usually many per cell, undergoing coordinated motility within a tissue. Cilia/flagella have asymmetries which contribute to generating the correct beat form – firstly those which keep bending in a plane, and secondly those which introduce asymmetries in the beat with the correct polarisation.

Typical asymmetric beats undergo a power-stroke, which drives fluid movement relative to the cell, followed by a recovery stroke, returning the cilium/flagellum to its starting configuration. A planar asymmetric beat has two possible polarizations, corresponding to which way the power stroke pushes fluid as the flagellum beats. The single flagellum on Leishmania parasites undergoes a symmetric tip-to-base beat (typical of flagella) for forward swimming and an asymmetric base-to-tip beat (typical of cilia) to rotate the cell. An asymmetric beat can arise when a flagellum has a large static curvature (underlying shape of the flagellum) in addition to the symmetric dynamic curvature (the propagating wave). It is unknown whether a single Leishmania flagellum has a fixed polarization for its asymmetric beat.

Using a mixture of novel microscopy tools and genetic modification, in this preprint Wang et al (1) addressed this question, and investigated whether polarization is fixed or switchable, and the structures involved.

Key findings and developments

Main developments

Wang et al developed high frame rate dual colour widefield epifluorescence of Leishmania parasites to observe the asymmetric internal cytoskeletal structure while also observing flagellum beatings.
Wang et al generated various Leishmania mutants targeting different features of the flagellum and cell-flagellum attachment.

Main findings

The authors showed that the flagellum has a fixed polarisation of the asymmetric flagellar beat.
Paralysed flagellum mutants which form a static curvature (eg. deletion mutants for axonemal proteins, or extra-axonemal and rootlet-like flagellum-associated structures) retain a fixed polarisation.
This asymmetry does not require the large paraflagellar rod structure or lateral attachment by the flagellum attachment zone.
They hypothesize rather, that asymmetry is likely intrinsic to either the nine-fold rotational symmetry of the axoneme structure, or due to differences between the outer doublet decorations.
These findings have important implications for understanding the mechanisms by which Leishmania achieves directed taxis.

Figure 1. Top: Known asymmetries in Leishmania flagellar beating. Middle: Key asymmetries in axoneme structure which may contribute to asymmetry of the base-to-tip beat. Bottom: Structure of the flagellar base and flagellar pocket by EM. (Adapted from Ref1).

Specific findings and developments (to know more details)

The asymmetric Leishmania beat occurs with a constant polarisation

The dynamic curvature of both the tip-to-base and base-to-tip beats are near-symmetric, however the base-to-tip beats often do not propagate along the entire flagellum.
While in other organisms ciliary beating is highly polarised, whether this is the case for Leishmania parasites was unknown. To determine a possible preferential direction, intracellular structures were used as reporters of cell orientation during flagellar beating -these included a flagellum membrane marker (SMP1) and a marker of the asymmetric microtubule-based cytoskeletal structure comprising the microtubule quartet and the lysosomal microtubule(s) (SPEF1).
The latter allows inferring the orientation of the flagellum axoneme, paraflagellar rod, and flagellar attachment zone.

The paraflagellar rod is on the inside of the tightly curved recovery stroke

The paraflagellar rod sits on the leading side of the flagellum during the asymmetric beat power stroke which corresponds to the inside of the static curvature. The paraflagellar rod was consistently offset from the axoneme.
The authors concluded that the paraflagellar rod experiences greater compression during the recovery stroke/reverse bend than extension during the power stroke/principal bend.

Mutants only able to form a static bend have inverted polarization to asymmetric beats.

In Leishmania, deletion mutants of many conserved axoneme proteins have a paralysed flagellum, and are unable to undergo a beat because they lack the dynamic bending component. In a subset of these, the paralysed flagella retain some capacity for bending and have a curled configuration. These mutants can form a static but not a dynamic flagellar beat.
The authors tested whether the static curvature of some of these mutants retains a preferred polarisation. The curved flagella in two mutants (for inner dynein arms, and central pairs) had a preferred bend direction towards the side of the cell with the lysosomal microtubule with the paraflagellar rod on the outside of the coil. The curvature was also much larger than in WT parasites.

Disruption of asymmetric extra-axonemal flagellum structures does not alter polarisation

PFR2 is a major structural component of the paraflagellar rod and its deletion leads to loss of almost all of the paraflagellar rod.
In Leishmania this leads to a flagellar beat which is still dominantly tip-to-base but with a shorter wavelength and lower amplitude, leading to slower forward swimming.
The authors found that the tip-to-base beat was still near-symmetrical, while the more infrequent base-to-tip beat was asymmetric with normal polarity, albeit with less pronounced asymmetry than in WT parasites.
FAZ5 is vital for lateral attachment between the flagellum and the flagellar pocket neck. Its loss results in a motility defect. The authors determined whether this defect was due to altered asymmetry of the base-to-tip beat.
Almost all flagella underwent a base-to-tip beat, suggesting a reduced ability for a tip-to-base beat is the origin of the previously observed swimming defect.
The base-to-tip beats appeared normal and consistently occurred with normal polarization.

What I like about this preprint

My main interests are biophysics and its use to understand parasitology. This preprint focuses precisely on this, and carefully dissects a topic that might be key to understand parasite behaviour, including pathology in hosts. Moreover, the authors designed various tools that made this work possible.

References

Wang et al, The single flagellum of Leishmania has a fixed polarisation of its asymmetric beat, 2020, bioRxiv, doi: 10.1101/2020.03.21.001610

doi: https://doi.org/10.1242/prelights.18159

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Author's response

Richard Wheeler shared

Open questions

1. You mention in your discussion that Leishmania parasites are an excellent system for understanding the fundamental biology of flagella/cilia and how flagellar beating is controlled. Evolutionarily, what do you think are the advantages for Leishmania’s motility basis compared to other organisms?

To me, the most notable feature of Leishmania swimming is their use of a tip-to-base waveform during forward swimming while most cilia/flagella undergo base-to-tip waves. It’s this which give rise to the distinctive “backwards” swimming of Leishmania and related species in comparison to sperm. Perhaps the flagellum tip is a key point of sensation for interaction (there’s already some support for this) and our simulation suggests that cell morphology will promote flagellum/surface interaction. They’re not constrained to this single movement mode though, and can change direction easily with their asymmetric beat.

2.Other work on cilia has shown that there is a high degree of coordination of cilia, and that the waves generated affect dynamics of particles or liquid dynamics around those cilia. How does the Leishmania flagellar beating occur when there are high number of parasites (as would be the case in a natural infection)? Would the same behaviour occur when the parasite is within tissues?

Recent evidence from other labs working on related parasites, mostly Trypanosoma brucei, have seen that high densities of cells which do indeed naturally occur in the vector do end up synchronised – this is likely through hydrodynamic coupling. I would certainly expect to see similar synchronisation in high Leishmania parasite loads in the vector gut or mouthparts. Interestingly, the Leishmania vector, the sandfly, is very small so it will be very interesting to see if synchronised flagellum beating generates significant aberrant gut fluid motion.

3.Research on other parasites (eg. Plasmodium) has suggested that the specific curvature polarization is related to, and is advantageous for, parasite circulation within the mammalian vasculature. Do you think this is the case for Leishmania?

Leishmania transition to a life cycle stage with a very short immotile flagellum in the host, so during the majority of the infection they are immotile. However, the Leishmania infectious life cycle stage, the metacyclic promastigote, has a long and actively beating flagellum. It is still essentially unknown how this contributes to dissemination through the host following innoculation by a fly bite, different beating behaviours (and the specific polarisations underlying that) certainly could contribute.

4.Are the findings you made regarding the biology of flagellar beating in Leishmania, applicable to other parasites, including Trypanosoma ?

I certainly hope so! However, one motivation for analysing Leishmania was its comparatively simple morphology – Trypanosoma have the unusual characteristic that the flagellum lies laterally attached to the cell body. This complexity makes direct translation of these results to Trypanosoma difficult. However, the proportion of the flagellum that is attached depends on the morphology of the life cycle meaning Leishmania are likely more informative for the motility of epimastigote. At the molecular level there will also be a range of conserved and different features we can use to understand Trypanosoma movement.