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

Article activity feed

1. 

   Review Commons

   May 15, 2025

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   Manuscript number: RC-2025-02887

   Corresponding author(s): Philippe Bastin

   1. General Statements [optional]

   We thank the reviewers for their constructive suggestions. We are delighted to see that they appreciated our work and its interest for the broad cell biology community, as well as the potential impact of the inducible expression of tagged tubulin as a new tool to investigate microtubule assembly at large.

   We are now providing a full revision that contains two major modifications and that addresses all the minor points detailed below. The two major modifications are:

   • A simplification and a shortening of the text as requested by reviewers 1 and 3
   • Th…

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   Manuscript number: RC-2025-02887

   Corresponding author(s): Philippe Bastin

   1. General Statements [optional]

   We thank the reviewers for their constructive suggestions. We are delighted to see that they appreciated our work and its interest for the broad cell biology community, as well as the potential impact of the inducible expression of tagged tubulin as a new tool to investigate microtubule assembly at large.

   We are now providing a full revision that contains two major modifications and that addresses all the minor points detailed below. The two major modifications are:

   • A simplification and a shortening of the text as requested by reviewers 1 and 3
   • The addition of a new experiment evaluating the role of the locking protein CEP164C to gain insight into the mechanism, as suggested by reviewers 1 and 2 Briefly, CEP164C is a protein localised to the transition fibres (structures that dock the basal body of the flagellum to the membrane) of only the old flagellum. Its depletion leads to an excessive elongation of the old flagellum and the production of a shorter new flagellum, suggesting competition between the two flagella for tubulin incorporation (Atkins et al., 2021). In the new figure 5, we have expressed tagged tubulin in the CEP164CRNAi cell line and formally demonstrated simultaneous incorporation in both flagella. Unexpectedly, the new flagellum incorporated more tubulin than the old one, suggesting a bias of tubulin targeting in favour of the new flagellum and the existence of additional contributors to the Grow-and-Lock model.

   2. Point-by-point description of the revisions

   This section is mandatory. *Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. *

   Reviewer #1

   Evidence, reproducibility and clarity

   The manuscript by Daniel Abbühl on "A novel approach to tagging tubulin reveals MT assembly dynamics of the axoneme in Trypanosoma brucei" uses an innnovative approach to label tubulin, which allows the authors to unveil new mechanisms in flagellar length regulation.

   The manuscript is very nice and will be very interesting for the cell biology community and therefore should be accepted. In some parts it becames a bit complex with all the models and complex phrasing, I wonder whether the text could be simplified to be more appealing. I have a few minor comments:

   We agree that some of the explanations are lengthy and complex. We have simplified the explanations and hopefully made the models more accessible. Complexity comes from the fact that trypanosomes do not have a synchronized cell cycle.

   -From the model the authors show in Figure 8- there should be a way of pulsing the cells in G1 for a short amount of time -2 hours- and getting both flagella tips labelled. But the authors seem to require longer labelling to get that result. This should be better explained.

   We are not quite sure what is meant here with both flagella as in G1-phase, all cells are mono-flagellated. We do see mono-flagellated cells with a labelled tip after 2 hours, both with the HALO-tag or the Ty-1-tubulin system.

   In regard to bi-flagellate cells, we believe that incorporation in the OF happened at the beginning of G1-phase when the cell was mono-flagellated. If tubulin is present at that point, it will be incorporated at the tip. This cell then approaches the end of G1-phase and starts to initiate NF assembly. Since tagged tubulin is already present it will be incorporated along the whole length of the NF.

   A short induction of 2h would not suffice as it wouldn't cover the duration of the G1-phase and the initiation of a NF (duration of G1-phase is ~4h). We attempted to explain this in Fig. 4 and reworked the text to make this clearer.

   -Why do some cells not express the construct? Weren´t they all selected?

   We never managed to get a cell line where inducible expression is present in 100% of cells. Here, around 95% of cells were positive for Ty-1-tubulin after 24h of induction. Non-expression is not a phenomenon restricted to this tubulin cell line but also observed with other ectopically expressed proteins (e.g. Sunter et al. JCS 2015, Bastin et al. MCB 1999). All these cell lines represent clonal populations and are resistant to antibiotic treatment, however not all cells express the respective protein. For each experiment where we believed the number of expressing cells matter (for example the washout), we quantified in how many cells Ty-1-tubulin was present in the cell body microtubules.

   -"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". However, in the authors data the delay may amount to one hour (western analysis- S4). Shouldn´t they use their data.

   Indeed, the western blot shows expression after 1-hour, however we did not take a 45-minute timepoint, so we don't know if the protein was detectable at that time. In addition, IFA is more sensitive than western blot. We cannot say exactly when the average cell starts to express the induced protein.

   -Fig 3: To measure the timepoints of flagella growth, wouldn´t it be better to do it with NF that started to grow before induction, rather than starting to grow after induction, to be sure that the timing of incorporation is fully accounted for?

   We indeed did consider only NFs, which started to grow before induction, as suggested by the reviewer. In the revised version the description of the experiment can be found on page 9 line 22 - 28.

   -Although it is not the focus of the manuscript it would have been very interesting to use the CEP164C mutant to see whether it would change the dynamics of incorporation and fully test their model and discussion.

   This is a great suggestion, so we performed some experiments to address this issue. When CEP164C was knocked down before Ty-1-tubulin expression, integration is seen at the distal tip of both NF and OF. This is coherent with the idea of removal of the locking protein from the OF. However, lengths of the green segments in NF and OF do not have the same length (NF ~6 µm, OF ~2 µm), which indicates that CEP164C might not be the only protein involved in regulating flagellum length. A new figure explaining this experiment was added (Fig. 5, Fig. S6). We believe this data provides novel insight on the locking mechanism and strengthens the manuscript.

   -In some parts of the manuscript/supplemental material the authors say they insert the Ty-1- tag one aminoacid after the acetylated lysine- other parts they say two aminoacids after- this should be consistent.

   We thank the reviewer for spotting these mistakes, we have changed the text accordingly.

   -Fig. S1: 'Binding epitope of the TAT-1 antibody is highlighted in red'. There is no highlighting in red in this figure?

   This sentence was removed.

   -Fig. S2: Western blots are not very clear. What is the 'X' present in the C (first lane)? Weight of markers should be shown also in S4.

   Molecular weight markers have been added. X is an empty lane, we have now indicated this in the figure legend.

   -Fig 5: 'C: Frequency of bi-flagellated cells grouped by the different types of' The authors didn't finish the sentence.

   Previous Fig. 5 is now Fig. 6. Sentence has been completed. "Frequency of bi-flagellated cells grouped by different types of old flagella"

   -Fig. S7: The 'B' is missing in both picture and legend.

   This has been added

   ---

   Significance

   This study advances our knowledge of flagellar length regulation and maintenance. Moreover, the tools designed in this work will be very useful for the cell biology community in general.

   ---

   Reviewer #2

   Evidence, reproducibility and clarity

   Summary: The length of the old flagellum of Trypanosome is constant during G1 phase as well as during cell cycle progression when the new flagellum is assembled. The authors have previously proposed a "Grow and Lock" model for the flagellar length control in which no flagellar building blocks are incorporated. To test this hypothesis, the authors used a tagging strategy for alpha-tubulin and tracking its incorporation. The authors showed that the new flagellum incorporates new tubulins, as is expected. For the mature flagellum, tubulins are incorporated at the flagellar tip and only when the cells start to assemble the new flagellum. Thus, it shows that old flagellum is stable but not completely locked for the incorporation of tubulins.

   Major comments: The study is methodologically rigorous, integrating fluorescence microscopy, biochemical approaches, and proteomic analyses to validate the functionality of the tagged tubulin. The use of both inducible expression and endogenous protein tagging (HaloTag) strengthens the conclusions. This study has supported the "Grow-and-Lock" model" that the authors previously proposed. In addition, they have revealed that the stability of the old flagellum is temporally controlled.

   The data showed that brief incorporation of tubulins at the tip of the old flagellum occurs when the cells start to form the new flagellum. I thought the assembly of the new flagellum occurs during the cell division. However, in the abstract, it says that "The restriction is lifted briefly after the bi-flagellated cell has divided." Is my understanding wrong?

   We believe incorporation at the tip of the "OF" occurred after the cell has divided, when the OF daughter is mono-flagellated. It happens before this daughter cells starts assembling its new flagellum is formed. Of course, when looking at biflagellated cells, the NF as well as the tip of the OF will be green, but our data supports that incorporation happened in G1-phase and not during the biflagellated stage as the lock seals the OF before the NF emerges. To clarify on terminology: The bi-flagellate stage begins when basal bodies are duplicated, shortly after the beginning of S-phase and ends with cytokinesis. This means G1-phase and the mono-flagellated stage are nearly the same (Woodward and Gull, JCS1990) and occupy ~40% of the cell cycle.

   P12, "The cartoon in Fig. 5A illustrates the progression of the cells in scenario 2 (Fig. 4A) over the duration of one cell cycle (~9 hours)" I thought that one cell cycle should start with cell with only one flagellum, followed by assembly of a new flagellum during cell division, the cell then divides when the new flagellum is almost completely assembled. If my understanding is correct, perhaps the cartoon should be modified accordingly.

   Indeed, the cell cycle starts with a cell in G1-phase. Here, we have chosen the initiation of a NF assembly as our starting point because we focused the investigation on bi-flagellated cells. We have now illustrated the cell cycle (adapted from Woodward and Gull 1990) and when cells are biflagellated in Fig. 6A (revised version).

   Minor comments:

   1. Several references are not correctly formatted. P3: (Flavin and Slaughter, 1974) (Rosenbaum 1969). P10, (Sherwin et al., 1987)(Sheriff et al., 2014)
   2. In several places there are no space between the number and the unit. For eample, P3, 9 - 24µm/h. 7, 1μg/m; P8, 50kDa; P9, 1M; 8-9h; P11, 2.9µm/h and etc.
   3. P11, Flagella were extracted. I thought the cells were extracted.

   Thank you for pointing these out, we have changed these in the text.

   ---

   Significance

   Cilia and eukaryotic flagella are considered dynamic structures in which the flagellar components especially tubulins under constant turnovers even in steady state. This work demonstrates that in Trypanosome the stable old flagellum is temporally controlled for tubulin turnovers, suggesting a tight regulation of microtubule dynamics. Future elucidation of the regulatory mechanism will be more interesting. This work will be interesting to the field of cilia and microtubules. In addition, the new technique used for tracking tubulins will also be interesting.

   I am an expert on ciliary biology.

   Reviewer #3 (Evidence, reproducibility and clarity (Required)):

   Summary:

   This study seeks to investigate the mechanism by which the length of an eukaryotic cilium is set and maintained in a constant state. The flagellated protist Trypanosoma brucei serves as the study model and the authors take advantage of the genetic tools that allow precise modification and tagging of flagellar proteins and they build on prior knowledge about the well-characterised flagellar assembly cycle, which allows tracking the assembly of a new flagellum alongside an existing old one in the course of one cell cycle. The group of Bastin has previously reported a very interesting "Grow-and-Lock Model for the Control of Flagellum Length in Trypanosomes" and this current manuscript provides a test of this model, and a refinement. Key to this is an advance in technique, reported here, namely expression of an epitope tagged version of alpha tubulin. The epitope is inserted in an internal loop, which apparently for the first time provides a traceable tubulin that is reliably incorporated into the cytoskeleton (subpellicular array, spindle and cilium). Expressing an inducible version of this Ty-1-tubulin allows for a set of experiments that measure the place and timing of tubulin incorporation into cilia. The results are largely confirmatory of previous findings (incorporation exclusively into the new flagellum, at the distal end, linear growth rate that matches previous estimates). Examination of tubulin incorporation patterns then reveal additional information about the old flagellum: evidence from Ty-1-tubulin labelling, corroborated by incorporation patterns of another ciliary protein (RSP 4/6) suggest that the "lock" on the old flagellum is relieved for short periods after cell division, leading to a refined model presented in Figure 8.

   Major comments:

   This study provides an elegant test of the grow-and-lock model and the major conclusions are supported by the data. I have no major concerns.

   Minor comments:

   There are several minor points that could be addressed to make the manuscript easier to follow (and adding line numbers to the manuscript would help with reviewing).

   The introduction is quite long. Some of the well-established background information on the T. brucei cell cycle could be shortened. If the paper is intended for a broader audience, it would be valuable instead to cite studies that have succeeded in tagging tubulin and tracing its incorporation in other cilia. Could the Ty-1-tubulin approach be relevant more broadly or are simpler methods already established?

   The introduction has been shortened, we now also cite two published studies that tracked tubulin integration in Chlamydomonas and C. elegans respectively.

   On p.6 the rationale for endogenous tagging was to "reduce the risk of artifacts portentially due to untimely expression or unnatural protein levels". However most of the experiments were done with ectopically expressed inducible Ty-1-tubulin. For the experiments it is crucial to use an inducible system but the authors may wish to comment why the risk of artifacts was no longer a concern.

   The reasoning here was that in case the Ty-1-tubulin would not have been incorporated into MTs, we could have attributed it solely to the presence of the tag and no other factors, but this was not the case. This therefore allowed us to move to the inducible expression system.

   On p.7 / Fig S2A-B there appears to be a mistake in the presentation. Spindles are mentioned in the text - I can't see any in the figure. Fig S2A and B both show cytoskeletons, but the text suggests only B is about cytoskeletons. None of the blot shows BB2 staining of different cell fractions, contrary to statements in the text. The letter codes in the panel (T, C, D) don't match the codes in the legend (T, P, S).

   We thank the reviewer for spotting the mistakes. A panel with the spindle was added in Fig. S2. We did not stain fraction blots of the in-situ tagged cell lines with BB2. However, this was done with the inducible cell line and is shown in Fig. 1D. Letter code in the legend was adapted to match the figure.

   Figure 1. The evidence for incorporation into spindles is not strong. The structure indicated by the arrive could be a spindle but it's not very clear. There is a great example of a labelled spindle only in figure S5A. Here, at the start, it would be good to show a panel of cells in successive cell cycle stages (best, whole cells and cytoskeletons) to clearly show the structures that are labelled with Ty-1-tubulin.

   The current Fig. 1B (Fig. 1A before) depicts whole cells of an induced and a non-induced culture; we show whole cells to provide a complete picture of tubulin integration. A panel with detergent extracted cytoskeletons from the in situ tagged cell line has been added to Fig. 1A. We chose to show cytoskeletons or isolated flagella instead of whole cells because (1) the flagella are easier to see and (2) it formally demonstrates that tagged tubulin is incorporated in MTs.

   In general, tubulin labelling of the spindle was more consistently observed in whole cells as we did not use spindle preserving extraction buffers when preparing cytoskeletons. However, we did observe clear spindles in cytoskeletons as well (see Fig. S5 for example). The same was observed for the beta-tubulin specific KMX1 antibody in the past which is the gold standard to visualize the spindle (Sasse and Gull JCS1988). Regardless, a panel depicting spindle progression through mitosis using staining of Ty-1-tubulin has been added in Fig. S2 (The panel is a mix of whole cells and cytoskeletons).

   On p.8 (end of first paragraph) there is reference to cell cycle analyses, but no data is shown. Also on p.8, please clarify what the evidence is that "a fraction of cells did not respond to tetracycline". The fact that they remain unstained by Ty-1-tubulin is not in itself evidence they did not respond to tetracycline.

   We did not show the cell cycle data as it was similar to non-induced and does not provide any new information in our opinion. Hence, the sentence has been removed.

   The reviewer is correct that we do not have evidence that these cells did not respond to tetracycline. Some cells remained completely devoid of Ty-1-tubulin even after multiple days of induction. This was typically between 5-10% of cells. In experiments where the exact number is important, we counted the amount of "non-expressers" in whole cells.

   Figure S4A. The blot for the soluble fraction is not of great quality. I don't see how the conclusion was reached that the Ty-1-tubulin bands were faint.

   The blot of the soluble fraction that was stained with BB2 had to be exposed a lot longer compared to the blot stained with TAT-1. The soluble blots were repeated with the same result (lots of background noise when using BB2, a clear blot with TAT-1). In the TAT-1 blot only the endogenous tubulin band is clearly visible, with some very faint signal above corresponding to the Ty-1-tubulin. Soluble Ty-1-tubulin with BB2 or TAT-1 is visible in Fig. 1D after longer inductions.

   On p.11, it would be interesting to compare measured elongation rates with previously measured estimates for flagellum growth, comparing the growth rates, and relating them to cell cycle times in the corresponding experiments (which vary slightly between labs and studies).

   We attempted to address this in the discussion by comparing our experiments to the assembly rate measured with the PFR as reporter (Bastin et al. 1999). We could mention the corresponding doubling times in correlation to how many cells are bi-flagellated, but this was only done with the Ty-1-tubulin cell line and not with the PFR. In our experiments the average doubling time was ~9 hours with 52% of cells being bi-flagellated. This was measured with FTZC (marker of the transition zone at the base of the flagellum) and Mab25 (marker of the axoneme of the flagellum) which will lead to a slight underestimate of the real number of bi-flagellated cells, as the NF is initially very close which makes it difficult to notice/differentiate from the old one.

   Figure S6. I find the presentation of this figure confusing. It should be revised with clearer labelling of "cell cycle 1", "cell cycle 2", and the precise meaning of "type 3" should be clarified. There are two instances of "type 1" in the drawing, but one of these seems to fulfil the criteria of "type 3" (OF 1-4µm).

   We agree with the reviewer and therefore decided to remove this figure. We also considered the comments of the other two reviewers about complexity of the manuscript and changed the text of figure 5 to make it more approachable. This includes a simpler explanation for the expected amounts of flagella.

   Figure 7. In panel A, the absence of label at the NF distal end is not total, a purple line is still visible. Was any quantitation attempted (signal intensity, changes in length of labelled fragments over time?). Minimally, say how many cells were analysed for the numbers in panels D and E, and how many times this experiment was done.

   We agree with the reviewer that the decrease in the TMR signal in the NF of the cell in the original Fig. 7A (currently Fig. 8A) is gradual and not abrupt. Similarly to the Ty-1-tubulin experiments where the tagged protein becomes progressively more available (increasing intensity), the intensity of TMR-ligand becomes progressively less abundant (gradually decreasing intensity) as new (not TMR labelled) protein gets synthesized during the period of NF construction, progressively diluting the initially fully labeled population of RSP4/6. The slope of the gradient may differ between axonemal constituents, as it reflects the kinetics of protein synthesis, degradation, its incorporation into the axoneme, as well as the size of the soluble protein pool in the cytosol. We classify this type of signal as gradients, as opposed to the sharp decrease. At initial times after TMR-ligand washout (e.g. 4 hours in Fig. 8C), this long gradient is observed at the distal end of NFs and in some uniflagellated cells (NF-inheriting daughters). The distal ends of OFs in these experiments (if not fully labelled) display a sharp decrease, as do frequent uniflagellated cells, likely OF-inheriting daughters. The existence of these two different patterns demonstrates that two different mechanisms are responsible for incorporation of fresh RSP4/6 into the NF and OF axoneme, respectively. While incorporation into the NF is gradual, incorporation into the distal region of the OF is stepwise (restricted in time). Numbers of cells quantified for the table in Fig. 8 have been added. The NFs and OFs displaying the patterns of the gradient and sharp decrease, respectively, were observed in multiple experiments.

   Reviewer #3 (Significance (Required)):

   • General assessment: strengths and limitations

   Strengths: Trypanosoma brucei is a powerful model system in which to ask detailed questions about the assembly dynamics and hierarchy of microtubule-based cytoskeletal structures in general and cilia in particular. This elegant and well-designed study overcomes a previous technical limitation by allowing for the direct labelling of alpha tubulin, one of the main building blocks of the ciliary axoneme. The study sets out to test a specific hypothesis (grow-and-lock model) and provides evidence in support, leading to a refined model for cilia length regulation in trypanosomes.

   Limitations: With this system, visualisation of new tubulin incorporation requires de novo synthesis. There is a time lag between inducing expression of Ty-1-tubulin with tetracycline and being able to visualize the tagged proteins that needs to be taken into consideration. This time lag was estimated based on previous studies and the relatively quick appearance of Ty-1-tubulin on Western blots (within hours). This inevitably creates a situation where levels of tagged tubulin change rapidly, creating gradients of signal intensity (and variations in levels) that lead to some uncertainty in estimations of length of labelled microtubule fragments. Furhtermore, the epitope label is not compatible with live cell imaging, restricting analyses to fixed cells. The Ty-1-tubulin data is well ducmented; the RSP4/6 data appear to corroborate these findings but are less extensively documented.

   • Advance: The results succeed in integrating several recent findings from different research groups into a refined coherent model about cilia length regulation in trypanosomes. The tubulin tagging method could be gainfully transferred to other systems (although the state of the field in tubulin tagging in other systems is not clearly laid out in the paper).

   This paper could be of interest to a broad cell biology community interested in cilia and cytoskeletal dynamics.

   Read the original source
2. 

   Review Commons

   May 15, 2025

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #3

   Evidence, reproducibility and clarity

   Summary:

   This study seeks to investigate the mechanism by which the length of an eukaryotic cilium is set and maintained in a constant state. The flagellated protist Trypanosoma brucei serves as the study model and the authors take advantage of the genetic tools that allow precise modification and tagging of flagellar proteins and they build on prior knowledge about the well-characterised flagellar assembly cycle, which allows tracking the assembly of a new flagellum alongside an existing old one in the course of one cell cycle. The group of Bastin has previously reported a very interesting "Grow-and-Lock Model for the Control of …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #3

   Evidence, reproducibility and clarity

   Summary:

   This study seeks to investigate the mechanism by which the length of an eukaryotic cilium is set and maintained in a constant state. The flagellated protist Trypanosoma brucei serves as the study model and the authors take advantage of the genetic tools that allow precise modification and tagging of flagellar proteins and they build on prior knowledge about the well-characterised flagellar assembly cycle, which allows tracking the assembly of a new flagellum alongside an existing old one in the course of one cell cycle. The group of Bastin has previously reported a very interesting "Grow-and-Lock Model for the Control of Flagellum Length in Trypanosomes" and this current manuscript provides a test of this model, and a refinement. Key to this is an advance in technique, reported here, namely expression of an epitope tagged version of alpha tubulin. The epitope is inserted in an internal loop, which apparently for the first time provides a traceable tubulin that is reliably incorporated into the cytoskeleton (subpellicular array, spindle and cilium). Expressing an inducible version of this Ty-1-tubulin allows for a set of experiments that measure the place and timing of tubulin incorporation into cilia. The results are largely confirmatory of previous findings (incorporation exclusively into the new flagellum, at the distal end, linear growth rate that matches previous estimates). Examination of tubulin incorporation patterns then reveal additional information about the old flagellum: evidence from Ty-1-tubulin labelling, corroborated by incorporation patterns of another ciliary protein (RSP 4/6) suggest that the "lock" on the old flagellum is relieved for short periods after cell division, leading to a refined model presented in Figure 8.

   Major comments:

   This study provides an elegant test of the grow-and-lock model and the major conclusions are supported by the data. I have no major concerns.

   Minor comments:

   There are several minor points that could be addressed to make the manuscript easier to follow (and adding line numbers to the manuscript would help with reviewing).

   The introduction is quite long. Some of the well-established background information on the T. brucei cell cycle could be shortened. If the paper is intended for a broader audience, it would be valuable instead to cite studies that have succeeded in tagging tubulin and tracing its incorporation in other cilia. Could the Ty-1-tubulin approach be relevant more broadly or are simpler methods already established?

   On p.6 the rationale for endogenous tagging was to "reduce the risk of artifacts portentially due to untimely expression or unnatural protein levels". However most of the experiments were done with ectopically expressed inducible Ty-1-tubulin. For the experiments it is crucial to use an inducible system but the authors may wish to comment why the risk of artifacts was no longer a concern.

   On p.7 / Fig S2A-B there appears to be a mistake in the presentation. Spindles are mentioned in the text - I can't see any in the figure. Fig S2A and B both show cytoskeletons, but the text suggests only B is about cytoskeletons. None of the blot shows BB2 staining of different cell fractions, contrary to statements in the text. The letter codes in the panel (T, C, D) don't match the codes in the legend (T, P, S).

   Figure 1. The evidence for incorporation into spindles is not strong. The structure indicated by the arrive could be a spindle but it's not very clear. There is a great example of a labelled spindle only in figure S5A. Here, at the start, it would be good to show a panel of cells in successive cell cycle stages (best, whole cells and cytoskeletons) to clearly show the structures that are labelled with Ty-1-tubulin.

   On p.8 (end of first paragraph) there is reference to cell cycle analyses, but no data is shown. Also on p.8, please clarify what the evidence is that "a fraction of cells did not respond to tetracycline". The fact that they remain unstained by Ty-1-tubulin is not in itself evidence they did not respond to tetracycline.

   Figure S4A. The blot for the soluble fraction is not of great quality. I don't see how the conclusion was reached that the Ty-1-tubulin bands were faint.

   On p.11, it would be interesting to compare measured elongation rates with previously measured estimates for flagellum growth, comparing the growth rates, and relating them to cell cycle times in the corresponding experiments (which vary slightly between labs and studies).

   Figure S6. I find the presentation of this figure confusing. It should be revised with clearer labelling of "cell cycle 1", "cell cycle 2", and the precise meaning of "type 3" should be clarified. There are two instances of "type 1" in the drawing, but one of these seems to fulfil the criteria of "type 3" (OF 1-4µm).

   Figure 7. In panel A, the absence of label at the NF distal end is not total, a purple line is still visible. Was any quantitation attempted (signal intensity, changes in length of labelled fragments over time?). Minimally, say how many cells were analysed for the numbers in panels D and E, and how many times this experiment was done.

   Significance

   General assessment: strengths and limitations

   Strengths: Trypanosoma brucei is a powerful model system in which to ask detailed questions about the assembly dynamics and hierarchy of microtubule-based cytoskeletal structures in general and cilia in particular. This elegant and well-designed study overcomes a previous technical limitation by allowing for the direct labelling of alpha tubulin, one of the main building blocks of the ciliary axoneme. The study sets out to test a specific hypothesis (grow-and-lock model) and provides evidence in support, leading to a refined model for cilia length regulation in trypanosomes.

   Limitations: With this system, visualisation of new tubulin incorporation requires de novo synthesis. There is a time lag between inducing expression of Ty-1-tubulin with tetracycline and being able to visualize the tagged proteins that needs to be taken into consideration. This time lag was estimated based on previous studies and the relatively quick appearance of Ty-1-tubulin on Western blots (within hours). This inevitably creates a situation where levels of tagged tubulin change rapidly, creating gradients of signal intensity (and variations in levels) that lead to some uncertainty in estimations of length of labelled microtubule fragments. Furhtermore, the epitope label is not compatible with live cell imaging, restricting analyses to fixed cells. The Ty-1-tubulin data is well ducmented; the RSP4/6 data appear to corroborate these findings but are less extensively documented.

   Advance: The results succeed in integrating several recent findings from different research groups into a refined coherent model about cilia length regulation in trypanosomes. The tubulin tagging method could be gainfully transferred to other systems (although he state of the field in tubulin tagging in other systems is not clearly laid out in the paper).

   This paper could be of interest to a broad cell biology community interested in cilia and cytoskeletal dynamics.

   Read the original source
3. 

   Review Commons

   May 15, 2025

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   Summary:

   The length of the old flagellum of Trypanosome is constant during G1 phase as well as during cell cycle progression when the new flagellum is assembled. The authors have previously proposed a "Grow and Lock" model for the flagellar length control in which no flagellar building blocks are incorporated. To test this hypothesis, the authors used a tagging strategy for alpha-tubulin and tracking its incorporation. The authors showed that the new flagellum incorporates new tubulins, as is expected. For the mature flagellum, tubulins are incorporated at the flagellar tip and only when the cells start to assemble the new …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   Summary:

   The length of the old flagellum of Trypanosome is constant during G1 phase as well as during cell cycle progression when the new flagellum is assembled. The authors have previously proposed a "Grow and Lock" model for the flagellar length control in which no flagellar building blocks are incorporated. To test this hypothesis, the authors used a tagging strategy for alpha-tubulin and tracking its incorporation. The authors showed that the new flagellum incorporates new tubulins, as is expected. For the mature flagellum, tubulins are incorporated at the flagellar tip and only when the cells start to assemble the new flagellum. Thus, it shows that old flagellum is stable but not completely locked for the incorporation of tubulins.

   Major comments:

   The study is methodologically rigorous, integrating fluorescence microscopy, biochemical approaches, and proteomic analyses to validate the functionality of the tagged tubulin. The use of both inducible expression and endogenous protein tagging (HaloTag) strengthens the conclusions. This study has supported the "Grow-and-Lock" model" that the authors previously proposed. In addition, they have revealed that the stability of the old flagellum is temporally controlled.

   The data showed that brief incorporation of tubulins at the tip of the old flagellum occurs when the cells start to form the new flagellum. I thought the assembly of the new flagellum occurs during the cell division. However, in the abstract, it says that "The restriction is lifted briefly after the bi-flagellated cell has divided." Is my understanding wrong?

   P12, "The cartoon in Fig. 5A illustrates the progression of the cells in scenario 2 (Fig. 4A) over the duration of one cell cycle (~9 hours)" I thought that one cell cycle should start with cell with only one flagellum, followed by assembly of a new flagellum during cell division, the cell then divides when the new flagellum is almost completely assembled. If my understanding is correct, perhaps the cartoon should be modified accordingly.

   Minor comments:

   1. Several references are not correctly formatted. P3: (Flavin and Slaughter, 1974) (Rosenbaum 1969). P10, (Sherwin et al., 1987)(Sheriff et al., 2014)
   2. In several places there are no space between the number and the unit. For eample, P3, 9 - 24µm/h. 7, 1μg/m; P8, 50kDa; P9, 1M; 8-9h; P11, 2.9µm/h and etc.
   3. P11, Flagella were extracted. I thought the cells were extracted.

   Significance

   Cilia and eukaryotic flagella are considered dynamic structures in which the flagellar components especially tubulins under constant turnovers even in steady state. This work demonstrates that in Trypanosome the stable old flagellum is temporally controlled for tubulin turnovers, suggesting a tight regulation of microtubule dynamics. Future elucidation of the regulatory mechanism will be more interesting. This work will be interesting to the field of cilia and microtubules. In addition, the new technique used for tracking tubulins will also be interesting.

   I am an expert on ciliary biology.

   Read the original source
4. 

   Review Commons

   May 15, 2025

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   The manuscript by Daniel Abbühl on "A novel approach to tagging tubulin reveals MT assembly dynamics of the axoneme in Trypanosoma brucei" uses an innnovative approach to label tubulin, which allows the authors to unveil new mechanisms in flagellar length regulation.

   The manuscript is very nice and will be very interesting for the cell biology community and therefore should be accepted. In some parts it becames a bit complex with all the models and complex phrasing, I wonder whether the text could be simplified to be more appealing. I have a few minor comments:

   • From the model the authors show in Figure 8- there should be a way of …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   The manuscript by Daniel Abbühl on "A novel approach to tagging tubulin reveals MT assembly dynamics of the axoneme in Trypanosoma brucei" uses an innnovative approach to label tubulin, which allows the authors to unveil new mechanisms in flagellar length regulation.

   The manuscript is very nice and will be very interesting for the cell biology community and therefore should be accepted. In some parts it becames a bit complex with all the models and complex phrasing, I wonder whether the text could be simplified to be more appealing. I have a few minor comments:

   • From the model the authors show in Figure 8- there should be a way of pulsing the cells in G1 for a short amount of time -2 hours- and getting both flagella tips labelled. But the authors seem to require longer labelling to get that result. This should be better explained.
   • Why do some cells not express the construct? Weren´t they all selected?
   • "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". However, in the authors data the delay may amount to one hour (western analysis- S4). Shouldn´t they use their data.
   • Fig 3: To measure the timepoints of flagella growth, wouldn´t it be better to do it with NF that started to grow before induction, rather than starting to grow after induction, to be sure that the timing of incorporation is fully accounted for?
   • Although it is not the focus of the manuscript it would have been very interesting to use the CEP164C mutant to see whether it would change the dynamics of incorporation and fully test their model and discussion.
   • In some parts of the manuscript/supplemental material the authors say they insert the Ty-1- tag one aminoacid after the acetylated lysine- other parts they say two aminoacids after- this should be consistent.
   • Fig. S1: 'Binding epitope of the TAT-1 antibody is highlighted in red'. There is no highlighting in red in this figure?
   • Fig. S2: Western blots are not very clear. What is the 'X' present in the C (first lane)? Weight of markers should be shown also in S4.
   • Fig 5: 'C: Frequency of bi-flagellated cells grouped by the different types of' The authors didn't finish the sentence.
   • Fig. S7: The 'B' is missing in both picture and legend.

   Significance

   This study advances our knowledge of flagellar length regulation and maintenance. Moreover the tools designed in this work will be very useful for the cell biology community in general.

   Read the original source
5. 

   Version published to 10.1101/2025.01.27.634986 on bioRxiv

   Jan 27, 2025