# A novel approach to tagging tubulin reveals MT assembly dynamics of the axoneme in Trypanosoma brucei

Daniel Abbühl, Martina Pružincová, Luděk Štěpánek, Eleonore Bouscasse, Rita Azevedo, Mariette Matondo, Vladimir Varga, Serge Bonnefoy, Philippe Bastin

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## To cite this version:

Daniel Abbühl, Martina Pružincová, Luděk Štěpánek, Eleonore Bouscasse, Rita Azevedo, et al.. A novel approach to tagging tubulin reveals MT assembly dynamics of the axoneme in Trypanosoma brucei. Journal of Cell Science, 2025, 138 (20), pp.jcs.264145. 10.1242/jcs.264145. pasteur-05101008

HAL Id: pasteur-05101008

https://pasteur.hal.science/pasteur-05101008v1

Submitted on 6 Jun 2025

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# A novel approach to tagging tubulin reveals MT assembly dynamics of the axoneme in *Trypanosoma brucei*

Daniel Abbühl¹,², Martina Pružincová³,‡, Luděk Štěpánek³, Eleonore Bouscasse⁴,
Rita Azevedo⁴, Mariette Matondo⁴, Vladimir Varga³, Serge Bonnefoy¹, Philippe Bastin¹,*

¹Trypanosome Cell Biology Unit, Institut Pasteur, Université de Paris Cité, INSERM U1201, Paris, France

²Sorbonne Université, école doctorale complexité du vivant, ED 515, 7, quai Saint-Bernard, case 32, 75252 Paris Cedex 05, France

³Laboratory of Cell Motility, Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 14220, Prague, Czech Republic

⁴Institut Pasteur, Proteomics Core Facility, Mass Spectrometry for Biology unit, UAR CNRS 2024, Université Paris Cité, Paris, France

**Keywords:** Trypanosoma brucei, tubulin, flagellum, Grow-and-Lock model, CEP164C

## Abstract

The protozoan parasite *Trypanosoma brucei* assembles a new flagellum while maintaining the existing one in the same cell. Our group proposed a model where the mature flagellum is locked after construction to full length (Bertiaux et al. 2018). To test this hypothesis directly, we monitored flagellum assembly dynamics by inducible expression of tubulin, marked with an intragenic tag. We found that addition of new tubulin occurs at the distal flagellum tip at a linear rate and is indeed restricted to the new flagellum in bi-flagellated cells. Depleting the locking protein CEP164C prior to induction resulted in simultaneous integration of new tubulin in both flagella. This is direct evidence that trypanosomes avoid competition between the two flagella by allowing tubulin incorporation only in the new organelle. However, by tracing flagella over several cell cycles we also found that flagella do not remain locked forever. An orthogonal approach with HaloTag-tagged radial spoke protein 4/6 supported these findings. Since flagellum length in trypanosomes is stable, this indicates regular events of transient disassembly, followed by assembly, of the entire axoneme at the distal tip.

<footnote>*Corresponding author: pbastin@pasteur.fr</footnote>

<footnote>†Present address: Department of Molecular Life Sciences, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland</footnote>
Summary statement

The protozoan parasite *Trypanosoma brucei* assembles a new flagellum while maintaining an existing one in the same cell. A model was proposed whereby the mature flagellum would be locked after construction and so could not elongate. To test this directly, the authors broke a 25-year-old technological barrier by successfully tagging trypanosome tubulin, allowing them to monitor flagellum assembly dynamics following inducible expression. They discovered that addition of new tubulin occurs at the flagellum tip at a linear rate and is indeed restricted to the growing flagellum in bi-flagellated cells. Depleting the locking protein CEP164C resulted in simultaneous integration of new tubulin in both flagella, but with a preference for the new one. Moreover, by tracing tagged tubulin over several cell cycles, the authors found that flagella do not remain locked forever, suggesting regular events of transient disassembly in locked flagella that need to be compensated after cell division. An orthogonal approach with HaloTag-tagged radial spoke protein 4/6 supported these findings. Therefore, this work combines technological and conceptual advances that could be of interest to people studying microtubules and organelles.

**Introduction**

The flagellum is a membrane wrapped cell protrusion involved in motility, cell morphogenesis and signaling, that is found throughout the eukaryotic lineage. The structural core called the axoneme is made from microtubules (MT), formed by tubulin dimers. In addition to tubulins, the flagellum contains hundreds of other proteins (Billington et al., 2023; Pazour et al., 2005; Subota et al., 2014). Structure, molecular composition and many processes required for its assembly and maintenance, are highly conserved. As ribosomes are absent from the flagellum, its components are synthesized in the cell body, actively transported along the flagellum and then incorporated at the distal tip (Johnson and Rosenbaum, 1992). This is facilitated by protein complexes that carry cargo towards and back from the flagellum tip. This intraflagellar transport (IFT) is driven by kinesin and dynein motors (Kozminski et al., 1995, 1993).

Despite the common basic architecture, cilia and flagella exhibit variability in structure and
length, in relation to their function. Flagellum length can substantially vary between species
or even cell types. Several models have been proposed to explain how cells control flagellum
length. At their heart, they aim to describe how cells manage the availability, transport and
incorporation of the flagellum's most important building component, tubulin. Alpha and beta-
tubulin are globular proteins that form heterodimers which are assembled into filaments that get laterally connected to form the hollow tubes called microtubules (MTs) (Nogales et al., 1998; Tilney et al., 1973). The flagellum extends by the addition of new tubulin at the distal tip (Johnson and Rosenbaum, 1992), or shortens when tubulin building blocks are removed. The balance-point model describes how the interplay between assembly and disassembly rates governs flagellum length. If the assembly rate exceeds the dis-assembly rate, the flagellum elongates, contrarily shortening occurs when the assembly rate is slower (Marshall and Rosenbaum, 2001) and at equilibrium, flagellum length is stable ("balance point"). The green algae *Chlamydomonas reinhardtii* regulates flagellum length by maintaining a balance between a constant dis-assembly rate and a variable assembly rate, ranging between 9 – 24 µm/h in elongating flagella (Flavin and Slaughter, 1974; Rosenbaum et al., 1969). Another model postulates that the length of flagella can be regulated by limiting the pool of building blocks available for elongation (limited pool model). Once a cell runs out of free tubulin, elongation halts and flagellum length is stable. This mode of assembly is employed for example by *Naegleria fowleri* (Fulton and Walsh, 1980; Goehring and Hyman, 2012). Uncovering the timing and the rate of tubulin addition in the flagellum is therefore crucial to understand how cells regulate flagellum length (Hao et al., 2011; Marshall and Rosenbaum, 2001). Here we investigated the regulation of flagellum length in *Trypanosoma brucei*. Every life cycle stage of this protozoan parasite is flagellated and has a characteristic flagellum length (Rotureau and Van Den Abbeele, 2013). *T. brucei* poses as a formidable model organism to study flagellum assembly due to its exceptional amenability for reverse genetics, relative ease of culturing, conserved IFT machinery and the fully sequenced genome (Langousis and Hill, 2014). The protein content (~750 proteins) (Subota et al., 2014) and protein localization inside the flagellum are well understood (Billington et al., 2023; Dean et al., 2017; Sunter et al., 2023).

Alongside the axoneme, the trypanosome flagellum contains the paraflagellar rod (PFR), a paracrystalline network, attached to axonemal microtubule doublets 4 and 7. Due to the coordinated assembly with the axoneme, the PFR has served as a model for studying flagellum dynamics. (Alves et al., 2020; Bastin et al., 1999, 1998; Hughes et al., 2012).

The *Trypanosoma brucei* flagellum is attached to the cell body along almost its length, apart from the distal tip. Before cell division, trypanosomes assemble a new flagellum (NF), while maintaining the length of the old flagellum (OF) at ~20µm. When the NF reaches ~80% of the OF's length, the cell divides, with each daughter inheriting one flagellum (Abeywickrema et al., 2019). The NF keeps elongating post cell division to ultimately reach the full length (Robinson et al., 1995). To explain how growth is restricted to one flagellum despite stemming for the same cytoplasm, (Bertiaux and Bastin, 2020) proposed the Grow-and-Lock
model postulating that the OF becomes "locked" at full length, preventing further elongation or shortening.

A first insight into the molecular mechanism governing this process has been revealed with the discovery of the CEP164C protein. It is found at the transition fibers that connect the basal body to the flagellar pocket membrane. Upon knockdown of CEP164C, OFs of bi-flagellated cells are abnormally long (up to 30 µm) while NFs are made too short. As CEP164C is exclusively found at the base of the OF, the excessive elongation can be explained by the removal of the locking mechanism conferred by CEP164C. As this occurs at the cost of NF elongation, the lock seemingly prevents competition between the two organelles (Atkins et al., 2021).

The Grow-and-Lock model is supported by indirect evidence: reducing IFT frequency via RNAi against IFT kinesins 2A/B resulted in shorter flagella (Bertiaux et al., 2018), but restoring IFT did not increase flagellum length. Additionally, in cells where cell division is blocked, NFs reach 100% of the parental length but do not exceed it, suggesting trypanosomes halt flagellum elongation at a certain point (Bertiaux et al., 2018).

To test this model directly, we wanted to study if incorporation of new building blocks is prevented in the OF by a locking mechanism and if OFs stay locked indefinitely, by monitoring tubulin incorporation and turnover. Tubulin dynamics have been investigated in the past for example in sensory cilia of Caenorhabditis *elegans* by fluorescently tagging different tubulin isoforms and then monitor their trafficking after photobleaching (Hao et al., 2011). The role of IFT in tubulin turn over has been also described in *C. reinhardtii* by mating HA-tubulin expressing cells with non-expressers and then selectively tracking the incorporation in previously unlabeled flagella (Marshall and Rosenbaum, 2001). Other research in *Chlamydomonas*, addressed the dynamics of diffusion and active transport during flagellum growth and maintenance by utilizing GFP-tagged tubulin (Craft et al., 2015). In *T. brucei*, the YL1/2 antibody, which detects tyrosinated alpha-tubulin, has been so far the best tool for tracking newly assembled microtubules (Kilmartin et al., 1982; Sherwin et al., 1987). However, its use is limited by the short time window between tubulin polymerization and its de-tyrosination (Sherwin et al., 1987; van der Laan et al., 2019). For long-term tracking over multiple cell cycles (~9 hours each), developing an inducible system for tagged tubulin expression is essential. Unfortunately, tagging tubulin in *T. brucei* has proven challenging, with most tagged tubulin remaining in the soluble pool and not being incorporated into microtubules (Sheriff et al., 2014).

We have now overcome this problem by tagging *T. brucei* alpha-tubulin with an internal tag inside the acetylation loop situated within the lumen of the microtubule (Sirajuddin et al., 2014; Soppina et al., 2012). After inducible ectopic expression, the incorporation of tagged tubulin could be followed over different periods of time in NFs and OFs during the cell cycle.
These experiments demonstrated that tubulin is incorporated at the distal tip of growing flagella at a linear rate and that tubulin integration in bi-flagellated cells indeed follows the locking model. Furthermore, we could corroborate the importance of CEP164C for establishing the locking mechanism. In addition, we managed to track flagella over several cell cycles after their emergence, revealing novel aspects of tubulin incorporation in monoflagellated cells. Moreover, we reached a similar conclusion using an orthogonal approach (a Halo-tag system) for another axonemal constituent, the radial spoke protein RSP4/6.

## Results

### Tagged tubulin is incorporated into MTs of the cell body, flagellum and mitotic spindle

Alpha-tubulin was tagged with one or two copies of the Ty-1- epitope (a sequence of 10 amino acids derived from a yeast viral particle) (Bastin et al., 1996) inside the acetylation loop of alpha-tubulin (Schatz et al., 1987; Sirajuddin et al., 2014; Soppina et al., 2012). Fig. S1 illustrates the insertion of the Ty-1-epitope, one amino acid after the acetylated lysine (Fig. S1A, K40 in cyan). In the diploid *T. brucei* genome, alpha and beta-tubulin are found in ~20 alternating, identical tandem repeats. First, the tagging strategy was tried by *in situ* tagging alpha-tubulin at one of the ~40 loci (Berriman et al., 2005; Ersfeld et al., 1998; Seebeck et al., 1983) using *in situ* recombination (Kelly et al., 2007). This was done with the endogenous alpha-tubulin UTR to attribute a potential lack of incorporation solely to the presence of the tag. The tagged protein will be referred to as Ty-1-tubulin. Fig. 1A depicts immunofluorescence assays (IFA) with an *in-situ* Ty-1-tagged alpha tubulin cell line. To test whether Ty-1-tubulin was integrated into MTs, we subjected cells to detergent treatment, which retains components integrated into the cell cytoskeleton (Sherwin and Gull, 1989). Detergent-extracted cytoskeletons were co-stained with antibodies recognizing the Ty-1 tag (BB2) and alpha-tubulin (TAT-1). Ty-1-tubulin is found in the entire cell body, the NF as well as the OF across all cell cycle stages, a localization that matches the one revealed by the TAT-1 antibody (Fig. 1A). Furthermore, tagged tubulin could be observed in the mitotic spindle (Fig. S2A).

Western blotting analysis with the BB2 antibody revealed a single band that migrates slightly above the 50 kDa marker (Fig. S2D). In all clones tested, alpha-tubulin tagged with two epitopes migrated slightly but discernably slower, despite being only 1kDa heavier. To confirm cytoskeletal association, samples of whole cells, cytoskeleton extracts and detergent-soluble fraction were run on an SDS-PAGE gel and membranes were stained with the TAT-1 antibody (Fig. S2 E-G). In the WT sample, alpha-tubulin was found predominantly in the cytoskeletal fraction, with only a minimal amount found in the soluble fraction. In the
tagged cell lines, Ty-1 containing alpha-tubulin could be discriminated from the endogenous alpha-tubulin (expected size of alpha tubulin: ~49.8kDa) as an extra band detected by TAT-1 (Fig. S2 E-G), despite being only 1kDa heavier. The apparent size difference of endogenous versus tagged tubulin was more pronounced in samples with two Ty-1-epitopes (Fig. S2 D-G). The distribution of the tagged tubulin between the soluble and detergent resistant fractions matched the one observed for endogenous protein. These results prove that cells readily incorporated alpha-tubulin that contains a Ty-1-epitope inside the acetylation loop into MTs of the cell body, mitotic spindle and the flagellum making it a suitable tubulin tag in *T. brucei*.

To modulate the timing of gene expression, we then developed a cell line that allowed inducible ectopic expression of Ty-1-tubulin (Fig. S1D), which is induced by the addition of tetracycline (Wirtz and Clayton, 1995).

Figures 1 and 2 depict a set of validation experiments performed with the derived cell line. Whole cells were grown in the absence (Fig. 1A, non-induced, upper panel) or in the presence of 1µg/ml tetracycline for 16 h (Fig. 1A, induced, lower panel) and treated for IFA with the BB2-antibody as well as DAPI. In the absence of tetracycline, there is no signal in almost all cells (Fig.1A, top right). By contrast, in induced cells tagged tubulin is observed in the entire cell body (Fig. 1B) and the flagellum (Fig. 1B, white arrows).

To evaluate if the inducible expressed Ty-1-tubulin remains associated to the cytoskeleton, cell fractionation was performed and analysed by western blotting. In Fig. 1C, two membranes are shown with samples from the wild type cell line (WT), from the non-induced control (-tet), and from cells that were grown for 16 hours with tetracycline (+tet). With the BB2 antibody, no signal is detected for WT (Fig. 1C, upper, WT) or non-induced samples (Fig. 1C, upper, -tet). In the induced samples (Fig. 1C, upper, +tet), intense bands are visible for the whole cell as well as the cytoskeleton fraction and a band of relatively lower intensity is visible for the soluble pool fraction, as expected (Sherwin et al., 1987). All bands migrated slightly above the 50kDa band of the marker. In the blot stained with the alpha-tubulin antibody, (Fig. 1C, TAT-1) a band that migrated at the size of the 50kDa-marker is visible for all three fractions of all conditions, with the band in the soluble protein fraction being fainter, as expected. Under induced conditions (Fig. 1C, lower, +tet) an additional set of bands is visible slightly above the 50kDa marker, which corresponds to Ty-1-tubulin. Importantly, their distribution profile is comparable with that of untagged tubulin, confirming that the majority of Ty-1-tubulin had integrated into MTs and behaves as endogenous tubulin (Fig. 1C). Finally, this ectopic expression of Ty-1-tubulin does not affect cell growth even when cells were induced for several days (Fig. 1D).

To complement the data obtained from western blots, Fig. 2 depicts two IFA experiments with cells that were harvested 24 h after the addition of tetracycline. Whole cells were treated
with 0.5% Nonidet P-40 to obtain detergent extracted cytoskeletons and processed for IFA
with the BB2 antibody in combination with the mAb25 antibody (anti-TbSAXO protein), which
is a marker of the axoneme (Dacheux et al., 2012), and the anti-FTZC antibody (Flagellum
Transition Zone Component), a marker of the flagellum base (Bringaud et al., 2000). Ty-1-
tubulin remained associated to the cytoskeleton (Fig. 2A). To better visualize the flagellar
skeleton, cytoskeletal extracts were further treated with 1 M NaCl, which de-polymerizes the
MTs of the corset while leaving the ones of the axoneme intact (Robinson et al. 1990). Ty-1-
tubulin is indeed associated to the flagellum throughout its length, further confirming its tight
incorporation (Fig. 2B). A minority of cells did not show any BB2 signal, indicating that they
did not express Ty-1-tubulin and some flagella remained unstained (Fig. 2B, asterisk). This
could be a consequence of the semi-conservative mode of flagella inheritance (Sherwin and
Gull 1989), but a low proportion of cells remained negative even after longer induction times
(see below). We also noted some heterogeneity in signal intensity between cells. As we did
not observe this when Ty-1-tubulin was constitutively expressed, this is most likely the
consequence of increased Ty-1-tubulin levels with time elapsed after induction.

Lastly, we investigated tagged alpha-tubulin for the presence of classic post-translational
modifications, especially acetylation, since the Ty-1 tag is positioned very close to the
acetylated lysine K40 (Fig. S3A). To assess the acetylation status of Ty-1-tubulin, protein
lysates of a 24h-induced culture were compared to a non-induced control using western
blotting. Membranes were stained with TAT-1 and YL1/2 (tyrosinated alpha-tubulin) and both
revealed the presence of endogenous and tagged tubulin (arrows in Fig. S3A). Contrarily, the
two antibodies that recognize acetylated alpha tubulin (C3B9 and 6-11B-1 (Piperno and
Fuller, 1985; Woods et al., 1989)) only detected the endogenous tubulin (Fig. S3A).
According to literature, however, virtually all incorporated tubulin is acetylated (Schneider et
al., 1997). As Ty-1-tubulin behaves in other respects indistinguishable from untagged tubulin,
the absence of this modification was unexpected. We reasoned that the insertion of the tag
just one amino acid after lysine 40 could destroy the binding epitopes of C3B9 and 6-11-B1.
Therefore, the Ty-1-tubulin cell line was induced for 7 days, cells were harvested and their
flagellar skeletons isolated by detergent treatment followed by exposure to 1 M NaCl as
above (Robinson et al., 1991). These isolates were then subjected to analysis by mass
spectrometry after digestion with AspN. Thanks to the close presence of the tag (Fig. S3B),
peptides derived from Ty-1-tagged and endogenous tubulin can be discriminated. These
experiments detected acetylated peptides (K40) on peptides derived from both endogenous
and tagged tubulin (four replicates shown in Fig. S3C), confirming that Ty-1-tubulin is
acetylated and detection by antibodies likely failed due to the alteration of their binding
epitope.
Lastly, the timing of Ty-1-tubulin expression upon tetracycline addition was evaluated by
western blotting, revealing that it could be observed as early as one hour post induction, with
a rapid increase over time (Fig. S4A-B). On the blot stained with TAT-1, the bands
corresponding to the soluble pool of Ty-1-tubulin are barely visible when compared to the
ones of endogenous tubulin below, indicating that the majority of tagged protein is found in
the cytoskeleton fraction and therefore incorporated into MTs, even at early time points of
induction. As a full cell cycle lasts around 8-9 h, this is sufficient to measure assembly
dynamics of the axoneme during NF construction. We conclude that we have succeeded to
tag alpha-tubulin in a way it gets properly incorporated into all the MTs of trypanosomes.
Combined with the rapid expression provided by the inducible system, this offers the
possibility to directly monitor tubulin incorporation into trypanosome flagella for the first time.

Tagged tubulin is distally integrated into the axoneme at a linear rate

The assembly rate of the *T. brucei* axoneme has so far been estimated indirectly, based on tubulin de-tyrosination (Sherwin and Gull, 1989), cell cycle duration (Woodward and Gull, 1990) or the measurement of an extra-axonemal component (Bastin et al., 1999). To analyse the kinetics of tubulin integration into the axoneme, we performed time courses by inducing expression of Ty-tubulin and taking samples at various points after induction.

At first, Ty-1-tubulin expression was induced for short periods of 2-4 hours and incorporation
was monitored at the level of the entire cell. From previous studies, it is known that newly
synthesized tubulin incorporates into subpellicular microtubules at the posterior cell body end
(Sheriff et al., 2014; Sherwin et al., 1987) and the same pattern was observed upon short
induction times of Ty-1-tubulin (Fig. S5A). In addition, newly synthesized Ty-1-tubulin is
clearly present in the growing flagellum (Fig. S5A, arrows) and in the mitotic spindle (Fig.
S5A, stars). To visualize flagella more clearly, subpellicular MT were depolymerised with 1 M
NaCl treatment (Fig. S5B). Conveniently, procyclic trypanosomes possess a structure called
the flagella connector (FC), that links the tip of the NF axoneme to the side of the OF
axoneme (Briggs et al., 2004). The FC is resistant to detergent and 1 M NaCl treatments
upon which the connection of the NF to the OF is maintained (Moreira-Leite et al., 2001)(Fig.
3A). To highlight the proximal end of the axoneme, the transition zone marker FTZC was
used. It can be observed as a dot that marks the very proximal portion of the flagellum. The
new flagellum is found in a posterior position, is shorter and is connected at its tip to the OF
axoneme. In these flagella isolates, Ty-1-tubulin is found integrated exclusively in the distal
portion of the new flagellum. This is a direct proof that the addition of the new tubulin occurs
at the distal tip of a growing flagellum in *T. brucei*.

As Ty-1-tubulin localization after tetracycline addition followed the expected patterns, we
were able to determine the kinetics of Ty-1-tubulin integration into the axoneme of the new
flagellum during its assembly. This was done by measuring the length of flagellar segments that had incorporated Ty-1-tubulin after different periods of induction. Because trypanosomes divide asynchronously and all cell cycle stages are present in a culture (Sherwin and Gull, 1989; Woodward and Gull, 1990), we focused our measurements on cells with a new flagellum that had integrated Ty-1-tubulin in a distal segment. Time courses were performed, where tagged tubulin expression was induced between 2-4 hours and samples were then taken for analysis every 30 minutes. We decided to analyze the assembly rate with 1 M NaCl-extracted flagella, as tubulin signal in flagella is sometimes hard to distinguish from signal of the MTs in the cell body.

A cartoon of flagella of a cell that had started assembly of its new flagellum before addition of tetracycline is depicted in Fig. 3A, 0 h. Once Ty-1-tubulin is produced, it is added at the distal tip of the growing NF (Fig. 3A, 2-4 h, Fig. S5B). Individual examples are shown for each timepoint in Fig. 3B, the measured Ty-1-tubulin segment is indicated by a white bracket. A gradient of increasing intensity of Ty-1-tubulin signal towards the distal tip was noted. This was expected as Ty-1-tubulin becomes relatively more abundant during the course of induction (Fig. S4, Fig. 3B).

The length of the Ty-1-tubulin containing segment of these NFs was measured and the data of three separate experiments (70 - 100 flagella per timepoint and experiment, ~1500 total) was pooled (Fig. 3C). A linear regression was performed for the five timepoints (2-4 hours, 30-minute intervals). This revealed that tagged tubulin was added to the distal tip of NF at a linear rate of ~2.9 µm/h. The linear regression line in Fig. 3C was corrected by subtracting 45 minutes from each timepoint due to the previously reported delay between addition of tetracycline and the expression of the respective protein (Wirtz and Clayton, 1995). We conclude that the flagellum in procyclic trypanosomes is constructed at a constant rate and that our expression system is a suitable tool to dynamically track flagellum assembly during the cell cycle.

Old flagella of bi-flagellated cells do not incorporate tagged tubulin at early timepoints

We next investigated in which flagella tagged tubulin had incorporated during 2-4 hours of induction. According to the Grow-and-Lock model, OF are supposedly locked and not expected to integrate Ty-1-tubulin (Bertiaux et al., 2018; Bertiaux and Bastin, 2020). To test this, cells were induced for 2-4 h and harvested every 30 minutes. Flagella were then isolated as above and bi-flagellated cells analysed for the presence of integrated Ty-1-tubulin in their flagella. When investigating early timepoints (2 h and 2.5 h post induction), Ty-1-tubulin integration was observed exclusively in the NF (Fig. 4A). After 3-4 h, increasingly more bi-flagellated cells were observed where Ty-1-tubulin had integrated in the OF as well (Fig. 4B, C). These cells could be distinguished by several characteristics: 1) the portion at
the distal tip of the OF was always short (~1 µm in length); 2) the NF was relatively short (~2-5 µm) (Fig. 4D) and 3) the NF was always labelled with Ty-1-tubulin along its entire length. Notably, cells with fully labeled NF longer than 5 µm were present, but Ty-1-tubulin was absent in their OF.

When we analysed cells where Ty-1-tubulin had integrated only in the distal portion of the NF (Fig. 3 and 4E), the corresponding OF was always devoid of tagged tubulin (Fig. 4F) at all timepoints. The proximal part of their NF not containing tagged tubulin indicates that these NF had emerged before the induction. Since these cells were biflagellate at the point of their fixation, any incorporation of Ty-1-tubulin in the OF would therefore need to happen during NF construction. Simultaneous incorporation in both organelles is not compatible with the idea of a locked OF. This was never observed, providing the first direct evidence that OF are locked.

One explanation why we observed cells with Ty-1-tubulin in the distal part of their OF (and a short NF containing tagged tubulin along its entire length) (Fig. 4B-D) is that these distal parts correspond to the last elongation step of a NF formed in previous cell cycle. Indeed, assembly of the flagellum in the daughter cell is only completed after cell division (Abeywickrema et al., 2019; Bertiaux et al., 2018; Bertiaux and Bastin, 2020; Farr and Gull, 2009). We investigated the exact origin of this signal and what it might represent further in the section "Old flagella frequently integrated tagged tubulin at the distal tip after cell division".

## Tubulin is integrated in both flagella when CEP164C is depleted

As a next step we tested if we could correlate these findings to the presence of the locking protein CEP164C. For this purpose, we employed the same CEP164C RNAi cell line that was used by Atkins and colleagues (Atkins et al., 2021) to express Ty-1-tubulin in the absence of CEP164C. Here, we relied on transient episomal expression of Ty-1-tubulin to circumvent the limitation that expression of double stranded RNA against CEP164C is also induced by tetracycline. First, RNAi against CEP164C was induced for 48 h, cells were then harvested and subjected to transfection with the circular 1xTy-1-tubulin pHD430 plasmid. After 4 h, cells were harvested and isolated flagella analyzed for the presence of Ty-1-tubulin. First, we analysed cells that were only subjected to transient transfection (-tet), and noted that Ty-1-tubulin was expressed, albeit in a low number of cells than in the cell lines expressing Ty-1-tubulin from a construct integrated in the genome (Fig. S6). Nonetheless, the incorporation matched with was observed for inducible expression of Ty-1-tubulin after 3 - 4 h (see Fig. 4). Most importantly, when distal integration was observed in the NF, the corresponding OF was always devoid of Ty-1-Tubulin (Fig. 5A) in agreement with Fig. 4F.
Next, we analysed flagella from cells that were subjected to tetracycline for 48 h prior to transfection. We immediately noted the presence of longer than usual OF, reaching up to 30µm in length, indicating that CEP164C depletion took effect, consistent with previous observation (Atkins et al., 2021).

Focusing on cell where the NF had incorporated Ty-1-tubulin distally (Fig. S6, white arrows) we now frequently observed that the OF had done so as well (Fig 5A). This supported the role of CEP164C in preventing simultaneous access to both flagella. In addition, we could derive two more observations: First, even in the absence of CEP164C, NF elongation occurred at a faster rate, as the Ty-1-tubulin segment in the NF was significantly longer compared to the one in the OF (Fig. 5B). Secondly, there was a negative correlation between the length of the OF and the length of its Ty-1-tubulin containing region (Fig. 5C).

With the above observations, we provide the first direct evidence that OF are sealed from tubulin incorporation and can corroborate the important role of CEP164C in the locking mechanism.

Old flagella frequently integrated tagged tubulin at the distal tip after cell division

In Fig. 4B we described how Ty-1-tubulin had integrated in the distal tip of the OF. We hypothesized that this segment in the OF could correspond to the last elongation step of a NF formed in the preceding cell cycle. If this is true we expected to see segments with increased length when cells were induced for longer. The Ty-1-tubulin cell line was therefore incubated for 9 h with tetracycline, cells then harvested and 1 M NaCl extracted flagella were stained with BB2, Mab25 and α-FTCZ as above. Fig. 6A illustrates a 9-hour induction as predicted from the Grow-and-Lock model. As this time span is equal to ~1 cell cycle, we assume that all flagella at induction start (0 h) will become OF at harvest (9 h). More precisely, 50% of these flagella will be an OF for the first time and 50% will correspond to OF from earlier generations and should therefore be locked.

After 9 h of induction, Ty-1-tubulin is incorporated along the entire length of the NF in biflagellate cells (compare Fig. 3 and 4E). As expected, distal incorporation is now observed in OF. In a first category of cells, the Ty-1-tubulin containing segment displays a clear gradient of increasing intensity towards the tip (Fig. 6B, type 1). We attribute this to the increasing relative amount of Ty-1-tubulin over the course of flagellar elongation, similar to the NF described in Fig. 3. These observations are consistent with the idea that these OF at harvest were NF at the beginning of induction (Fig. 6A). The Grow-and-Lock model predicts that 50% of OF should correspond to these type 1 flagella. However, only 30% of OF display this pattern (Fig. 6C). In a second type of cells, Ty-1-tubulin was entirely absent from the OF (type 2). Assuming the Grow-and-Lock model, this should be the case for all OF present at
the time of induction, due to them being locked, hence they should represent 50% of the population (Fig. 6A). Yet, only 33% of OF are devoid of Ty-1-tubulin (Fig. 6C).

Surprisingly, a third category of OFs was discovered in which Ty-1-tubulin is integrated only in the very distal tip. This Ty-1-tubulin containing segment has a mean length of ~1 µm (Fig. 6B, type 3) and the transition between it and the preceding segment made of untagged tubulin was abrupt (Fig. 6B type 3, white arrows) rather than in a form of a gradient, similar to the OF we observed after 3 – 4 h of Ty-1 tubulin induction (Fig. 4B). These type 3 cells represent 37% of the population (Fig. 6C). Assuming the Grow-and-Lock model and linear growth, the integration of 1 µm at the tip should correspond to the last ~5% of elongation (maximum length ~20µm). Hence, a much lower number of flagella with such a short segment is expected in the population. This is indicative of a distinct event that occurs more frequently.

As OF are locked and stop elongating before an NF assembly is initiated, we tested if there is a relationship between the Ty-1-tubulin segments in the two flagella. For each cell, the length of the Ty-1-tubulin containing segment in the OF (Fig. 6D, segment 2) was plotted against the length of the NF (Fig. 6D segment 1). A clear negative correlation can be observed (Fig. 6D, green dots, segment 1 vs. 2). Additionally, the average cumulative length of the NF and the labeled segment in the OF was ~18 µm. This means that an average cell spent approximately 6 hours assembling a NF at a linear assembly rate of 3 µm/h (~3 µm/hour = ~18 µm), including the elongation that occurs after cytokinesis. The total length of the OF (Fig. 6D, segment 3) however did not show a correlation with that of the NF (Fig. 6D, segment 1).

The question when new flagella reached their final full length after cell division remains open (Farr and Gull, 2007). To assess if 1st-gen OF (type 1) have reached full length before the bi-flagellate stage, their length was compared to the length of the two other types of OF (Fig. 6E). On average the length of type 1 OF is slightly but significantly (ordinary one-way Anova) shorter when compared to OF that had integrated Ty-1-tubulin at the distal tip (type 3). This length difference of ~0.8 µm was similar to the average length of the green tip (~1 µm, Fig. 6B, type 3, right panel).

As assembly of NF is linear (at least during the bi-flagellated stage, Fig. 3C), the length of the NF is a reliable indicator of how advanced an individual trypanosome is in its cell cycle (Robinson et al., 1995). Fig. 6F shows the average length of the NF in cells sorted by the type of their corresponding OF. As seen in Fig. 6D, type 1 cells possess a NF of any length, representing a normal distribution (Fig. 6F). This is expected as NF length at any given point in time in an exponentially growing culture follows Gaussian distribution (Woodward and Gull, 1990), in agreement with the linear growth rate measured here. In OF where no tagged tubulin integrated (type 2), the corresponding NF was on average significantly longer
(ordinary one-way ANOVA), which indicates that integration might be linked to an event at
the very beginning of G1 after cytokinesis. NF length of type 3 flagella was broadly
distributed but nonetheless tending to be shorter compared to NFs in cells type 1 and type 2
OFs.

Together, these results suggest that integration of tagged tubulin in the distal OF tip occurs frequently but is restricted to a certain window in the cell cycle. Judging by the distribution of NF lengths in type 2 (absence of incorporation) and type 3 (presence), this might occur early on in G1-phase.

Integration of Ty-1-tubulin during the G1-phase

In the previously described experiments, we observed that ¹st-genOF (type 1, Fig. 6) are on average slightly shorter compared to the other two types (Fig. 6E) and that ~37% of OF had integrated Ty-1-tubulin only at the very distal tip (Fig. 6C, type 3). It is conceivable that this integration corresponds to a distinct elongation step that ¹st-genOF (type 1) would have to undergo in order to reach maximum length. Another possibility is that the integration at the tip corresponds to a more frequently occurring event, that happens in all flagella, presumably when cells are monoflagellated. This would entail that the locking mechanism is briefly removed to allow integration during G1-phase. It would then have to be re-applied before the cell initiates the assembly of a NF. This is coherent with the localization pattern of CEP164C, which is absent in 60% of monoflagellated cells (Atkins et al., 2021).

In the following experiments, we tracked flagella for longer periods to investigate if they were
subject to integration of tubulin over multiple cell cycles following their emergence. A pulse-
chase experiment was designed in which cells were initially pulsed with a 24-hour induction
with tetracycline corresponding to ~2-3 cell cycles and then subjected to a 48 h de-induction.
These pulse-chase experiments are presented in Fig. 7 (flagellar skeletons) and Fig. S8
(detergent extracted cytoskeletons).

After 24 h in tetracycline cells were harvested and whole cells, detergent extracted
cytoskeletons and flagella were isolated and subjected to IFA. The samples were stained
with BB2, Mab25, α-FTZC antibodies and DAPI as above. We observed that ~95% of cells
had integrated Ty-1-tubulin into MTs of the cell body or flagella and were therefore
responding to tetracycline (Fig. S7A).

In preparations of extracted flagella, we scored OFs of bi-flagellated cells and the single
flagellum of monoflagellated cells. We observed that a majority (~63%) of them had
integrated tagged tubulin along the entire length, 20% had incorporated Ty-1-tubulin at the tip
and ~11% showed a gradient (Fig. S7B). 7% of flagella did not incorporate any Ty-tubulin.
This is just marginally higher than the 5% whole cells not responsive to tetracycline,
indicating that ~98% of cells producing Ty-1-tubulin had indeed incorporated it into the flagellum after 24 h of induction.

Fig. 7A shows flagella extracts from a cell that had integrated Ty-1-tubulin along the entire length of the OF as well as the NF (Fig. 7A, +tet 24 h). These flagella that incorporated Ty-1-tubulin during the initial 24 h induction will be henceforth referred to as pulsed flagella. After 24-hour incubation with tetracycline, cells were thoroughly washed with medium devoid of tetracycline and then grown in the absence of tetracycline for 48 hours (5-6 cell cycles, "chase"). We then prepared samples for IFA and searched for flagella of cells that were still positive for Ty-1-tubulin, which they must have integrated during the initial 24 h induction. In bi-flagellated cells which expressed tagged tubulin, we found that Ty-1-tubulin was present in the OF but not the NF, as expected (Fig. 7A, middle panel, +tet 24 h, -tet 48 h). This pattern is the mirror image of a short induction (Fig. 4A, Fig. S5A), which indicates that the de-induction was successful. Strikingly, Ty-1-tubulin was absent from the distal tip in 95% (n=300) of chased OF (OF in Fig. 7A, middle panel). This meant that most flagella that had been constructed during the 24 h induction had integrated untagged tubulin at the distal tip during the 48 h washout period.

As a last step of this experiment, we subjected the cells that were grown in the absence of tetracycline for 48 h to another round of induction for 4 hours (re-induction). In the right panel of Fig. 7A a bi-flagellated cell from this timepoint is shown. It has a green OF due to the Ty-1-tubulin integration during the initial 24 h induction. The NF in this cell has integrated Ty-1-tubulin in its distal segment (see case 1 scenario 2 Fig. 4A), indicating its construction had been initiated before the re-induction, evident by the proximal untagged segment. This demonstrates that the chased cells were still capable of producing and integrating Ty-1-tubulin into the NF during the 4 h re-induction. However, the tagged tubulin was absent from the distal tip of the OF similar to what was observed 48 h post washout. This indicates that the OF is locked in these cells, again highlighting that simultaneous integration of tubulin in both NF and OF does not occur. After de-induction Ty-1-tubulin is absent in NF and the posterior MTs of the cell body (Fig. 7A, S8B, +24-48 h, magenta arrow) as well as the tip of the OF (magenta arrows). In bi-flagellated cells that were observed after re-induction, tagged tubulin was found integrated again in the NF as well as the MTs of the posterior cell body (Fig. 7A, S8C +24-48 h +4 h, green arrow).

From these experiments, we derived two important observations. First, 95% of chased OF that were labeled in the 24 h induction, had integrated untagged tubulin at their distal tip, indicating that incorporation occurs regularly, most likely in all flagella. Second, these experiments (Fig. 7A) confirm our previous observations that OF of bi-flagellate cells do not
incorporate tubulin at the same time as the NF (Fig. 4A), indicating that integration occurs
after cell division during G1-phase.

Therefore, mono-flagellated (G1-phase) cells with a green flagellum were analysed in the re-
induced conditions. Fig. 7B shows an example of a single flagellum in which multiple rounds
of integration are revealed. This flagellum most likely emerged shortly before the initial 24 h
induction (short untagged proximal segment, labeled “before”). It was then assembled for
most of its length in the presence of tetracycline (long green segment, “24 h induction”). A
short untagged portion then follows, which we attribute to the 48 hours in which the cells
were grown in the absence of tetracycline, post washout (“de-induction”). At the very distal
tip, following this untagged part, one can observe the integration of tagged tubulin again
(short green segment, “Re-induction”), corresponding to the 4 h re-induction. A detergent
extracted cytoskeleton of a mono-flagellated cell presenting a similar pattern is shown in the
right panel of Fig. S8D. Taken together, these experiments suggest that chased mono-
flagellated cells had integrated tubulin at the distal tip of their flagellum again during G1-
phase.

Above experiments suggest that integration of Ty-1-tubulin is observed frequently at the distal tip of OF and that this most likely occurs during the G1-phase, as it is not observed in OF of bi-flagellated cells.

Integration at the distal tip is also found for a HaloTag-tagged component of the axoneme

Next, we wanted to corroborate these findings and exclude that they are an artifact of tubulin overexpression due to the inducible system. Hence, we employed an orthogonal system that allowed for the dynamic tracking of other axonemal components. Radial spoke protein 4/6 was N-terminally *in situ* tagged with mNeonGreen followed by a HaloTag using the PCR - tagging approach (Dean et al., Open Biol 2015), generating a culture of cells all of which expressed the tagged protein. This was validated based on presence of the mNG signal, which serves as a general reporter for RSP4/6 in these cells, regardless of time of synthesis. Although the HaloTag itself is not fluorescent, the cells can be incubated with a fluorescently labelled ligand that covalently binds the tag (Dean et al., 2016; Los et al., 2008), enabling us to perform pulse chase experiments and hence trace populations of proteins synthesized in the cell at various time points.

The cells were incubated for 1 hour with TMR ligand that can conjugate with the HaloTag.
When imaged immediately after the unconjugated TMR ligand washout, both mNG signal
and TMR signal localized along all flagella from their proximal to distal ends (Fig. S9A),
demonstrating that the tagged protein efficiently binds the TMR-ligand. We then performed a
chase experiment, when the cells were grown in a medium for two hours after the ligand
washout followed by their fixation and imaging. Similarly, to the experiments with induction of
Ty-1-tubulin expression (Fig. 3B), we observed that the newly synthesized RSP4/6 was
incorporated into the distal portion of the new flagellum, which was manifested as a gradient
of decreasing TMR intensity towards the distal tip (Fig. 8A). The OF in these bi-flagellated
cells was fully labelled with TMR (Fig. 8A), supporting our earlier observations that the two
flagella do not compete for building blocks (case 1 scenario 2 in Fig. 4A). When cells were
fixed 4 hours after the ligand washout, bi-flagellated cells with a short NF and a decrease in
the TMR intensity at the distal tip of their OF were observed (Fig. 8B), resembling results of
4-hour induction of Ty-1-tubulin expression (Fig. 4C). Moreover, we noticed that in addition to
monoflagellated cells with a gradually decreasing TMR intensity along their flagellum, other
cells had a high TMR intensity along the flagellum with a sharp decrease in the distal part
(Fig. 8C, white arrows), mirroring the type 3 flagella in Fig. 8B.

Strikingly, comparing the number of fully labeled flagella after 4 h (~0.5 cell cycles, 36.5%) and after 16 h (~1.7 cell cycles, 0.7%) shows that almost no cells retain fully labeled flagella once they had passed the threshold of completing one cell cycle (~9 h) (Fig. 8D). When the distribution of different types of flagella after 24 h TMR washout was compared to the flagella type distribution after 24 h induction with the Ty-1-tubulin cell line, it revealed that corresponding flagella types are present in similar numbers in the two types of experimental systems (Fig. 8E).

Hence, we concluded that the results obtained using these two orthogonal systems, inducible
expression of Ty-1-tubulin and HaloTag-conjugated TMR ligand labelling of the radial spoke
protein RSP4/6 expressed from endogenous locus, are consistent. In both systems the
axonemal extension is restricted to the NF in bi-flagellated cells. However, after cells divide
and the two flagella are no longer competing, remodeling occurs at the distal tip in all flagella,
indicated by the disappearance of TMR-labeling, which is thereby a robust phenomenon.

Since flagellum length of *T. brucei* is remarkably stable, these results are only explicable with
the idea of cell cycle dependent dis-assembly and subsequent re-assembly of the distal part
of the axoneme.

Discussion

A fast response rate to tetracycline (~1h) and low level of leaky expression enabled us to
elucidate incorporation of tagged alpha-tubulin into the axoneme, with enough sensitivity to
determine the growth rate of the axoneme. Previously, the assembly rate of the flagellum has
been measured with an extra-axonemal structure, namely the PFR (Bastin et al., 1999). At a
speed of ~2.9 µm/h, the assembly rate of the axoneme measured by us using tagged tubulin
is similar, albeit a little slower, than the one shown for the PFR (~3.6 µm) (Bastin et al.,
1999). Similar to the PFR, we observed that the axoneme was built at a linear rate. The
delayed initiation of assembly of PFR in respect to the axoneme together with the
coincidental finish of their assembly is a possible explanation for the slightly higher rate at
which new PFR material is added. Alternatively, this difference may be the consequence of
variability between the two different cell lines in which the growth rates were measured.

In contrast to animal cells but like the vast majority of protists, trypanosomes maintain during
cell division a flagellum that was constructed in one of the previous cell cycles while
constructing a new one. This raises two questions: (1) how do they deal with flagella of
different age? (Bertiaux and Bastin, 2020) and (2) what is the fate of each flagellum? Thanks
to the data reported here, we can address both questions.

We can follow the fate of a flagellum in the models depicted in Fig. 9. First, we want to
illustrate flagellum assembly by assuming the Grow-and-Lock model for a flagellum that gets
locked and is then sealed from further tubulin incorporation forever (Fig. 9A). The assembly
of a NF, here represented with an orange line, occupies approximately 50-60% (~ 5 h) of the
cell cycle (Sherwin and Gull, 1989; Woodward and Gull, 1990). With an assembly rate of
~2.9 µm/h, this is sufficient for a flagellum to reach ~15 µm in length (~80% of full length).

The cell then divides and the NF is assembled to full length post division during G1-phase.
Based on our experiments in Fig. 6 we assume that elongation after cell division continues at
a linear rate and most likely concludes soon after cytokinesis and before the assembly of a
NF is initiated. (Abeywickrema et al., 2019). The flagellum then acquires CEP164C at its
base, before it becomes an OF for the first time, (Atkins et al., 2021) to establish the lock.
From that point on, it no longer elongates the axoneme (indicated by the red lock in Fig. 9).
OF length is maintained at ~20 µm and does not change, indicated by the straight orange
line. If these OF do not incorporate new building blocks for the rest of the lifespan of the cell,
their proportion in a culture at any given moment is at least 50% (OF from bi-flagellated cells
(~33.3%) plus locked flagella from monoflagellated cells (16.6%)).

This hypothesis could be tested with the inducible expression of Ty-1-tubulin. According to
the length of the cell cycle, after 9 h post tetracycline addition, 50% of flagella should be
devoid of Ty-1-tubulin, after 18 h it should be 25%, and after 27 h 12.5%, respectively.
However, in reality, 98% of flagella contained Ty-1-tubulin 24 hours after the addition of
tetracycline (Fig. S7). Furthermore, in the de-induction experiments, >95% of the pulsed
green flagella that were chased 48 h post washout had incorporated untagged tubulin again.

Should flagella stay locked indefinitely, this could only be possible for the 50% of flagella that
did not finish assembly during the last cell cycle of the Ty-1-tubulin labeling step. In re-
induced mono-flagellated cells, two segments could be observed at the distal flagellum tip
and correlated with timely distinct instances of integration (washout and re-induction, Fig. 7).
Multiple cell cycles must have passed since the emergence of these cells, showing that a flagellum does not stay locked indefinitely but instead undergoes regular integration of new tubulin specifically at its distal tip. Employing an orthogonal approach with the HaloTag system for tracing RSP4/6 integration demonstrated that after less than 2 cell cycles, virtually all flagella possessed a segment containing recently synthesized RSP4/6.

To account for these observations, we propose a refined model presented in Fig. 9B: A new flagellum is assembled at a linear rate to ~16 µm and then finishes assembly post cell division. It then gets locked and another NF emerges (indicated by the blue line, Fig. 9B). While this NF elongates, the OF can no longer incorporate new building blocks, as shown here in multiple experiments. *T. brucei* most likely employs this strategy to prevent competition between the two organelles (Atkins et al., 2021; Bertiaux et al., 2018).

Although locked for growth the OF could still be shortening during elongation of its NF counterpart. Measuring the length of flagella over the course of a cell cycle indicated that OF are on average shortest in post mitotic cells (Abeywickrema et al., 2019). This leads to a conundrum: if there is such shortening in each cell cycle, it would over the course of many cell cycles result in significantly shorter flagella, something that has not been described. We propose that the previously locked OF gets unlocked (green lock, Fig. 9B), granting access for new building blocks to facilitate re-assembly of the flagellum to its full length after shortening. This could happen in a short time-window around the beginning of G1-phase, when OF and NF are not competing. This idea is supported by absence of the locking protein Cep164C in 60% of mono-flagellated cells (Atkins et al., 2021) and our observation that simultaneous integration in NF and OF only occurs when CEP164C is depleted.

Taken together, our results demonstrate regular integration of new building blocks at the distal tip of the axoneme. As the length of the *T. brucei* flagellum is remarkably stable, this would require a disassembly mechanism to avoid excessively long flagella over multiple cell cycles. Although not previously reported for *T. brucei*, the flagellum shortening via disassembly of cytoskeletal structures was reported in related kinetoplastids, such as *Leishmania mexicana* (Wheeler et al., 2015), the firebug trypanosomatid *Leptomonas pyrrhocoris* (He et al., 2019), and the closely related *T. congolense* (Peacock et al., 2018). In *T. brucei*, there are certainly protein candidates located at the flagellum tip, which belong to families capable of tubulin removal. *T. brucei* kinesin 13-2 is found at the tip of both NF and OF. RNAi mediated knockdown has resulted in flagella that were constructed too long (Blaineau et al., 2007), but this result could however not be reproduced when the protein was knocked out (Chan and Ersfeld, 2010). The *T. brucei* genome encodes five putative proteins of the katanin family, which are generally known as MT severing enzymes, although this function was not yet demonstrated in *T. brucei*. Here they are localized to the distal tip of the flagellum (Tb 927.11.3870) or the posterior cell tip, where new tubulin is added to MTs
(Casanova et al., 2009). It is conceivable that these enzymes can effectively shorten the old flagellum after tubulin integration is inhibited by the locking mechanism. Alternatively, flagellum shortening could be restricted to the OF and even be cell cycle dependent, possibly explaining why tubulin integration between NF and OF was not equal when CEP164C was knocked down. However, an actor exclusive to the NF, that positively affects elongation, would also explain this result.

The biological relevance of this supposed turnover remains elusive: The integration of newly synthesized material is restricted to the flagellum part, that extends past the anterior cell body. This portion of the flagellum is likely more dynamic, as disassembly of the part attached to the cell body would involve remodeling of the flagellum attachment zone and might entail substantial rearrangement of parts of the cell body. The free part of the flagellum might be subject to physical forces of different magnitudes when compared to the attached part of the flagellum, which could lead to damage that requires repair by regular axonemal renewal. Moreover, the distal tip of the flagellum acts as a signaling hot spot (Bachmaier et al., 2022; Langousis and Hill, 2014; Lopez et al., 2015; Shaw et al., 2022, 2019). A cell cycle dependent restructuring of this region could allow trypanosomes to replenish components of signaling pathways that are depleted over a cell's lifespan. Furthermore, the trypanosomes could change the composition of the tip, to adjust to different environments and abide cell differentiation.

In addition to describing how flagellum growth is temporarily controlled, our investigation of CEP164C depleted cells also provides some insight into the locking mechanism. In the absence of CEP164C, NF and OF are competing for new tubulin, but do not elongate at the same rate, suggesting that other actors are involved. These may be present in the flagellum, possibly at its distal tip and disproportionally shorten the OF or elongate the NF. Alternatively, it is likely that the barrier at the OF base is formed by a network of different proteins. CEP164C, rather than establishing the lock on its own, might be the filler in a mesh to definitively seal the OF from access. Upon its depletion the barrier at the OF base is leaky, allowing some tubulin to enter.

Since integration in the OF was decreased in relatively longer OF, it is possible that proteins hindering elongation, accumulate over time and with increasing length. A simpler explanation is that overly long flagella might be too unstable to incorporate and retain new material at the tip. We hypothesize that trypanosomes use more than one tool to fine tune flagellum length, consistent with the large changes in the length of the organelle throughout the trypanosome life cycle.

In summary, we provide the first formal evidence for the Grow-and-Lock model in bi-flagellated cells. However, the flagellum is more dynamic than initially thought, as we also observed cell cycle dependent turnover at the distal tip of the flagellum, which occurs most
likely during G1-phase. We propose a model where procyclic trypanosomes lock the OF
before assembling a NF to avoid competition for building blocks. Towards the end of NF
elongation, the OF slightly shortens followed by a short phase of re-assembly in G1-phase
after cytokinesis. This repeats every cell cycle. The cells therefore maintain flagellum length
in a manner that resembles a balance-point model but with timely distinct (cell cycle
dependent) instances of disassembly and re-assembly, which are restricted to the free
portion of the flagellum at its distal tip. In contrast, Chlamydomonas combines a constant dis-
assembly rate with a variable assembly rate to regulate flagellum length. The balance-point
is reached when dis-assembly and assembly rates are equal.

Finally, we provide a robust tool to measure assembly dynamics of MTs with tubulin directly. Flagellum assembly was the main focus of this work; however, inducible expression of Ty-1-tubulin can also be used to track tubulin integration into the MTs of the cell corset and the mitotic spindle.

**Material and methods**

**Cell culture**

Procyclic trypanosome strain 427 were cultured at 27° in SDM-79 medium (GE Healthcare Cat No.: SH3A8023.01) pH 7.4, complemented with 10% fetal bovine serum and Hemin (7.5 mg/l). For growth curves, cells were counted every 24 h with a Luminex® Guava® Muse® flow cytometer and then diluted to 2 x 10⁶ cells/ml.

To generate the mNG-HaloTag tagged cell line the SmOxP927 strain (Poon SK, Peacock L, Gibson W, Gull K, Kelly S. A modular and optimized single marker system for generating Trypanosoma brucei cell lines expressing T7 RNA polymerase and the tetracycline repressor. Open Biol. 2012 Feb;2(2):110037.) was used.

**Vectors and transfection**

For in situ tagging of alpha-tubulin, one (1xTy-1) or two (2xTy-1) epitopes of the Ty-1-tag
were inserted after threonine 41 inside the first 500bp of the alpha-tubulin ORF. The
fragment was synthesized with Xhol and Apal flanking sites and cloned into p2675 (reviewed
in Kelly et al. 2007) by GeneCust (Boynes, France) (see Fig. S1). The resulting vectors were
linearized with FspAI (4891-4898bp of p2675-alpha-TubTy1) before transfection. For
inducible ectopic expression, one (1xTy-1) epitope of the Ty-1-tag was inserted after
threonine 41 inside the full length alpha-tubulin ORF. (Sirajuddin et al., 2014). This fragment
was synthesized and cloned between HindIII and BamHI sides of a pHD430-mNeonGreen-
Ty1-IFT172 (pHD 430 derivative, Wirtz and Clayton 1995) by GeneCust (Boynes, France)
(see Fig. S1). Prior to transfection the plasmid was linearized with NotI. For transfection, 4 x
10⁷ cells were harvested and then transfected with 10 µg of linearized plasmid using an Amaxa nucleofector II (program X-014) (Burkard et al. 2011). To obtain the α-Ty1-tubulin cell line, two rounds of transfection were carried out. 4 x 10⁷ cells were transfected with the pHD430 1xTy1-tub and then selected with phleomycin (2.5 µg/ml) for 10 days. The upcoming wells were harvested and screened for the presence of tagged tubulin by IFA using the BB2 antibody (Bastin et al., 1996). Positive wells were cloned out with serial dilutions and screened in similar fashion. Clones with expression of the tag in >90% of cells were then transfected as described above with the pHD360 (Wirtz and Clayton, 1995) to provide the tet repressor.

Knock down of CEP164C was achieved in the SmOxP9 T.brucei procyclic cell line, harbouring an inducible CEP164C RNAi construct (pQuadra system), and expressing Ty::mNG::CEP164C as well as Ty::mScarlet::SAS6. This cell line was provided by the lab of Sue Vaughan, Oxford, UK (Atkins et al., 2021). Depletion of CEP164C was achieved by the addition of 1 µg/ml of tetracycline for 48 hours. Transient transfection was performed with 10 µg of circular pHD430 1xTy1-tub plasmid with an Amaxa nucleofector II (program X-014).

## Tagging

To generate the cell line expressing RSP4/6 (Tb927.11.4480) N-terminally tagged at an endogenous locus with mNG-HaloTag the PCR only tagging approach was employed (Paterou et al., 2023) using pPOTv4 containing a sequence for mNG-HaloTag and blasticidin resistance as a template and the following long primers:

Tb927.11.4480 FW
GTGAAGTGGAGGTGGAAAGGAGAGCCAGTTGGAGAGAAAAACCACGTACACAAGTTTT
ACAGAATAAATCAAGGTCAGATgtataatgcagacctgctgc

Tb927.11.4480 RV
TCCTTATTCGCGCACATGAGATATGCCTTTGTCCTTGCGAACATCGCCTCAAGATCCTGT
GGTTCAGGATTTACTGGCATactacccgatcctgatcc

Cells were electroporated with the PCR product using the BTX ECM 830 electroporator and 16 hours later blasticidin was added to the final concentration of 20 µg/ml. After selection of blasticidin-resistant clones genomic DNA was isolated and correct integration of the PCR product validated by PCR.

## TMR labelling and imaging

Live cells expressing mNG-HaloTag tagged RSP4/6 were incubated for 1 hour at 28°C with HaloTag TMR Ligand (Promega, G8251) 1000-fold diluted in medium. Subsequently, three washes with fresh medium were performed to remove ligand not bound to the HaloTag.
Thereafter, cells were incubated in medium for a desired length of time, washed with PBS, settled on glass slides, and fixed in -20°C methanol for 20 minutes. The cells were then rehydrated in PBS for 20 minutes, mounted into 90% glycerol supplemented with 1% 1,4-antioxidant diazabicyclo[2.2.2]octane and 100 ng/ml of 4',6-diamidino-2-phenylindole (DAPI) and imaged using the Zeiss AXIOPLAN 2 microscope with a 40X Plan-NEOFLUAR, 1.3 Oil, and 100X Plan-APOCHROMAT, 1.4 Oil objective lenses. Image acquisition was performed using a Zyla 4.2 sCMOS camera (ANDOR) and Micro-manager software. Images were processed and analysed using the Java based program ImageJ (Schindelin et al., 2012).

## Immunofluorescence assays

Procyclic cells were harvested at densities between $5 \times 10^6 – 1 \times 10^7$. For whole cell analysis, cells were transferred onto poly-L-lysine coated slides (epredia, REF J2800AMNZ, Gerhard Menzel GmbH), left to air-dry for 10 – 15 minutes and then fixed with methanol at -20°C for 5 min. To extract cytoskeletons, cells were settled on poly-L-lysine slides for 10 min, then treated with 0.5% Nonidet P-40 in PEM buffer (2 mM EGTA; 1 mM MgSO$_4$; 0.1 M, pH6.9, PIPES) for 5 minutes and fixed with methanol at -20°C for 5 minutes (Robinson et al., 1991). Extraction of flagella was achieved by treating detergent extracted cytoskeletons with 1.1 M NaCl twice for 5 – 10 minutes before fixation. Post fixation samples were rehydrated in PBS for 15 minutes. Primary antibodies: BB2 mouse monoclonal antibody (Bastin et al. 1996, undiluted) against Ty-1 tagged alpha-tubulin, FTZC rabbit polyclonal antibody ((Bringaud et al., 2000), 1:2000 in 0.1%BSA in PBS or BB2 hybridoma) as transition zone marker, Mab 25 mouse monoclonal IgG2a (Dacheux et al., 2012), 1:4 in 0.1% BSA in PBS or BB2 hybridoma) as an axonemal marker. Secondary antibodies: α-mouse IgG1 coupled to Alexa Fluor488 (AF488) (Jackson ImmunoResearch Laboratories, #Cat: 115-545-205), α-mouse IgG2a coupled to Cy5 (Jackson ImmunoResearch Laboratories, #Cat: 115-175-206), α-rabbit coupled to AF647 (Jackson ImmunoResearch Laboratories, #Cat: 711-605-152). DNA was stained with 4',6-diamidino-2-phenylindole (DAPI, 10 µg/ml in PBS) for 1 minute. Glass cover slips were mounted with ProLong™ Gold antifade reagent (P36930, invitrogen by Thermo Fisher Scientific). Images were acquired with a 100x objective of the Leica DMI4000 microscope (NA=1.4, photometrics PRIME 95B™ back illuminated scientific CMOS camera) and analyzed with ImageJ (Schindelin et al., 2012).

## Western blot analysis

Procyclic cells were harvested at densities between $5 \times 10^6 – 1 \times 10^7$. To obtain whole cell lysates cells were resuspended in 100 µl hot Laemmli buffer (Laemmli, 1970) and then boiled at 100°C for 5min. For cytoskeletal fraction the cell pellet was treated with 1% NP-40 in PEM
buffer containing protease inhibitor (Roche cOmplete Mini EDTA free, #Cat: 11826153001) for 2 minutes. The pellet was harvested by centrifugation and then resuspended and boiled in Laemmli buffer for 5min at 100°C. To obtain the soluble fraction, the supernatant after whole cell lysis and removal of the cytoskeletal fraction was mixed with 2x hot Laemmli buffer and then boiled at 100°C for 5 minutes. Samples were separated by Sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in Mini-PROTEAN TGX Precast gels from BIO-RAD (7.5% cat: #4561021 or 4 – 15% gradient gel cat: #4561083) with the BIO-RAD running system in Tris / Glycine / SDS – buffer (BIO-RAD cat: #1610772). Proteins were then transferred onto Nitrocellulose or PVDF membranes (Trans-Blot Turbo Transfer Pack BIO-RAD cat: #1704158 and #1704156) using the Trans-Blot Turbo transfer system of BIO-RAD. Membranes were blocked at room temperature in 5% milk in PBS for 1 hour. Primary antibodies diluted in 2.5% powder milk in PBS: BB2 to detect Ty-1-tubulin (mouse monoclonal, 1:10), Y11/2 to detect tyrosinated tubulin (rat monoclonal 1:1000, (Kilmartin et al., 1971) obtained from Thermo Fisher scientific cat: # MA1-80017); TAT-1 to detect alpha-tubulin (mouse monoclonal 1:500, (Woods et al., 1989); C3B9 1:50 (Woods et al., 1989), and 6-11B-1 (mouse monoclonal IgG2b, invitrogen Thermo Fisher Scientific cat:# 32-2700) to detect acetylated tubulin. Incubation for 1 hour at RT or 4°C overnight. Thereafter membranes were washed 3x in 0.1% PBS-Tween. Secondary antibodies were added in 2.5% milk in PBS: ECL™ Anti-mouse IgG, Horseradish Peroxidase-Linked (Amersham™ from Cytiva, #Cat: NA931-100 µl, 1 : 10'000), Anti-rabbit IgG, Horseradishperoxidase-Linked (Amersham™ from Cytiva, #Cat: NA934-100 µl, 1 : 10'000), Anti-rat IgG, Horseradish Peroxidase-Linked (Amersham™ from Cytiva, #Cat: NA935-1 ml, 1 : 10'000) and the blots incubated at room temperature for 1 hour. Membranes were then washed in 0.1% Tween in PBS 3 times for 10 minutes. The blots were then incubated with ECL Select™ Western Blotting Detection Reagent for 5 minutes and revealed with an Amersham™ ImageQuant 800. Protein amounts were estimated with the ImageQuantTL® software.

## Tetracycline induction and washout of the inducible Ty-1-tubulin cell line

To induce Ty-1-tubulin expression tetracycline (1µg/ml) was added to a culture flask containing cells at densities between 2 x 10⁶ – 8 x 10⁶. For time courses tetracycline was added at different time intervals to a separate flask per timepoint. Samples were then harvested at the same time by centrifugation and subject to analysis by IFA and western blot. To monitor the disappearance of Ty-1-tubulin over several cell cycles de-induction experiments were performed. A culture in logarithmic growth phase was induced with tetracycline and diluted to 2 x 10⁶ every 24 h with new medium containing tetracycline. After 48 h de-induction was started: the cells were washed with medium (supplemented with 10% FBS and Hemin) four times and then placed into fresh medium devoid of tetracycline. Cells
were diluted every 24 h to 2 x 10⁶ cells/ml and monitored by IFA at 24 h, 48 and 72 h post de-induction. From every time point whole cells, cytoskeletal and flagella extracts were harvested as described above and subjected to IFA.

## LC-MS/MS to assess Ty-1-tubulin acetylation

To purify flagella 3 x 10⁷ cells were harvested and washed in SDM-79 without serum for 3 times. They were then resuspended in 300 µl of 1% NP-40 in PEM buffer containing protease inhibitor (Roche cOmplete Mini EDTA free, #Cat: 11826153001) and 1 µl benzonase and then incubated for 15 min at RT. After that they were spun down and resuspended again in 1% NP-40 in PEM buffer containing protease inhibitor without benzonase. Samples were incubated at RT for 2 minutes and then spun down and washed in PEM buffer. Incubation in 1% NP-40 in PEM buffer followed by washing was repeated twice more and the samples spun down at 15000g. They were then resuspended in 1 ml of 1 M NaCl and incubated for 15 min. The samples were then spun down and washed in PBS and the NaCl treatment repeated one more time. After a last washing step, the samples were resuspended in water and the purity of the preparation was assessed at the microscope. The samples were then spun down and all supernatant was removed. The pellet containing purified flagella was stored at -80°C.

## Protein digestion and peptide cleanup

Proteins were denatured by adding 8 M urea in 100 mM Tris buffer (pH 7.5). Proteins were reduced using 5 mM DTT for 30 minutes at room temperature. Alkylation of the reduced disulfide bridges was performed using 20 mM iodoacetamide for 30 minutes at room temperature in the dark. Urea was diluted to below 1.5 M with 50 mM ammonium bicarbonate. Each sample was divided into three aliquots, each digested with a different enzyme. For one aliquot, sequencing-grade chymotrypsin was added at an enzyme-to-protein ratio of 1:50 (wt:wt) in 100 mM ammonium bicarbonate, and digestion was performed overnight at 37°C. For the second aliquot, thermolysin was added at an enzyme-to-protein ratio of 1:20 (wt:wt) in 0.5 mM CaCl₂, and digestion was performed for 6 hours at 65°C. For the third aliquot, sequencing-grade Asp-N was added at an enzyme-to-protein ratio of 1:10 (wt:wt) in 10 mM Tris-HCl (pH 8.0), and digestion was performed overnight at 37°C. Digestion was stopped by adding 1% formic acid. Samples were centrifuged at 16,000 × g for 10 minutes, and the supernatant containing peptides was collected. Peptide clean-up was performed using an Agilent AssayMap Bravo robot on C18 tips according to the supplier's protocol. Peptides were resuspended in 2% acetonitrile/0.1% formic acid prior to LC-MS injection.
## LC-MS/MS analysis

The peptides were analyzed by nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a Proxeon EASY-nLC 1200 system (Thermo Fisher Scientific) coupled to an Orbitrap Q Exactive Plus Mass Spectrometer (Thermo Fisher Scientific). One microgram of peptides was injected onto a homemade 38 cm C18 column (1.9 µm particles, 100 Å pore size). Column equilibration and peptide loading were performed at 900 bar in buffer A (0.1% formic acid). Peptides were separated using a multi-step gradient: 3–6% buffer B (80% acetonitrile, 0.1% formic acid) in 3 minutes, 6–31% buffer B in 67 minutes, 31–62% buffer B in 25 minutes, 62–100% buffer B in 5 minutes, followed by 100% buffer B for 5 minutes and re-equilibration at 3% buffer B for 3 minutes, at a flow rate of 250 nL/min. The column temperature was maintained at 60°C. Mass spectrometry (MS) data were acquired using the Xcalibur software in data-dependent acquisition mode. Full MS scans were acquired at a resolution of 70,000, while MS/MS scans (fixed first mass at 100 m/z) were acquired at a resolution of 17,500. The AGC target and maximum injection times were set to 3 × 10⁶ and 20 ms for the survey scans, and 1 × 10⁶ and 60 ms for the MS/MS scans, respectively. The 10 most intense precursor ions were selected for fragmentation (Top 10 method) with a dynamic exclusion of 45 seconds. The isolation window was set to 1.6 m/z, and a normalized collision energy of 28 was used for higher-energy collision dissociation (HCD) fragmentation. The underfill ratio was set to 1.0%, corresponding to an intensity threshold of 1.7 × 10⁵. Precursor ions with unassigned charge states, or with charge states of 1, 7, 8, or >8, were excluded. The peptide match option was disabled.

## Data analysis

Acquired raw files were analyzed using MaxQuant software version 2.1.4.0 (Cox and Mann, 2008) with the Andromeda search engine (Cox et al., 2011; Tyanova et al., 2016). Samples were grouped by the type of experiment. The MS/MS spectra were searched against the *Trypanosoma brucei brucei* database (8,832 entries). All searches included the oxidation of methionine, protein N-terminal acetylation, and lysine acetylation as variable modifications, while cysteine carbamidomethylation was set as a fixed modification. Chymotrypsin, Asp-N, or thermolysin were selected as proteases, allowing for up to two missed cleavages. The minimum peptide length was set to five amino acids, and the peptide mass was limited to a maximum of 8,000 Da. The false discovery rate (FDR) for both peptide and protein identification was set to 0.01. The main search peptide tolerance was set to 4.5 ppm, and the MS/MS match tolerance was set to 20 ppm. The "second peptides" option was enabled to account for co-fragmentation events. Protein identification required at least one unique peptide assigned to the protein group. A false discovery rate cutoff of 1% was applied at both the peptide and protein levels. The mass spectrometry proteomics data have been deposited
in the ProteomeXchange Consortium via the PRIDE partner repository under the dataset identifier PXD.

## Acknowledgements

Antibodies were kindly provided by the following sources: Melanie Bonhivers, Université de Bordeaux, provided the Mab25 antibody, Frederic Bringaud, Université de Bordeaux, provided the anti-FTZC antibody. We thank Luke Rice (University of Texas Southwestern) for the suggestion of tagging tubulin in the acetylation loop. We thank Parul Sharma for the helpful discussions and critical reading of the manuscript. We thank Dr. Sue Vaughan, University of Oxford Brookes, for providing the CEP164C RNAi cell line. We thank Dr. Samuel Dean, University of Warwick, for providing the pPOTv4-blast-mNG-HaloTag vector.

## Competing interests

The authors declare no competing or financial interests.

## Author contributions

Conceptualization: D.A., S.B., V.V., P.B.; Methodology: D.A., M.P., L.S., E.B., R.A., M.M., V.V. S.B. P.B.; Validation: D.A., M.P., L.S., E.B., R.A., M.M., V.V. S.B. P.B.; Formal analysis: D.A., M.P., L.S., E.B., R.A., M.M., V.V. S.B. P.B.; Investigation: D.A., M.P., L.S., E.B., R.A.; Resources: P.B., M.M., V.V.; Data curation: V.V., P.B.; Writing – original draft: D.A., V.V., P.B.; Writing – review + editing: D.A., M.M., V.V., S.B., P.B.; Visualization: D.A., M.P., L.S., S.B., V.V., P.B.; Project administration: S.B., P.B.; Funding acquisition: V.V., P.B.

## Funding

The work carried out in the laboratory of VV was supported by the Czech Science Foundation (GA CR) project no. 23-07370S. The work carried out in the laboratory of PB was funded by La Fondation pour la Recherche Méicale (EQU202203014654), the ANR (ANR-18-CE13-0014-01), a French Government Investissement d'Avenir programme, Laboratoire d'Excellence "Integrative Biology of Emerging Infectious Diseases" (ANR-10-LABX-62-IBEID) and a PTR grant from the Institut Pasteur (PTR-474-21).

## Data and resource availability

Plasmid sequences and samples can be provided upon request. All other relevant data and details of resources can be found within the article and its supplementary information.
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<figure><img src="image_3.png" /><figcaption>Fig. 1. Tagged tubulin is incorporated into MTs.</figcaption></figure>

A: IFA with detergent extracted cytoskeletons of *in situ* tagged Ty-1-tubulin cell line. Cells were extracted with 0.5% NP-40, fixed with methanol and stained with BB2 (Ty-1-tag), TAT-1 (alpha-tubulin) and DAPI. K: kinetoplast, N: nucleus, F: flagellum. B:
IFA with whole cells of a cell line capable of an inducible expression of tagged tubulin. Cells were fixed with methanol and stained with the BB2 antibody (Ty-1-tubulin, white) and DAPI (cyan). Top: non-induced, Bottom: Induced with tetracycline for 16 hours. C: Zoom in of induced cells highlighting the presence of tagged tubulin into cell body, mitotic spindle (red arrow) and the flagellum (white arrows). D: Western blots with protein samples of different fractions from the wild-type cell line, the non-induced (-tet) and induced (+tet) tagged tubulin cell line. Top: membrane was stained with BB2 (tagged tubulin). Bottom: membrane was stained with TAT-1 (alpha-tubulin). T: whole cell isolates, C: detergent extracted cytoskeletons, S: soluble proteins. E: Growth curve of non-induced (black circles) and induced (red squares) tagged tubulin cell line. Scale bar = 10 µm.
<figure><img src="image_4.png" /><figcaption>Fig. 2. Ty-1-tubulin is incorporated into MTs of the cell body and the flagellum</figcaption></figure>

IFA with cells 24 h after the addition of tetracycline. Samples were incubated with
antibodies targeting the axonemal component TbSAXO (Mab25, magenta), the
transition zone protein FTZC (anti-FTZC, magenta dot), the antibody that recognises
the Ty-1-epitope inside tagged tubulin (BB2, green), and DAPI. A: Whole cells were
treated with 0.5% NP40 to obtain detergent extracted cytoskeletons. Ty-1-tubulin is
present inside the subpellicular MT corset as well as the flagellum. B: Cytoskeleton
extracts were further treated with 1 M NaCl to depolymerise the corset MTs to obtain
1 M NaCl extracted flagella. Ty-1-tubulin is still present inside flagella and therefore
integrated into the MTs of the axoneme. Green arrow: NF, white arrow: OF, blue
arrow: cell corset, white asterisk: tetracycline non-responders. Scale bar = 10 µm.
<figure><img src="image_5.png" /><figcaption>Fig. 3. Tagged tubulin is incorporated at a linear rate into the new flagellum.</figcaption></figure>

A: Cartoon depicting isolated flagella before and after induction of Ty-tubulin expression (2-4 h). This cell has started NF assembly before induction. The dot in magenta marks the transition zone (proximal portion of the flagellum) and the line in magenta is the axoneme. Top: A cell before induction with a NF during assembly and with an OF. Bottom: The same cell after induction. If integration takes place at the distal end as expected, the tagged tubulin synthesized after the addition of tetracycline should integrate as highlighted in green. BioRender license: HJ289VQ8SH. B: Example images of flagella isolated from five timepoints post induction. Flagella were extracted with 1 M NaCl concentration and incubated with antibodies targeting the axonemal component TbSAXO (Mab25, magenta), the transition zone protein FTZC (anti-FTZC, magenta dot), the antibody that recognises the Ty-1-epitope inside tagged tubulin (BB2, green). White spacers highlight the portion of the NF in which tagged tubulin has integrated. C: The length of the green portion highlighted in Fig. 2B was measured. Three biological replicates (70 - 100
flagella each) were pooled and plotted. Timepoints were corrected by subtracting 45 minutes from the induction time, which is an estimated response time for tetracycline addition to protein expression (Wirtz and Clayton 1995). Regression line shows linear growth with a 99% confidence interval. Whiskers indicate the mean with standard deviation. Simple linear regression: non-zero slope = significant (P value = 0.0002), Slope = 2.937, R² = 0.9949. Scale bar = 10 µm.
<figure><img src="image_6.png" /><figcaption>Fig. 4. At early timepoints of induction tagged tubulin is incorporated only in NF of bi-flagellated cells.</figcaption></figure>

A: Illustration of a cell that incorporated Ty-1-tubulin only in the NF. These cells were found throughout the entire time course, with the average length of the NF increasing over time. The image below is an example of a bi-flagellated cell isolated after 2h of induction (+). B: Illustration of a cell that incorporated Ty-1-tubulin along the entire length of the NF and at the distal tip of the OF (*). These cells were found only after 3 hours of induction. Image below depicts an example cell that was isolated after 4 h.

C: Pie charts depicting the fraction of bi-flagellated cells where Ty-1-tubulin was present in the OF tip (green, *) or absent (magenta, +) D: NF length of cells where Ty-1-tubulin was present at the tip of the OF (*). Bar and whiskers indicate median with interquartile range. E: Illustration of a cell where the NF had emerged before
induction, evident by the absence of Ty-1-tubulin in the proximal part. Here, Ty-1-
tubulin is absent in the OF. These cells were found from 2 – 4 h of induction. An
example of such a cell isolated after 2 h is shown below. F: Illustration of a cell where
the NF had emerged before induction. Ty-1-tubulin is incorporated in the tip of the NF
and also the OF. No such cells were found throughout the time course. BioRender
license: OZ289VR2IV. Scale bar = 10 µm.
<figure><img src="image_7.png" /><figcaption>Fig. 5. Upon CEP164C depletion Ty-1-tubulin is incorporated in both flagella.</figcaption></figure>

A: Illustration of the CEP164C knockdown followed by transient transfection of the Ty-1-tub pHD430 plasmid. When CEP164C knockdown is not induced, the protein is present at the base of the OF but not the NF (-tet). Upon the addition of tetracycline for 48 h, CEP164C is also absent at the OF (+tet). In non-induced conditions (-tet), 4 h after addition of the Ty-1-tub plasmid, tagged tubulin is incorporated at the distal NF tip, but never in the OF. When CEP164C knockdown was first induced for 48 h and then cells transiently transfected with the Ty-1-tub pHD430, tagged tubulin was found at the tip of both NF and OF. BioRender license: UI289VO6D9. B: Length measurements of the Ty-1-tubulin segment in the OF and the NF. Bar and whiskers indicate mean with SD. C: Length of the Ty-1-tubulin segment in the OF was plotted against the total length of the OF. On average longer OF possessed a shorter Ty-1-tubulin-containing region. Note that the used cell line also expresses Ty::mNG::CEP164C and Ty::mScarlet::SAS-6. Both are recognised by the BB2 antibody and localised to the flagellum base. Simple linear regression: non-zero slope = significant (P value = <0.0001). Scale bar = 10 µm.
<figure><img src="image_8.png" /><figcaption>Fig. 6. Tagged tubulin is frequently incorporated into the OF tip.</figcaption></figure>

A: Cartoon shows the progression of a cell that had started the assembly of a NF before induction over the length of one cell cycle (9-hour induction). While the NF is assembled, tagged tubulin is integrated in the distal portion. Over time the cell will divide and one cell will inherit the NF (top, 6 and 9 h) and one cell will inherit the OF (bottom, 6 h and 9 h). Both daughter cells will then start to assemble a NF again. These NF will be fully green. The OF that was a NF (1st gen OF, 9 h, upper cell) has a green gradient of tagged tubulin with increasing intensity towards the distal tip. The illustration of the cell cycle that indicates when cells are bi-flagellated was adapted
from Woodward and Gull, 1990. S = synthesis, M = Mitosis, C = cytokinesis, BB = basal body duplication. BioRender license: CY289VNVVI. **B:** Examples of cells with a green NF grouped by the different types of OF. 1: A new 1st-gen OF (Cartoon A 9 h top,). 2: An old flagellum of an OF daughter (Cartoon A 9 h bottom,). 3: OF that had integrated tagged tubulin exclusively at the very distal tip (average length ~1 µm). White arrows = Ty-1-tubulin in OF. **C:** Frequency of bi-flagellated cells grouped by the different types of old flagella **D:** Black squares: the total length of type 1 cell OF (Y-Axis, segment 3) plotted versus the length of the corresponding NF (X-Axis, segment 1) in the same cell. Simple linear regression: non-zero slope = not significant (P value = 0.7115). Green dots: the length of the green portion in the OF (Y-axis, segment 2) plotted versus the length of the NF (X-Axis, segment 1). Simple linear regression: non-zero slope = significant (P value = <0.0001). **E:** Comparison of the length of OF grouped by the different OF types described in B. Significance was tested with Tukey's multiple comparisons, adjusted P values: ns = 0.5750, * = 0.0336, *** = 0.0007. Bar indicates the mean. **F:** Comparison of the length of NF grouped by the different types of the OF. Significance was tested with Tukey's multiple comparisons, adjusted P values: * = 0.0223, **** (1 vs. 2) = <0.0001, **** (2 vs. 3) = <0.0001. Bar indicates the mean.
<figure><img src="image_9.png" /><figcaption>Fig. 7. Tagged tubulin is frequently incorporated into the OF tip in cells that are in G1-phase.</figcaption></figure>

A: Flagella extracts of cells that were subject to a pulse-chase experiment. Cells were first induced with the addition of tetracycline to the medium for 24 h (left panel, +tet 24 h). Tetracycline was then washed out and the cells were harvested after being grown in absence of tetracycline for 48 h (middle panel, +tet 24 h, -tet 48 h). After that the cells were re-induced with tetracycline for 4h and harvested for IFA (right panel, +tet 24 h, -tet 48 h, +tet 4h). Samples were stained with BB2, Mab25 and anti-FTZC. Green arrows highlight the presence of Ty-1-tubulin at the flagellum base and tip. Magenta arrows highlight the absence of Ty-1-tubulin. B: An example of flagella from mono-flagellated cells (G1-phase). Arrows indicate the times during the pulse chase experiments in which the corresponding part was most likely assembled. Scale bar = 10 µm.
<figure><img src="image_10.png" /><figcaption>Fig. 8. Incorporation at the tip is found for other axonemal component visualized using a HaloTag</figcaption></figure>

A: A biflagellar cell fixed **2 hours** after TMR labelling and ligand washout. Unlabelled RSP4/6 is incorporated in the new flagellum exclusively at its distal end. HaloTag TMR-ligand signal (magenta), mNG-HaloTag::RSP4/6 (green), DAPI (cyan). B: A biflagellar cell fixed **4 hours** after TMR labelling and ligand washout. Unlabelled RSP4/6 is incorporated at the distal tip of the OF and along the entire length of the NF. C: A field of fixed cells **4 hours** after TMR labelling and ligand washout. White arrows: monoflagellated cells with a visible sharp decrease of TMR ligand at the distal tip of the flagellum. Green arrows: integration of unlabelled RSP4/6 into short NF and the OF tip. + = case 2 scenario 1 cell (similar to Fig. 4A); * = case 1 scenario 1 cell (similar to Fig. 4B/C). D: The proportion of different types of flagella found after various timepoint post TMR ligand labelling + washout. Between 4 and 16 h the proportion of flagella that did not incorporate unlabelled RSP4/6 drops to 0.7% (highlighted by the black rectangle). E: Comparison between Ty-1-tubulin cell line **24 h** after the addition of tetracycline (n = 265) and mNG-Halo::RSP4/6 **24 h** after TMR ligand labelling + washout (n = 253). In all experiments, mono-flagellated and bi-flagellated cells were scored together. Scale bar = 5 µm.
<figure><img src="image_11.png" /><figcaption>Fig. 9. Flagellum dynamics over multiple cell cycles</figcaption></figure>

Model describing flagellum growth and maintenance in Trypanosoma brucei assuming the Grow-and-Lock model. A: In this graph the locking mechanism persists indefinitely. Once the maximum length is reached it remains stable and the flagellum neither shortens nor elongates. The orange line represents flagellum length, with a NF assembling at a linear rate that reaches maximum length in the following G1-phase and then gets locked. The blue line represents linear assembly of the NF inside the cell that carries the OF that is represented by the orange line. B: The flagellum assembles at a linear rate and gets locked before a NF is assembled in the same cell, represented by the blue line. While the NF assembles the OF (orange line) slightly shortens. After the cell divides the NF daughter (blue line) assembles its flagellum to full length, meanwhile the OF daughter is unlocked briefly, to allow the previously dis-assembled portion to re-assemble. BioRender license: OF289VOE06, UM289VOK8E.
<figure><img src="image_12.png" /><figcaption>Fig. S1. Alpha-tubulin tagging strategy</figcaption></figure>

A: Sequence of *T.brucei* alpha-tubulin. Insertion of the Ty-1-tag is highlighted in green, one amino acid after the acetylated lysine (K40, cyan). B: Positioning of the Ty-1-tag in the acetylation loop highlighting that the tag faces the lumen of the MTs. C: In situ tagging strategy with p2675. The first 500bp of the alpha-tubulin ORF that contains the Ty-1-tag were introduced between Xhol and Apal sites downstream of the puromycin selection marker followed by the beta-alpha-tubulin intergenic region. The tagged ORF will recombine in the genome with one of the ~40 alpha-tubulin loci under its endogenous 5'UTR (introduced by the plasmid) and its endogenous 3' UTR from the recombined tubulin locus. D: Inducible ectopic expression of Ty-1-tubulin in pHD430. The whole ORF containing the Ty-1-tag was cloned between HindIII and BamHI sites downstream of a procyclin promoter that contains two Tet operator sequences. In a first round of nucleofection 1xTy-1-tub-pHD430 was introduced. Followed by a second round of transfection with the pHD360 plasmid that introduced the Tet-repressor. BioRender license: WR289VORIP, SW289VQFBJ.
<figure><img src="image_13.png" /><figcaption>Fig. S2. In situ tagging of alpha-tubulin</figcaption></figure>

A: Incorporation of Ty-1-tubulin in the mitotic spindle progressing through mitosis from left to right. Cells with one nucleus are whole cells, with two nuclei cytoskeleton extracts. B: Detergent extracted cytoskeletons from a cell line where alpha-tubulin was tagged with one epitope of the Ty-1-tag inside the acetylation loop (1xTy). C: Cell line that was tagged with two epitopes of the Ty-1-tag (2xTy). Samples were stained with antibodies recognising the Ty-1-tag (BB2), alpha-tubulin (TAT-1) and DAPI. D-G: Western blots with the 1xTy and 2xTy cell lines. Stained with BB2 or TAT-1 antibody. T=whole cell samples P= cytoskeleton extracts, S= soluble proteins.
D: Whole cell samples from three clones of each cell line, stained with the BB2 antibody (tag only). E: T, P and S samples from two clones of the 1xTy cell line. Membrane was stained with the TAT-1 antibody. Two bands are indicated with arrows, the upper one for the tagged protein and the lower one for untagged. F: Same experiment as in D with the 2xTy cell line instead. G: T, P and S samples from one clone of the 1xTy and one of the 2xTy cell lines analysed on the same blot. Scale bar = 10 µm.
A

<figure><img src="image_14.png" /></figure>

B

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Ty-1-tag

AspN cleavage sites

Acetylated lysine (K40)

C

<figure><img src="image_16.png" /></figure>

D

<figure><img src="image_17.png" /><figcaption>Fig. S3. Acetylation is present on Ty-1-tubulin but cannot be detected with antibodies.</figcaption></figure>

A: Western blots with whole cell isolates from the Ty-1-tubulin cell line without (-) and 24 hours after the addition of tetracycline (+). Five individual membranes were stained with different antibodies. BB2 (Ty-1-tag), TAT-1 (alpha-tubulin), YL1/2 (tyrosinated alpha-tubulin), C3B9 (acetylated alpha-tubulin (K40)), 6-11B-1 (acetylated alpha-tubulin (K40)). Under induced conditions a second band corresponding to Ty-1-tubulin is visible for TAT-1 and YL1/2 but not for the two acetylation specific antibodies. B: Amino acid sequence of tagged tubulin with AspN cleavage sites marked in yellow, the acetylated lysine (K40) marked in red and the Ty-1-tag marked in green. C: MS-analysis of protein isolates from detergent extracted flagella of the Ty-1-tubulin cell line 7 days after induction. Presence of the acetylated lysine on the Ty-1-tagged peptide was found in all 4 replicates (R1-R4). D: Spectra of replicate 3 depicting different ions that were detected from the tagged peptide including the acetylated lysine (acK).
<figure><img src="image_18.png" /><figcaption>Fig. S4. Expression of Ty-1-tubulin can be detected early after induction</figcaption></figure>

Ty-1-tubulin cell line was induced for 4 hours and cells harvested in 30-minute intervals. A: Three membranes containing whole cell isolates, cytoskeleton extracts and soluble proteins from every timepoint were stained with the BB2 antibody. B: Three separate membranes with the same samples as in S4A were stained with the TAT-1 antibody. X = empty lane.
A
4h induction

<figure><img src="image_19.png" /></figure>

B
2h induction
phase

<figure><img src="image_20.png" /><figcaption>Fig. S5. Integration of new Ty-1-tubulin.</figcaption></figure>

A: Detergent extracted cytoskeleton of a bi-flagellate cell from the inducible Ty-1-tubulin cell line after 4 hours of induction. Samples were stained with BB2 (Ty-1-tubulin, green), FTZC (transition zone, magenta dot) and Mab25 (axoneme, magenta) as well as DAPI (blue). Ty-1-tubulin has integrated in the new flagellum (NF) but not the old flagellum (OF) as well as the posterior cell body and the mitotic spindle. spindle= mitotic spindle, N = nuclei, K = kinetoplast, TZ = transition zone. B: 1M NaCl extracted flagella of a cell from the inducible Ty-1-tubulin cell line, 2 hours after induction. Samples stained with BB2, Mab25 and FTZC. Ty-1-tubulin is incorporated at the distal tip of the NF but not the OF. Scale bar = 10 µm.
<figure><img src="image_21.png" /><figcaption>Fig. S6. Transient expression of Ty-1-tubulin in cells depleted of CEP164C.</figcaption></figure>

1M NaCl extracted flagella from the CEP164CRNAi cell line 4 h after transient transfection with the circular Ty-1-tub pHD430 plasmid. Staining with BB2 (Ty-1-tag), Mab25 (axoneme) and FTZC (transition zone). Left: In these cells RNAi was not induced before transient transfection. Right: In these cells CEP164C RNAi was induced with tetracycline for 48 h before the transient transfection. Arrows indicate cells where NF had emerged before transient transfection (untagged proximal portion). White arrow: NF, red arrow: OF. This cell line also expresses Ty::mNG::CEP164C and Ty::mScarlet::SAS-6. Both are recognised by the BB2 antibody and localise to the flagellum base.
<figure><img src="image_22.png" /><figcaption>Fig. S7. >95% of flagella incorporate Ty-1-tubulin after 24h</figcaption></figure>

A: 1M NaCl extracted flagella from the Ty-1-tubulin cell line 24h after the addition of tetracycline that were stained with BB2 (Ty-1-tag), Mab25 (axoneme) and FTZC (transition zone). Arrows indicate four types of flagella: green= flagella that incorporated Ty-1-tubulin along the entire length, red= flagella with a gradient, blue= flagella with Ty-1-tubulin at the distal tip, grey= flagella that did not incorporate Ty-1-tubulin. In whole cells from the same experiment 5% of cells (total n = 265) did not incorporate any Ty-1-tubulin in the cell body. B: The pie chart on the right depicts the relative amount of different types of flagella found after 24h of induction. Scale bar = 10 µm.
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<figure><img src="image_34.png" /><figcaption>Fig. S8. Detergent extracted cytoskeletons of de-induction experiments</figcaption></figure>

Detergent extracted cytoskeletons from the same experiment as in Fig. 6. Green arrows highlight presence of Ty-1-tubulin in posterior cell body, NF, and flagellum tip, magenta arrows indicate Ty-1-tubulin absence, respectively. A: After 24h Ty-1-tubulin is present in NF and OF (from base to tip). B: 48h post de-induction Ty-1-tubulin is absent from the NF but present in the OF, except the OF distal tip. C: 4h after re-induction Ty-1-tubulin is present in the NF and the OF, but absent from the OF distal tip. D: Detergent extracted cytoskeleton of a monoflagellated cell. Scale bar = 10 µm.
<figure><img src="image_35.png" /><figcaption>Fig. S9. mNG-HaloTag::RSP4/6 localizes to the flagellum and is efficiently labelled with TMR-HaloTag ligand.</figcaption></figure>

mNG-HaloTag::RSP4/6 cells were incubated with TMR-ligand for 1 hour. The ligand was then washed out and the cells immediately fixed. TMR signal is shown in magenta, mNG in green and DAPI in cyan. TMR and mNG signal colocalize along the entire length of both flagella. Scale bar = 5 µm.
<figure><img src="image_36.png" /><figcaption>Fig. 1D</figcaption></figure>
<figure><img src="image_37.png" /><figcaption>Fig. S2</figcaption></figure>

Blot transparency Fig. S3A

<figure><img src="image_38.png" /></figure>

Fig. S3 marker and chemi channel merged
<figure><img src="image_39.png" /><figcaption>Fig. S10. Blot transparency</figcaption></figure>