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© 2020. Published by The Company of Biologists Ltd | Journal of Science (2020) 133, jcs246637. doi:10.1242/jcs.246637

RESEARCH ARTICLE The single of has a fixed polarisation of its asymmetric beat Ziyin Wang1,‡,*, Tom Beneke1,‡, Eva Gluenz3 and Richard John Wheeler2,§

ABSTRACT undergoing coordinated movement across a . Ultimately, the Eukaryotic flagella undertake different beat types as necessary for correct choice of symmetric or asymmetric waveform, and the correct different functions; for example, the Leishmania parasite flagellum polarisation of the latter, must be used to achieve the necessary undergoes a symmetric tip-to-base beat for forward swimming and an biological function. Defects tend to cause defects in asymmetric base-to-tip beat to rotate the cell. In multi-ciliated tissues swimming cells, and , a range of mild to severe genetic or organisms, the asymmetric beats are coordinated, leading to , in . movement of the cell, organism or surrounding fluid. This coordination Typical strongly asymmetric beats undergo a power stroke, which involves a polarisation of power stroke direction. Here, we asked drives fluid movement relative to the cell, followed by a recovery whether the asymmetric beat of the single Leishmania flagellum also stroke, returning the /flagellum to its starting configuration. has a fixed polarisation. We developed high frame rate dual-colour A planar asymmetric beat has two possible polarisations, fluorescence microscopy to visualise flagellar-associated structures corresponding to which way the power stroke pushes fluid as the in live swimming cells. This showed that the asymmetric Leishmania flagellum beats. For example, in Fig. 1B the flagellum pushes fluid beat is polarised, with power strokes only occurring in one direction down and rotates the cell anticlockwise while the opposite relative to the asymmetric flagellar machinery. Polarisation of bending polarisation would push the fluid up and rotate the cell clockwise. was retained in deletion mutants whose flagella cannot beat but have The general assumption seems to be that this polarisation is fixed. In a static bend. Furthermore, deletion mutants for required for multi-ciliated systems, the polarisation of the power stroke tends to asymmetric extra-axonemal and rootlet-like flagellum-associated be organised to generate fluid flows [e.g. ciliated epithelia, structures also retained normal polarisation. Leishmania beat such as in brain ventricles (Faubel et al., 2016)] or to drive cell Paramecium polarisation therefore likely arises from either the nine-fold rotational swimming (e.g. and ) (Naitoh and symmetry of the structure or is due to differences between Kaneko, 1972). There are varied configurations in cells with the outer doublet decorations. fewer cilia/flagella; for example, uses asymmetric beating of two flagella with opposite polarisation to achieve forward KEY WORDS: Leishmania, Flagellum, Cilium, Cell motility, swimming (Ringo, 1967), while the unicellular eukaryotic parasite Asymmetry, Trypanosomatid Leishmania uses asymmetric beats of its single flagellum to rotate (Gadelha et al., 2007; Holwill and McGregor, 1975) (Fig. 1A,B). INTRODUCTION Cilia/flagella have asymmetries that contribute to generating the Motile flagella and cilia have essentially indistinguishable correct beat form – firstly those which keep bending in a plane, and ultrastructures but originally received different names based on secondly those which introduce asymmetries in the beat with the their biological function – a combination of where they are present in correct polarisation. Although both structural and functional (e.g. organisms, their structure and the motion they undergo (Takeda and signalling) asymmetries may contribute, far more is known about Narita, 2012). The tends to be called a flagellum when they structural asymmetries. undergo a planar near-symmetric near-sinusoidal beat (Fig. 1A) and Motile flagella/cilia canonically have nine outer there are few per cell. In contrast, the term cilium tends to be used doublets in a circular arrangement, around a central pair of when they undergo a planar strongly asymmetric wafting beat singlet (called the 9+2 arrangement). Their beating (Fig. 1B) and there are many cilia per cell or many ciliated cells is driven by coordinated activity of motors bound to the outer doublet microtubules, which drives sliding between adjacent doublets. To generate planar motion, some structural or regulatory 1Sir William Dunn School of Pathology, University of Oxford, Oxford, UK. 2Peter break in this rotational symmetry must exist. Depending on Medawar Building for Pathogen Research, Nuffield Department of Medicine, University of Oxford, Oxford, UK. 3The Wellcome Centre for Integrative Parasitology, the organism, this may involve addition of a fixed orientation Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, central pair complex (Ishijima et al., 1988), which does not have UK. ‡ reflectional or rotational symmetry (Carbajal-González et al., 2013). These authors contributed equally to this work *Present address: Faculty of Medicine, Imperial College, London, UK. Leishmania and related species have a fixed central pair orientation (Gadelha et al., 2006). Alternatively, it may involve the presence § Author for correspondence ([email protected]) of specialised bridges between particular microtubule doublets T.B., 0000-0001-9117-2649; E.G., 0000-0003-4346-8896; R.J.W., 0000-0002- to restrict where dynein-driven sliding occurs (Bui et al., 2009; 4270-8360 Gibbons, 1961), thus removing the nine-fold rotational symmetry of

This is an Open Access article distributed under the terms of the Creative Commons Attribution the outer doublets. Flagella with nine-fold rotational symmetry of License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, the outer doublets and no central pair or asymmetric extra-axonemal distribution and in any medium provided that the original work is properly attributed. structures can undergo three-dimensional rotating or helical

Handling Editor: David Stephens movement without a fixed beat plane, such as nodal cilia (Nonaka

Received 21 March 2020; Accepted 17 September 2020 et al., 1998).

1 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs246637. doi:10.1242/jcs.246637

Fig. 1. Known asymmetries in Leishmania flagellar beating and flagellum ultrastructure. (A,B) Schematic representations of the two types of Leishmania beat. (A) The symmetric beat, where waves propagate from the tip to the base with symmetric curvature (r). (B) The asymmetric beat, where waves propagate from the base to the

tip with asymmetric curvature for power and recovery strokes (rp and rr). (C) Schematic representation of key asymmetries in axoneme structure that may contribute to asymmetry of the base-to-tip beat. This includes asymmetries in the central pair and outer doublets of the axoneme, the PFR and (toward the flagellum base) the attachment to the cell body by the FAZ.

The nature of structural asymmetries that (1) give rise to includes proteins with Ca2+- and cAMP-associated functions which asymmetric beats and (2) defines their polarisation are less well may regulate beating (Oberholzer et al., 2007; Portman et al., 2009), understood; however, many asymmetries that correlate with while its structure suggests biomechanical effects to convert planar asymmetric beat polarisation are known. Within the 9+2 axoneme, into three dimensional movement (Hughes et al., 2012; Koyfman differences between the outer doublet decoration, particularly the et al., 2011), although we have previously argued against the latter inner dynein arms, are likely important (Bui et al., 2009, 2012). In (Wheeler, 2017). The PFR has a fixed asymmetric position and is addition to the asymmetric 9+2 axoneme, cilium-associated attached to outer microtubule doublets 4–6 (Fuge, 1969; Gadelha structures tend to be asymmetric. This includes the anchoring of et al., 2005). The anchoring of the flagellum base to the cell is also the to the cell by rootlet structures – such as the basal asymmetric, analogous to rootlet structures in other organisms, with body–basal body linkage mediated by the distal striated fibre in lateral attachment to the cell body in the flagellar pocket via the Chlamydomonas (Dutcher and O’Toole, 2016; Ringo, 1967), the flagellum attachment zone (FAZ) only near outer microtubule basal foot structure in ciliated animal epithelia cells (Gibbons, 1961) doublets 9, 1 and 2 (Wheeler et al., 2016). The 9+2 axoneme itself and the basal body-associated structures in Paramecium (Tassin likely also has asymmetries between outer microtubule doublets, et al., 2016). Mutations of rootlet proteins cause a loss of ciliated based on recent cryo-electron tomography of the related species tissue polarity. However, individual cilia rotate randomly while (Imhof et al., 2019). retaining their normal asymmetric structure (Clare et al., 2014; Analysing polarisation of Leishmania asymmetric beats has a key Kunimoto et al., 2012). Each individual cilium presumably retains challenge – Leishmania cells appear axially symmetric when viewed their asymmetric beat, although this has not been analysed in detail. though transmitted light illumination. To determine the cellular An asymmetric beat can arise when a flagellum has a large static orientation in swimming cells, we developed a high frame rate dual- curvature (the underlying ‘shape’ of the flagellum) in addition to the colour widefield epifluorescence technique for visualising symmetric dynamic curvature (the propagating wave) (Eshel and Leishmania promastigotes, allowing us to observe the asymmetric Brokaw, 1987; Geyer et al., 2016). In Chlamydomonas, several internal cytoskeletal structure (labelled with a fluorescent ) mutants that have a more symmetric beat are known, including pf2 while also observing flagellum beating as the cell undergoes different (Brokaw and Kamiya, 1987) and mbo2 (Segal et al., 1984). flagellum beat behaviours. By combining this with mutations in Generation of static and dynamic curvature are separable, and mbo2 different asymmetric features of the flagellum and cell–flagellum has a greatly reduced static curvature (Geyer et al., 2016). However, attachment, we showed (1) that the flagellum has a fixed polarisation no known mutants invert the bend of this static component to invert of the asymmetric flagellar beat, (2) paralysed flagellum mutants that the polarisation of the beat. form a static curvature retain a fixed polarisation, and (3) this It is unknown whether a single Leishmania flagellum has a fixed asymmetry does not require the large PFR structure or lateral polarisation for its asymmetric beat. We therefore asked whether attachment through the FAZ. This has implications for the polarisation is fixed or switchable, and what structures could be mechanisms by which this parasite may achieve directed taxis. involved in conferring asymmetry (Fig. 1C). In addition to the 9+2 axoneme, Leishmania have an extra-axonemal structure called the RESULTS paraflagellar rod (PFR). There are several possible functions of Leishmania flagellum beating can be readily analysed from high the PFR (Portman and Gull, 2010), which is required for normal frame rate (400 Hz) phase-contrast videos using automated tracing flagellum beating (Santrich et al., 1997). Its protein composition of the flagellum configuration (Walker and Wheeler, 2019; Walker Journal of Cell Science

2 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs246637. doi:10.1242/jcs.246637 et al., 2019). As often used for other organisms, this data can be ∼20 to 25 Hz for the tip-to-base and ∼5 Hz for the base-to-tip beats represented as plots of tangent angle at different distances along the (Gadelha et al., 2007; Wheeler, 2017). The tip-to-base beat is flagellum over time (Fig. 2; Movie 1), where tangent angle is the symmetric (typical of flagella), while the base-to-tip beat is angle of a section of flagellum relative to the flagellum base. A asymmetric with a power and recovery stroke (typical of cilia) higher rate of change in tangent angle along the flagellum (Gadelha et al., 2007; Holwill and McGregor, 1975). corresponds to higher flagellum curvature. Leishmania undergo In tangent angle plots, the wavefronts of the high frequency tip-to- two well-described types of flagellum beating, a high frequency tip- base symmetric beat appear as lines aligned top right to bottom left, to-base beat and a lower frequency base-to-tip beat. These occur at alternately with positive or negative tangent angles (Fig. 2A;

Fig. 2. See next page for legend. Journal of Cell Science

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Fig. 2. The symmetric and asymmetric beats of the Leishmania flagellum. the plane perpendicular to the central pair (Gadelha et al., 2006). (A–D) One example cell undergoing either (A,B) a symmetric tip-to-base Therefore, there are two possible directions for the power stroke flagellar beat or (C,D) an asymmetric base-to-tip ciliary beat visualised from a of the asymmetric beat. Ciliary beating in other organisms is 400 Hz high-frame rate phase-contrast video. (A) The symmetric beat, plotted as a kymograph of tangent angle along the flagellum, automatically traced highly polarised, suggesting a single preferred direction; however, from the phase-contrast video. Yellow indicates clockwise tangent angles the Leishmania cell is near-axially symmetric. The cell body can be relative to the flagellum base, and cyan anticlockwise tangent angles. The slightly curved; however, not every cell is curved and it is not known waves during the symmetric beat propagate from the flagellum tip to its base whether this curvature occurs in a consistent direction relative to (from top right to bottom left), alternating between waves with a negative flagellar structures. Therefore, the orientation of flagellar structures, ‘−’ ‘ ’ tangent angle (labelled ) or a positive tangent angle (labelled + ). Aberrant and thus polarisation, of the asymmetric beat cannot be inferred waves occasionally occur with a different frequency or with additional bends in the flagellum; one example is indicated. (B) Six frames illustrating the from transmitted light images such as phase-contrast images (Fig. 2). propagation of positive and negative tangent angle waves over one beat cycle. Whether there is a preferred direction can be tested by observing Phase-contrast images from the frames indicated with horizontal lines in A. asymmetric intracellular structures, which act as a reporter of cell (C) Kymograph of tangent angle for the asymmetric beat. The waves propagate orientation, during flagellum beating. We achieved this using high from the flagellum base to its tip (from top left to bottom right), alternating frame rate dual-colour widefield epifluorescence microscopy (Fig. between a wave for a power stroke (small tangent angles, negative in this S1) using a cell line expressing the well-characterised flagellum example) and a wave for a recovery stroke (large tangent angles, positive in this membrane marker SMP1::mCh (Tull et al., 2004; Wheeler et al., example). (D) Six frames illustrating the power and recovery strokes over one beat cycle as indicated in C. (E) Top, plotted tangent angles at different 2015) and a marker of the asymmetric microtubule-based cytoskeletal distances along the flagellum for one symmetric tip-to-base beat cycle from the structure, comprising the microtubule quartet and the lysosomal experiment in A,B. The mean tangent angle at each distance along the microtubule(s), mNG::SPEF1 (Gheiratmand et al., 2013; Halliday flagellum gives the static curvature of the flagellum, represented as the tangent et al., 2018; Wang et al., 2019) (Fig. 3A). angle, which is very near straight. Bottom, tangent angles with static curvature Through comparison to previously published electron subtracted. (F) Average static curvature for many flagella undergoing tip-to- tomography of the flagellar pocket (Wheeler et al., 2016), the base beats (grey), and their mean. (G) Tangent angle plotted as in E, except for one asymmetric base-to-tip beat cycle from the experiment in C,D. (H) Average relative orientation of the flagellum axoneme, PFR and FAZ can be static curvature for many flagella undergoing base-to-tip beats (grey), and their inferred from the appearance of the mNG::SPEF1 signal (Fig. 3B). mean. In Fig. 3B, the plane of beating is the plane of the image (perpendicular to the central pair). As the flagellum exits the cell Movie 1A). These correspond to the successive wavefronts through the flagellar pocket neck, it attaches to the microtubule propagating from the flagellum tip over time (Fig. 2B). The quartet via the FAZ on one side of the flagellum. This attachment wavefronts of the asymmetric base-to-tip beat appear as wider region is near where specialised -associated microtubule(s) diagonal lines aligned top left to bottom right, corresponding to also meet the microtubule quartet, on the opposite side of the lower frequency waves propagating from the flagellum base over flagellum to the start of the PFR (Wheeler et al., 2016). time (Fig. 2C; Movie 1B). Unlike the symmetric beat, the alternating In high frame rate videos, the flagellum movement can be positive and negative tangent angle wavefronts in the asymmetric visualised and traced from the SMP1::mCh signal. Identifying beat have different magnitudes of tangent angle (Fig. 2C,D). example cells where the beat switches from symmetric to The conformation of the flagellum can be mathematically asymmetric over the course of the video is relatively easy, separated into two components – a moving component (dynamic illustrated for two example cells in Fig. 3C–H and Movie 2. The curvature) and a static component (static curvature). The tangent switch between flagellum beats is summarised by tangent angle at angles arising from the static curvature can be determined by different distances along the flagellum over time (Fig. 3C,F). In averaging the tangent angle over an integer number of beats or by these videos, the orientation of bending relative to the cellular taking the static/infinite frequency mode of a Fourier decomposition. ultrastructure can be inferred from the mNG::SPEF1 signal, both In Chlamydomonas, it has been shown that generation of dynamic while undergoing a symmetric beat (example frames and the traced and static curvature are biologically separable and not just beat shown in Fig. 3D,G) or an asymmetric beat (Fig. 3E,H). This mathematical constructs (Geyer et al., 2016). While undergoing a showed that the power stroke of the asymmetric beat pushed away symmetric tip-to-base beat the Leishmania flagellum static curvature from the side of the cell with the lysosomal microtubule(s), is low (Fig. 2E,F). In contrast, for asymmetric base-to-tip beats, the corresponding to a static curvature toward that side of the cell. tangent angle reaches large values (>π/2 rad, >90°) corresponding to We noted that the symmetric to asymmetric beat switching a high static curvature (Fig. 2G,H). Dynamic components of involved the following stages. First, a tip-to-base wave that stalls flagellum conformation can be determined by subtracting the static and then reverses, giving a bend that gradually propagates towards curvature. This corresponds to the propagating wave shape on the the flagellum tip. Second, high frequency tip-to-base waves that underlying static shape of the flagellum. Tangent angles arising from continue to initiate near the flagellum tip but fail to propagate past dynamic curvature of both the tip-to-base and base-to-tip beats are the stalled/reversed bend. Third, lower frequency asymmetric base- near symmetric; however, the base-to-tip beats often do not to-tip waves that start to initiate at the flagellum base but fail to propagate along the entire flagellum (Fig. 2G). For the proximal propagate past the stalled/reversed bend. This can give both base-to- portion of the flagellum, the tangent angle has a near-linear tip waves in the proximal and tip-to-base waves in the distal correlation with distance along the flagellum, corresponding to a domain of a single flagellum at the same time (which is unlike constant curvature (Fig. 2H), similar to what is observed in the whole previously described (Gadelha et al., 2007)). Finally, base-to-tip flagellum asymmetric in Chlamydomonas beats (Geyer et al., 2016). waves that can propagate along the whole flagellum and with no new tip-to-base waves initiated. Initiation of new tip-to-base waves The asymmetric Leishmania beat occurs with a constant can stop after after the first base-to-tip wave but may take several polarisation base-to-tip waves. The gradually propagating bend in the first stage Leishmania have a fixed axoneme central pair orientation (Fig. 1C), is likely associated with a base-to-tip establishment of the static and bending for flagellum beating is thought to occur only in curvature for the asymmetric beat. Journal of Cell Science

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Fig. 3. SPEF1 is an asymmetric reporter of Leishmania cell orientation during asymmetric flagellum beating. (A) The subcellular localisation of the microtubule quartet and lysosomal microtubule-associated mNG::SPEF1 relative to the flagellar membrane marker SMP1::mCh as determined by epifluorescence microscopy. (B) Structure of the flagellar base and flagellar pocket as determined by electron tomography, as previously published (Wheeler et al., 2016), indicating the SPEF1- and SMP1-associated ultrastructures. (C–H) Two examples of flagellum movement and cell orientation in cells expressing mNG::SPEF1 and SMP1::mCh switching from a symmetric tip-to-base to an asymmetric base-top-tip beat, derived from 100 Hz high frame rate dual-colour epifluorescence videos. (C–E) First example cell. (F–H) Second example cell. (C,F) Change in tangent angle over time, automatically traced from the SMP1::mCh signal. Yellow indicates tangent angles to the right, cyan to the left. (D,G) Four frames showing the symmetric tip-to-base beat. mNG::SPEF1 and SMP1::mCh fluorescence from the frames indicated “Flagellar” in C,F. The flagellum configuration over one beat cycle is shown at the bottom. (E,H) Four frames showing the asymmetric base-to-tip beat. mNG::SPEF1 and SMP1::mCh fluorescence from the frames indicated “Ciliary” in C,F. The asymmetric beat tends to bend away from the side of the cell with the lysosomal microtubule.

The PFR is on the inside of the tightly curved recovery stroke signal relative to background noise, allowing precise measurement The analysis of cell asymmetry using mNG::SPEF1 (Fig. 3C–H) in of red–green signal offset. This showed that there was a precise combination with the ultrastructure of the cell (Fig. 3B) indicates colocalisation of the two axoneme markers (mNG::PF16 and mCh:: that the PFR sits on the leading side of the flagellum during the RSP4/6; Fig. 4A–E; Movie 3A), both in straightened images from asymmetric beat power stroke, which corresponds to the inside of symmetric beats (Fig. 4D) and asymmetric beats (Fig. 4E). In the static curvature. To directly confirm this result, we analysed two contrast, the PFR was consistently offset from the axoneme (mNG:: cell lines, one expressing two axoneme markers (mNG::PF16 and PFR2 and mCh::RSP4/6; Fig. 4F–J; Movie 3B). The direction of the mCh::RSP4/6), and one expressing a PFR marker and an axoneme offset indicates the PFR is on the inside of the static curvature, marker (mNG::PFR2 and mCh::RSP4/6). In the former, the red and and the offset was consistent with the expected position of the green signal should precisely colocalise, while in the latter there PFR relative to the axoneme. should be a small offset – ∼150 nm based on flagellum By using many high frame rate dual-colour widefield ultrastructure (Fig. 1C). Again, it was relatively easy to find epifluorescence microscopy videos of either the mNG::SPEF1/ examples of cells where the beat switches from symmetric to SMP1::mCh (n=9) or mNG::PFR/mCh::RSP4/6 (n=10) cell lines, asymmetric (Fig. 4; Movie 3). The mCh::RSP4/6 signal was much we could determine the incidence of the two possible polarisations of weaker than SMP1::mCh; therefore, to analyse these videos, we the asymmetric beat – either with the power stroke bending away used automated tracing to determine the configuration of the from the side of the cell with the lysosomal microtubules with the flagellum, then digitally straightened the flagellum from each frame PFR sitting on the leading side of the flagellum or the inverse of the video. Multiple frames can then be averaged, to increase (Fig. 5A). The results indicate that there is a strong and consistent Journal of Cell Science

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Fig. 4. The position of the asymmetric PFR extra-axonemal structure in beating flagella. (A–E) Flagellum movement in a cell expressing mNG::PF16 and mCh::RSP4/6 (two axonemal proteins – central pair and radial spokes, respectively) switching from a symmetric tip-to-base to an asymmetric base-top-tip beat, derived from a 100 Hz high frame rate dual-colour epifluorescence video. (A) Change in tangent angle over time, as in Fig. 3C, automatically traced from the mNG::PF16 signal. (B) Four frames showing the symmetric tip-to-base beat and the flagellum configuration over the beat cycle. (C) Four frames showing the asymmetric base-to-tip beat and the flagellum configuration over the beat cycle. (D) Digitally straightened mNG::PF16 and mCh::RSP4/6 signal, averaged over 64 frames of tip-to-base symmetric beating, presented stretched in the transverse direction by 2×, and the transverse signal intensity profile. (E) As for D, but for 64 frames of tip-to-base asymmetric beating. There is no offset between mNG::PF16 and mCh::RSP4/6 signal. (F–J) As for A–E, except a cell expressing mNG::PFR2 and mCh::RSP4/6 (a PFR and an axonemal protein, respectively). The mNG::PFR2 signal is offset from mCh::RSP4/6 signal. During asymmetric beating, the flagellum curves with the PFR on the inside of the tighter curvature recovery stroke. polarisation of the asymmetric beat (Fig. 5B), with a tighter radius of asymmetric behaviour, we analysed flagellum bending in mutants curvature of the reverse bend (doublet 1 on the outside of the curve) of the axoneme components required for motility, PFR and the FAZ. than the principal bend (doublet 1 on the inside). This indicates that the PFR experiences greater compression during the recovery stroke/ Mutants only able to form a static bend have inverted reverse bend than extension during the power stroke/principal bend. polarisation to asymmetric beats To determine how the asymmetric structures of the Leishmania In Leishmania, deletion mutants for many conserved axoneme flagellum and associated structures (Fig. 1C) contribute to this proteins have a paralysed flagellum unable to undergo a beat.

Fig. 5. Asymmetric base-to-tip beating occurs in a consistent orientation. (A) Leishmania flagellum beating is near-planar, giving two possible polarisations for the asymmetric base-to-tip flagellum beat. Either polarisation (1), with the PFR on the inside of the narrow radius recovery stroke bend, or (2), with the PFR on the outside of the recovery stroke bend. (B) Instances of polarisation 1 or 2, counted from high frame rate fluorescence videos of cells expressing mNG::SPEF1 and SMP1::mCh (as in Fig. 3) or mNG::PFR2 and mCh::RSP4/6 (as in Fig. 4F–J). The asymmetric beat has a strong preference for the power stroke to bend away from the side of the cell with the lysosomal microtubule, positioning the PFR on the inside of the tightly curved recovery stroke. Journal of Cell Science

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However, in a subset of deletion mutants, the paralysed flagella retain losing the ability for dynamic curvature owing to dysfunction within some capacity for bending, and a large proportion have a curled the 9+2 axoneme. configuration – typically a few turns of a coil (Beneke et al., 2019). These mutants are not informative for understanding waveform Disruption of asymmetric extra-axonemal flagellum generation; however, they form a strong static curvature. We tested structures does not alter polarisation whether this static curvature retained a preferred polarisation using The PFR is a large extra-axonemal structure of comparable size to two mutants where curling is highly prevalent (Beneke et al., 2019) the axoneme. It is specific to the lineage of unicellular to determine whether polarised bending can occur in the absence of and is asymmetrically positioned in the flagellum next to flagellar beating. The first was a deletion of the inner dynein arm doublets 4 to 6 of the axoneme (Fig. 5C). PFR2 is a major structural intermediate chain protein IC140 (Hendrickson et al., 2013; Heuser component of the PFR and deletion of PFR2 leads to loss of almost et al., 2012). The second was a deletion of Hydin, a central pair all of the PFR. In Leishmania this leads to a flagellar beat that is still complex protein required for central pair microtubule stability and to dominantly tip-to-base but with a shorter wavelength and lower prevent central pair rotation (Dawe et al., 2007). Again, we used amplitude, leading to slower forward swimming (Santrich et al., mNG::SPEF1 as a reporter to allow determination of the direction of 1997). A similar motility defect also occurs upon PFR2 deletion in flagellum bending. Curled flagella in both the ΔIC140 and ΔHydin the related parasite Trypanosoma brucei (Bastin et al., 1999), where mutant remained in the normal beat plane and had a preferred bend it was also shown that PFR2 deletion does not affect the fixed direction towards the side of the cell with the lysosomal microtubule. central pair orientation (Gadelha et al., 2006). To determine whether This corresponds to the PFR lying on the outside of the coil, although loss of the PFR alters the base-to-tip asymmetric beat, we generated curling in both directions did occur (Fig. 6A–D). To confirm this a PFR2 deletion cell line expressing mNG::SPEF1 and SMP1::mCh result, we used scanning electron microscopy (SEM) of detergent- (Fig. 7; Movie 4). Plots of tangent angle over time show the multiple extracted of these two mutants. This allowed direct defects the flagellum beat experiences in the absence of the PFR visualisation of the PFR on the outside face of a large majority of (Fig. 7A,D). As previously described, flagella still predominantly coiled flagella (Fig. 6E–H). undergo tip-to-base waveforms, but examples of base-to-tip These curled flagella therefore have a strong static curvature waveforms were also readily identifiable. Tangent plots confirm in the opposite direction to the static curvature of the that bending tends to be lower amplitude and many wavefronts fail asymmetric base-to-tip beat. These flagella were strongly curved – to propagate along the entire length of the flagellum. The frequency the tangent angle at the flagellum tip could reach >2π rad, >360° was also variable. The lower amplitude flagellum movement also (Fig. 6A,C), with a tight curvature radius of 2.70±0.42 μm and 2.61 allows the cell to rotate more readily between the slide and coverslip, ±0.21 μm (mean±s.d.) for ΔHydin and ΔIC140, respectively. complicating analysis. Nonetheless, the tip-to-base beat still tends Interpreting this result is difficult as it is not clear that the to be near-symmetrical, if a little uncoordinated, and in the normal flagellum curling direction in these mutants originates from the beat plane (Fig. 7B,E). The more infrequent base-to-tip beat is same molecular mechanism as the asymmetry of the normal still often asymmetric with normal polarity and in the normal beat asymmetric beat. However, it is clear that upon disruption of either plane (Fig. 7C); however, in comparison to the parental line (Figs 3 the inner dynein arms (ΔIC140) or the central pair (ΔHydin), the and 5), there were also more cells with less pronounced asymmetry flagellum retains the ability to have polarised static curvature while (Fig. 7F,I).

Fig. 6. Axoneme protein deletion mutants with a paralysed ‘curled flagellum’ have a preferred curl direction. (A) Example fluorescence micrographs of a IC140, an inner dynein arm intermediate chain, deletion mutant (ΔIC140) expressing mNG::SPEF1, with the flagellum either curling toward the side of the cell with the lysosome microtubule (polarisation 1, PFR on the outside of the curve) or the opposite (polarisation 2). (B) Proportion of cells, assessed from light microscopy, with the flagellum curling with polarisation 1 or 2. (C,D) As for A,B, except for a deletion mutant of Hydin (ΔHydin), a central pair complex protein. (E) Example scanning electron micrograph of a detergent-extracted from an IC140 deletion mutant cell. The direction of curling is directly visible from the PFR next to the axoneme. (F) Proportion of cells, assessed from SEM, with the flagellum curling with polarisation 1 or 2. (G,H) As for E–F, except for a deletion mutant of Hydin. These mutants with dysregulation of flagellum beating still have a preferred bend direction, albeit for a non-beating flagellum. Journal of Cell Science

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Fig. 7. Neither disruption of the PFR nor the FAZ inverts asymmetric beat polarity. (A–F) Flagellum movement in two example cells of a PFR2 (a PFR protein) deletion mutant (ΔPFR2) expressing mNG::SPEF1 and SMP1::mCh switching from a symmetric tip-to-base to an asymmetric base-to-tip beat, derived from a 100 Hz high frame rate dual-colour epifluorescence video. (A) Change in tangent angle over time, as in Fig. 3C, automatically traced from mNG::PF16 signal. (B) Four frames showing the symmetric tip-to-base beat and the flagellum configuration over the beat cycle. (C) Four frames showing the asymmetric base- to-tip beat and the flagellum configuration over the beat cycle. (D–F) As for A–C except for the second example cell with a base-to-tip beat with no clear asymmetry. (G,H) Flagellum movement in two example cells of a FAZ5, a FAZ protein, deletion mutant (ΔFAZ5) expressing HaloTag::SPEF1 and labelled with Tetramethylrhodamine ligand. Both examples persistently underwent an asymmetric base-to-tip beat, summarised with four frames from a 100 Hz high frame rate green light brightfield and red fluorescence video. (I) Instances of asymmetric bending in the direction 1 or 2 (as defined in Fig. 5), counted from high frame rate fluorescence videos of either PFR2 or FAZ5 deletion mutants. The asymmetric beat retains a strong preference for the power stroke to bend away from the side of the cell with the lysosomal microtubule, with more examples of reverse beats with unclear asymmetry in the PFR2 deletion. (J) Curvature of the power and recovery strokes measured from fluorescence videos of PFR2 or FAZ5 deletion mutants relative to a cell line with no deletion.

Finally, we considered the asymmetrically positioned FAZ at the a chloroalkane with an amine-linked fluorophore – in this case base of the flagellum. Previous work has identified the protein Tetramethylrhodamine. HaloTag was used as the ΔFAZ5 cell line FAZ5 as vital for any lateral attachment between the flagellum and was, for an unknown reason, refractory to tagging of SPEF1 with the flagellar pocket neck, in turn giving rise to a motility defect conventional β-barrel fluorophores (mNG, eYFP and mCh). We (Sunter et al., 2019). To determine whether this defect was were also unsuccessful at generating a ΔFAZ5 cell line using a associated with altered asymmetry of the base-to-tip beat, we single selectable marker, leaving insufficient selectable markers to generated a FAZ5 deletion cell line expressing HaloTag::SPEF1, also tag SMP1. Therefore the cell line was visualised using a where HaloTag is a self-labelling protein tag which covalently binds combination of red fluorescence and bright field transmitted light Journal of Cell Science

8 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs246637. doi:10.1242/jcs.246637 using a green filter for simultaneous visualisation of the flagellum Rheotaxis and are complex behaviours with many and the orientation of the cell using HaloTag::SPEF1 (Fig. 7G,H; contributing mechanisms; however, a fixed polarisation of Movie 5). Flagella tended to undergo a base-to-tip beat, suggesting a asymmetric beat may restrict the possible responses Leishmania reduced ability for a tip-to-base beat is the origin of the previously can undergo to direct its swimming. This can be illustrated with a observed swimming defect (Sunter et al., 2019). Intriguingly, this hypothetical 2D situation: on approaching and sensing a barrier at suggests the FAZ represses the base-to-tip beat. The bright-field an oblique angle Leishmania could not chose to turn away from the videos had insufficient contrast for automated tracing of the wall by switching to an asymmetric beat, only to turn in the direction flagellum beat, but the base-to-tip beats appeared normal, the fixed polarisation asymmetric beat allows. Leishmania can consistently occurred with normal polarisation and consistently undergo directed taxis (osmotaxis) (Leslie et al., 2002); however, occurred in the normal beat plane (Fig. 7G,H; Movie 5). The how flagellum movement is modulated to do so is not yet clear. If combined evidence from the ΔPFR2 (n=17) and ΔFAZ5 (n=16) we assume that the only mechanism is the switch from the mutants suggest that the most prominent asymmetric flagellum- symmetrical tip-to-base beat to the asymmetric base-to-tip beat, associated structures in Leishmania are not required for achieving then the cell cannot simply turn to align itself with the osmotic asymmetric static flagellum curvatures (Fig. 7I, cf. Figs 3 and 5) and gradient making a run-and-tumble-like mechanism, with tumbles do not affect the curvature of the power and recovery strokes caused by the base-to-tip beat, most plausible. However, our results (Fig. 7J). do not preclude other modulations of the flagellar beat to achieve directed taxis. of many species have small asymmetries in the DISCUSSION flagellum beat leading to circular or helical swimming paths. By Leishmania are highly genetically tractable cells (Beneke et al., modulating this asymmetry, periodic changes to the curvature of the 2017) and the promastigote life cycle stages have a canonical 9+2 swimming path can give rise to robust chemotaxis (Kaupp et al., single flagellum that switches between a near-planar (Walker and 2008). Making detailed comparisons between chemotaxis/rheotaxis Wheeler, 2019) symmetric tip-to-base beat and an asymmetric base- mechanisms in sperm (a head first ‘pusher’)inLeishmania (a to-tip (Gadelha et al., 2007; Holwill and McGregor, 1975), making ‘puller’) may not be accurate. However, it is notable that sperm it an excellent system for understanding the fundamental biology of trajectories tend to constantly turn in the same direction and it seems flagella/cilia and control of their flagellar beating. In particular, new plausible that the small asymmetries in sperm flagella may also have opportunities to analyse the origin and switching of beat asymmetry a fixed polarisation. arise as the symmetric beat propagates from tip-to-base and the In Chlamydomonas, regulation of the symmetric dynamic asymmetric beat from base-to-tip. curvature of the flagellum to make a wave propagate and the static Leishmania have a near-axially symmetric cell shape, which makes curvature of the flagellum to introduce asymmetry are biologically it challenging to analyse cell orientation from transmitted light separable – mutant (demonstrated with mbo2) flagellum can fail to micrographs. To overcome this limitation, we developed the first dual- form a static curvature giving an aberrant symmetric waveform but colour high resolution and high frame rate fluorescence visualisation of otherwise normal flagellum beating (Brokaw and Kamiya, 1987; live swimming cells and used this to visualise proteins endogenously Geyer et al., 2016; Segal et al., 1984). In Leishmania, no mutants tagged with genetically encoded fluorophores (mCh or mNG) or that lose the asymmetry of their base-to-tip beat are yet known – fluorophore-binding proteins (HaloTag), through either simultaneous deletions of the outer dynein arm-associated proteins dDC2 and capture of red and green fluorescence, or red fluorescence along with LC4-like do promote asymmetric or symmetric beats, respectively, green transmitted light. This allowed us to analyse polarisation of but these beats occur in their normal respective base-to-tip and tip- flagellum beating relative to the orientation of flagellum and flagellum- to-base propagation directions (Edwards et al., 2018). However, associated intracellular ultrastructure (Fig. 3), despite the outward there are Leishmania deletion mutants (including PF16, Hydin and axially symmetric appearance of Leishmania. IC140) where the flagellum is paralysed but often curls up (Beneke Our analysis showed that the asymmetric Leishmania beat has et al., 2019) while, naïvely, paralysed flagella would be expected to a fixed polarisation (Fig. 5) that could be mathematically described be straight. Deletions of IC140 and Hydin are the most dramatic as a fixed direction for static curvature (Fig. 3). The fixed examples. The curling in these mutants is still polarised (Fig. 6); polarisation means the PFR and microtubule doublets 4 and 5 however, this polarisation is in the opposite direction to the static always lie on the inside of the static curvature and the inside of curvature in base-to-tip asymmetric beats (Fig. 5). This strongly tightly-curved power strokes. As Leishmania is a very early- suggests that flagellar polarisation is retained in the absence of the diverging , it is likely that that a fixed polarisation of central pair complex and in the absence of the IC140-associated strongly asymmetric beats is universal among eukaryotes, building inner dynein arms, although the precise correspondence of this on the lack of evidence for polarisation switching in other systems. mutant phenotype to normal symmetric or asymmetric beats is For example, Ca2+-mediated regulation of ciliated epithelia unclear. beating involves an arrest in beating or change in frequency rather Leishmania has additional asymmetric flagellum-associated than switching polarisation and Ca2+-mediated regulation of structures that may have been important for conferring flagellum C. reinhardtii beating involves a switch from an asymmetrical to beat polarisation. However, loss of lateral flagellum attachment by a symmetrical beat rather than switching polarisation (Inaba, 2015). the FAZ did not reduce asymmetry or invert the polarisation of the The only possible example of a switch in asymmetric beat asymmetric base-to-tip beat (Fig. 7). Loss of almost the entire bulk polarisation we identified is the ‘ciliary reversal’ described for of the PFR also did not invert the polarisation of the asymmetric Paramecium (Eckert, 1972; Naitoh and Kaneko, 1972). However, base-to-tip beat (Fig. 7). The FAZ is specific to the Leishmania the C. reinhardtii switch to symmetrical movement is also branch of life, but asymmetric rootlet structures analogous to the confusingly described as ‘flagellar reversal’ (Schmidt and Eckert, FAZ are often present in other organisms. Similarly, flagella in other 1976) despite not involving reversal of either waveform direction or species can have extensive extra-axonemal structures analogous to polarity, leaving the true nature of the waveform change in the PFR, such as the outer dense fibres and thickened fibrous sheath

Paramecium ciliary reversal unclear. regions of metazoa sperm. Together, this suggests that asymmetric Journal of Cell Science

9 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs246637. doi:10.1242/jcs.246637 extra-axonemal structures and rootlet structures in other species are (Andor). A 2019 mm focal length lens was used in the red light path for not likely to be responsible for the polarisation of asymmetric focus correction. flagellum beats. Both the PFR2 and FAZ5 deletion mutants did, Synchronously recording red fluorescence and brightfield microscopy however, perturb the flagellum beat in other ways. Interpreting these (in green) was possible because the samples do not emit green fluorescence. beat defects is unfortunately complex as each deletion leads to Therefore, when using white epi-illumination and trans-illumination through a green filter (ThorLabs, Astronomy Green), this set-up resulted disruption of a large complex cytoskeletal structure with many in the splitter projecting a green brightfield and red fluorescence image onto (likely >100) components (Portman and Gull, 2010; Sunter and the two halves of the camera. Gull, 2016; Sunter et al., 2019). The red and green images must be aligned to generate composite images. Taken together, this work greatly constrains the molecular origin Images for calibration of position and /magnification were captured of asymmetry in Leishmania base-to-tip beats. Our previous work using multi-wavelength fluorescent beads (TetraSpeck 0.1 µm indicated that outer dynein arm-associated factors are likely Microspheres, Invitrogen T7279); then the red and green images were important for switching between symmetric tip-to-base and aligned using the same approach we previously used for multi-focal plane asymmetric base-to-tip beats (Edwards et al., 2018); however, microscopy (Walker and Wheeler, 2019). For microscopy of cells adhered to glass, a sample of cells from late controlling switching is distinct from the actual generation of 7 7 −1 asymmetry. Having clearly excluded the FAZ and PFR, and logarithmic growth (0.5×10 to 1.0×10 cells ml ) were taken from culture, washed and placed on a slide then imaged live as previously described provided some evidence against the axoneme central pair and (Halliday et al., 2018). IC140-containing inner dynein arms being responsible for For microscopy of free-swimming and naturally behaving cells, samples asymmetry, the most likely remaining candidate is differences were taken from cultures in late logarithmic growth. To reduce cell between the outer doublet decorations – in particular in the region of adherence, glass slides were blocked with BSA prior to use by immersion in inner arm dynein b, based on cryo-electron tomography of T. brucei 1% BSA in distilled water for 30 s, followed by three washes in distilled (Imhof et al., 2019). However, the mechanical properties of the water and air drying. 1 μl cells from culture in normal culture medium nine-fold asymmetric outer microtubule doublets of the axoneme were applied to a 2 cm by 1 cm rectangle marked on a slide and coverslip itself could also be important. Specialised inner dynein arm are also using a hydrophobic pen, resulting in a liquid layer 2 μm thick. As the μ implicated in Chlamydomonas flagellum movement asymmetries flagellum beat amplitude is typically >2 m, this reduces the chance of the (Bui et al., 2009), perhaps indicating that this is the eukaryote-wide cell rotating such that the beat plane is perpendicular to the focal plane. This is, however, an artificial confinement and the confining surfaces introduce origin for asymmetry of flagellum movement. skin friction drag. In order to generate the plots of tangent angle at different distances along the MATERIALS AND METHODS flagellum over time, flagellawere automatically traced from videomicrographs Procyclic promastigote L. mexicana expressing Cas9 and T7 polymerase, using ImageJ. We used the intensity thresholding, skeletonisation and tracing derived from WHO strain MNYC/BZ/62/M379 (Beneke et al., 2017) were scheme we previously developed for phase-contrast videos (Walker and grown in M199 medium with Earle’s salts and L-glutamine (Life Wheeler, 2019). The midline is on the pixel grid; this was then −1 Technologies) supplemented with 26 mM NaHCO3,5μgml haemin, sampled at 3 pixels. The angle between the first and fifth points were taken as 40 mM, HEPES-NaOH (pH 7.4) and 10% fetal calf serum (FCS). the cell orientation in each frame and graphs of tangent angle are shown with a L. mexicana were grown at 28°C and maintained at culture densities 17-pixel rolling average. For tracing the fluorescence in high speed videos, the between 105 and 107 cells ml−1. approach was adapted such that red fluorescent signal (either SMP1::mCh or For endogenous tagging of L. mexicana genes, constructs and sgRNAs mCh::RSP4/6) was subject to intensity thresholding following a correction for were generated using the PCR-based approaches previously described photobleaching. Flagella were digitally straightened using the ImageJ (Beneke et al., 2017; Dean et al., 2015), using the pLPOT (also straighten tool using the flagellum midline from the red fluorescence as the called pLrPOT) (Edwards et al., 2018) series of plasmids as the PCR line selection for straightening both the green and red fluorescence images. template. L. mexicana were transfected and subjected to drug selection as For SEM cytoskeleton preparations, cells were harvested from culture by previously described (Dean et al., 2015). For endogenous tagging with centrifugation (800 g for 3 min), washed three times in PBS, settled on HaloTag, a new pLPOT variant with HaloTag and blasticidin deaminase coverslips in 24-well plates, washed with 0.1% NP-40 in PBS, washed three was generated. times in PBS and then fixed in 2.5% glutaraldehyde in PBS for 2 h at room The L. mexicana cell lines with deletion of both alleles of flagellum and temperature and then at 4°C overnight. Fixed cells were washed three times FAZ proteins were generated using the PCR-based approach previously for 5 min in PBS and incubated in 1% osmium tetroxide in PBS at 4°C for described, using the pT series of plasmids as the PCR template (Beneke 1 h in darkness. Samples were then washed three times with water for 5 min et al., 2017). All deletion cell lines have been previously been characterised: and ethanol dehydrated. Sample drying was completed by critical point ΔPFR2, ΔIC140 and ΔHydin in Beneke et al. (2019) and ΔFAZ5 in Sunter drying using an Autosamdri-815 (Tousimis). Coverslips were then sputter et al. (2019). gold coated for 60 s. Samples were imaged at around 20,000 to 35,000× on a Microscopy was performed with an Axio Observer A1 (Zeiss) JSM-6390 SEM (JEOL) with an Everhart-Thornley secondary electron microscope with incubator chamber using a 63× NA 1.4 Ph3 objective or detector using an EHT target of 2 kV and a 20 µm aperture. a 100× NA 1.4 without phase ring using a 120 V metal halide fluorescence light sources (Zeiss, HXP 120 V). Standard fluorescent microscopy was Acknowledgements performed with an mRFP (Zeiss, 63HE) or GFP (ThorLabs, MDF-GFP2) I would like to thank Jack Sunter for kindly providing the FAZ5 deletion mutant filter cube. and Keith Gull for his support, guidance and laboratory space. To synchronously record red and green fluorescence at high frame rate, we used an OptoSplit II (Cairn Research) optical splitter. A custom Competing interests microscope filter cube was fitted with a dual band (green and red) dichroic The authors declare no competing or financial interests. (Chroma Technology, 59022bs) and a dual band (blue and yellow) excitation filter (Chroma Technology, 59022x). The optical splitter was Author contributions Conceptualization: R.J.W.; Methodology: Z.W., T.B., R.J.W.; Software: R.J.W.; fitted with a filter cube with a 565 nm dichroic filter (Chroma Technology, Validation: Z.W.; Formal analysis: Z.W., T.B., E.G., R.J.W.; Investigation: T.B., T565lpxr) and green reflected and red transmitted light filters (Chroma R.J.W.; Resources: Z.W.; Data curation: R.J.W.; Writing - original draft: R.J.W.; Technology, ET520/40 m and ET632/60 m). When using white epi- Writing - review & editing: Z.W., T.B., E.G., R.J.W.; Visualization: R.J.W.; illumination, this results in the splitter projecting a green and red Supervision: E.G., R.J.W.; Project administration: R.J.W.; Funding acquisition: fluorescence image onto two halves of a single camera, a Neo 5.5 E.G., R.J.W. Journal of Cell Science

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Funding Geyer, V. F., Sartori, P., Friedrich, B. M., Jülicher, F. and Howard, J. (2016). R.J.W. and Z.W. were supported by the Wellcome Trust (211075/Z/18/Z, 104627/Z/ Independent control of the static and dynamic components of the chlamydomonas 14/Z). E.G. was supported by the Royal Society (UF100435 and UF160661). T.B. flagellar beat. Curr. Biol. 26, 1098-1103. doi:10.1016/j.cub.2016.02.053 was supported by the Medical Research Council (15/16_MSD_836338). Open Gheiratmand, L., Brasseur, A., Zhou, Q. and He, C. Y. (2013). Biochemical access funding provided by University of Oxford. Deposited in PMC for immediate characterization of the bi-lobe reveals a continuous structural network linking the release. bi-lobe to other single-copied in Trypanosoma brucei. J. Biol. Chem. 288, 3489-3499. doi:10.1074/jbc.M112.417428 Gibbons, I. R. (1961). The relationship between the fine structure and direction of Supplementary information beat in gill cilia of a lamellibranch mollusc. J. Cell Biol. 11, 179-205. doi:10.1083/ Supplementary information available online at jcb.11.1.179 https://jcs.biologists.org/lookup/doi/10.1242/jcs.246637.supplemental Halliday, C., Billington, K., Wang, Z., Dean, S., Sunter, J. D. and Wheeler, R. J. (2018). Cellular landmarks of Trypanosoma brucei and Leishmania mexicana. Peer review history Mol. Biochem. Parasitol. 230, 24-36. doi:10.1016/j.molbiopara.2018.12.003 The peer review history is available online at Hendrickson, T. W., Goss, J. L., Seaton, C. A. and Rohrs, H. W. (2013). The https://jcs.biologists.org/lookup/doi/10.1242/jcs.246637.reviewer-comments.pdf IC138 and IC140 intermediate chains of the I1 axonemal dynein complex bind directly to . Biochim. Biophys. Acta 1833, 3265-3271. doi:10.1016/j. References bbamcr.2013.09.011 Heuser, T., Barber, C. F., Lin, J., Krell, J., Rebesco, M., Porter, M. E. and Bastin, P., Pullen, T. J., Sherwin, T. and Gull, K. (1999). Protein transport and Nicastro, D. (2012). 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