ERK pathway activation inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

  1. Larissa L Dougherty
  2. Soumita Dutta
  3. Prachee Avasthi

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Mitogen-activated protein kinase (MAPK) pathways are well known regulators of the cell cycle but they have also been found to control ciliary length in a wide variety of organisms and cell types from Caenorhabditis elegans neurons to mammalian photoreceptors through unknown mechanisms. ERK1/2 is a MAP kinase in human cells that is predominantly phosphorylated by MEK1/2 and dephosphorylated by the phosphatase DUSP6. We have found that the ERK1/2 activator/DUSP6 inhibitor, (E)-2-benzylidine-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), inhibits ciliary maintenance in Chlamydomonas and hTERT-RPE1 cells and assembly in Chlamydomonas . These effects involve inhibition of total protein synthesis, microtubule organization, membrane trafficking, and partial kinesin-2 motor dynamics. Our data provide evidence for various avenues for BCI-induced ciliary shortening and impaired ciliogenesis that gives mechanistic insight into how MAP kinases can regulate ciliary length.

Article activity feed

  5. Review Commons

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

    Learn more at Review Commons

    Reply to the reviewers

    Manuscript number: RC-2022-01501

    Corresponding author(s): Prachee Avasthi

    [The “revision plan” should delineate the revisions that authors intend to carry out in response to the points raised by the referees. It also provides the authors with the opportunity to explain their view of the paper and of the referee reports.

    The document is important for the editors of affiliate journals when they make a first decision on the transferred manuscript. It will also be useful to readers of the reprint and help them to obtain a balanced view of the paper.

    If you wish to submit a full revision, please use our "Full Revision" template. It is important to use the appropriate template to clearly inform the editors of your intentions.]

    1. General Statements [optional]

    This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.

    We thank the reviewers for their careful reading and evaluation of our manuscript. The reviewers have emphasized the need for several important changes which we plan to address.

    First, they request better evidence and specificity of the BCI target in Chlamydomonas. We have created double mutants between the dusp6 ortholog mutants and found severe defects in ciliogenesis similar to what we see with BCI treatment. We plan to include this data in the paper as well as the subsequent analyses we performed with the single dusp6 ortholog mutants. This data will provide stronger evidence that this pathway regulates ciliary length in *Chlamydomonas *aside from the other potential off target effects that could be impacting this pathway that we may be seeing through the use of BCI.

    Second, the reviewers have requested more consistency and clarity both in statistics and descriptions of the data and to expand upon our findings in the discussion. We will create a clear guideline for our use of statistics and adjust the descriptions of the data to fit this guideline more strictly and prevent overstating/oversimplifying results. We will also add more discussion and information related to off target effects of BCI, the importance of the subtle defects in NPHP4 protein expression in the transition zone, and the relevancy of the membrane trafficking data in light of this study.

    2. Description of the planned revisions

    Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

    __Reviewer #1 (Evidence, reproducibility and clarity (Required)):____ __

    __SUMMARY:____ __

    __The authors investigated the effects of an allosteric inhibitor of DUSP (BCI) on cilia length regulation in Chlamydomonas. Among seven conclusions summarized in Fig. 7, BCI is found to severely disrupt cilia regeneration and microtubule reorganization. Additionally, changes in kinesin-II dynamic, ciliary protein synthesis, transition zone composition and membrane trafficking are also explored. All these aspects have been shown to affect cilia length regulation. Findings from this body of work may give insights on how MAPK, a major player in cilia length regulation, functions in various avenues. Additionally, the study of BCI and other specific phosphatase inhibitors may provide a unique addition to the toolset available to uncover this important and complicated mechanism. __

    MAJOR COMMENTS

    __Major comment 1____ __

    __The addition of BCI increases phosphorylated MAPK in Chlamydomonas based on Fig 1B. However, the claim that BCI inhibits Chlamydomonas MKPs is not supported at all. SF1A shows CrMKP2, 3 and 5 are related to each other but distant from HsDUSP6 and DrDUSP6. At the same time, 2 out 3 predicted BCI interacting residues are different from the Hs and Dr DUSP6 in SF1B, contradicting "well conserved" in line 172. Consistently, mutants of these orthologs have little to no ciliary length and regeneration defects compared to BCI treatment (see major comment 6 about statistical significance). I am not convinced that BCI inhibits the identified orthologs or any MKPs in Chlamydomonas. It's possible that BCI inhibits a broad range of phosphatases including the ones listed and/or those for upstream kinases. But such a point is not demonstrated by the presented data. __

    While BCI is predicted to interact with these residues, it is also predicted to interact with the “general acid loop backbone” by fitting in between the a7 helix and the acid loop backbone (Molina et al., 2009).

    MKP2 has ciliary length defects compared to wild type, though it regenerates normally. In addition, we have crossed these mutants together and have found that cells (2x3 12.2 and 3x5 29.4) cannot generate cilia. We will include this data in the supplement and perform follow up analyses on these double mutants. Because these structures are not 100% conserved, and we have changed the text to “partially conserved” to reflect this, it is possible that BCI is hitting all of these DUSPs rather than just one, or the DUSPs may serve compensatory functions that rescue ciliary length.

    __Major comment 3____ __

    __The claims that "BCI inhibits KAP-GFP protein expression" (line 271) and "BCI inhibits ciliary protein synthesis" (line 286) are not convincingly demonstrated. Overlooking that only KAP is investigated instead of kinesin-II, none of the relative intensity from the WB in 30 or 50 µM BCI and the basal body fluorescence intensity indicates a statistically significant difference. The washout made no difference in any of the assay and it's not explained how phosphatase inhibition by BCI might affect overall ciliary protein synthesis. The claims about protein expression may need a fair amount of effort and time investment to demonstrate, therefore I suggest leaving these out for this manuscript. __

    __Though it's very interesting to see that in SF 2C cilia in 20 µM BCI treatment can regeneration slowly. Line 162, the author claimed "In the presence of (30 µM) BCI, cilia could not regenerate at all (Fig 1E)". Since Fig 1E only extends to 2 hours, I think it's important to clarify if in 30 µM BCI cilia indeed can not generate even after 6 or 8 hours. __

    We have altered the text to be more specific with our wording that KAP-GFP is investigated rather than kinesin-2, and we have added text to indicate that downstream phosphorylation events could impact transcription and translation of proteins necessary for ciliary maintenance. This interpretation of the data mentioned above is correct; KAP-GFP is not significantly altered at the basal bodies or in accordance with the steady state western blots. What we see here and demonstrated in Figure 2F-I is the depleted KAP-GFP protein which is not restored following a 2 hour regeneration in BCI. We likely do not see a difference in steady state conditions because the protein is not degraded, just being moved around in the cell. We can only see the difference when the majority of KAP-GFP, which the data suggests is mostly present in cilia, is physically removed through ciliary shedding. This protein is not replaced during a 2 hour regeneration which allows us to conclude that this protein is inhibited due to BCI.

    The washout made a small difference in the double regeneration whereby we begin to see cilia begin to form in washed out conditions, though this was not statistically significant. It is possible that BCI has a potent effect on the cell similar to how other drugs, such as colchicine, cannot be easily washed out. The purpose here is to show that regardless of the statistical significance, cells can begin to regenerate their cilia after BCI washout, though this occurs 4 hours after washout in doubly regenerated cells, and we do not see this potent effect on the singly regenerated cells in SF 2C. Though in SF2C, as mentioned, we do see slowly growing cilia, and this could, once again, be due to the potent inhibition BCI has on ciliary protein synthesis. We will confirm and clarify if 30 µM BCI cannot regenerate even after 6 or 8 hours.

    __Major comment 5____ __

    __It is very interesting that BCI disrupts microtubule reorganization induced by deciliation and colchicine. Data in Fig 6B and C are presented differently than those in SF 4C. For example, in SF 4C, BCI treatment for 60 min has close to 50 % cells with microtubule partially reorganized while in Fig 6C about 20% cells with microtubule fully (or combined?) reorganized. The nature of the difference is unclear to me without an assay comparing the two directly. Hence the implied claim that BCI affects colchicine induced microtubule reorganization differently than deciliation induced one is hard to interpret (line 398, line 388 vs line 403). __

    __The fact that taxol doesn't rescue cilia regeneration defect by BCI is very interesting. Here taxol treatment results in fully regenerated cilia while Junmin Pan's group (Wang et. al., 2013) reported much shorter regenerated cilia. It might be worthwhile to compare the experimental variance as this is a key data point in both instances. The relationship between cilia regeneration and microtubule dynamic is not in one direction. On one side, there's a significant upregulation of tubulin after deciliation. While many microtubule depolymerization factors such as katanin, kinesin-13 positively regulate cilia assembly (though not without exceptions). It is hard to determine that the BCI induced cilia regeneration defect can't be rescued by other forms of microtubule stabilization. Microtubule reorganization is one of the most striking defects related to BCI treatment. I suggest changing the oversimplified claim to a more limited one (such as "PTX stabilized microtubule ...") and an expansion on the discussion about microtubule dynamics and cilia length regulation beyond the use of taxol. Meanwhile, I strongly encourage authors to continue to investigate this aspect and its connection to the cilia regeneration. __

    We will remove data regarding “partially” formed cytoplasmic microtubules and only include fully formed for each of these experiments for clarity.

    It is important to note the different taxol concentration used here. While Wang et al., 2013 used 40 µM taxol to study ciliary affects, we use 15 µM where stabilization still occurs. There have been reports of varied cell responses to higher vs. lower doses of taxol (see Ikui et al., 2005, Pushkarev 2009, Yeung 1999) mostly with regards to the cell’s mitotic/apoptotic response. We could be seeing altered responses at this lower concentration because Chlamydomonas cells also behave differently in higher vs. lower taxol concentrations. Thank you for your suggestions. We have adjusted the text to be more specific to PTX treatment as opposed to general stabilization.

    __Major comment 7:____ __

    __There are several places where the technical detail or presentation of the data are missing or clearly erroneous. __

    __Fig 1B: pMAPK and MAPK antibodies used in the WB are not described in the Material and methods. It's not clear if the same #9101, CST antibody used for RPE1 cell in Fig 1J is used. __

    We have updated the materials and methods to include that this antibody was used for both RPE1 and Chlamydomonas cells.

    __line 260 and Fig 3A state 20 µM BCI was used while Fig 3 legend repeatedly states 30 µM until (J). Also 30 µM in SF 2A. __

    We have corrected the text to 20 µM BCI in the mentioned places.

    __Fig 6C, the two lines under p value on top mostly likely start from the second column (B) instead of the first (D). Fig 6G, the line is perhaps intended for the second and fourth columns? __

    __We will make these comparisons more clear. We had performed a chi-square analysis and were comparing the difference between DMSO and BCI before PTX stabilization or MG132 treatment to after. We will add brackets to more clearly show these comparisons. __

    __Fig 6C, legends indicate bars representing each category. But only one bar is shown for each column. Same for 6G? __

    __This is the same as the previous comment for the way we represented the statistics. We will make this clearer with brackets to show the comparisons. __

    __Minor comments:____ __

    1. __ A number of small errors in text were noted above. __ Done.

    __ "orthologs" is misused in place of "ortholog mutants": line 176, 352, 421 (first), 879, 882, 898, 902, 938 , 939. __

    Done.

    __ Capital names is misused as mutant names (e.g. "MKP2"should be "mkp2"): line 178, SF 1C, 1D and 1E, SF 3C, SF 6A __

    Done.

    __ At several places such statistical analysis lines indicated are chosen confusingly. A simplest example is in Fig 1D, the comparison between 0 to 45 is less important than 0 to 30. Same as in Fig 1H, 1I. The line ends are inconsistent as well. They either end in the middle or the edge of the columns/data points (such as in SF 4B) and some with vertical lines (SF 2B, SF 4A, SF 6B). I suggest adding vertical lines pointing to the middle to indicate the compared datasets clearly. __

    Thank you for this suggestion. We agree and will update the figures to reflect this and provide clarity for statistical comparisons.

    __ line 101 remove "the" __

    Done.

    __ line 120 "modulate" to "alter" __

    Done.

    __ line 198 "N=30" should be "N=3" __

    Done.

    __ line 212. The legend for p value is likely for (G) __

    Done.

    __ line 284, "singly" should be "single" __

    Done.

    __ The dataset for "Pre" and "0m" in Fig 6D and 6E are clearly the same. Consider combining the two as in Fig 6C. __

    __This is correct. We will combine the data sets. __

    __ Fig 6E, "BCI" on the X-axis should be "DMSO". __

    __This is correct. We will correct this. __

    __ line 685, remove "?". __

    Done.

    __ line 894: "Fig 3J" instead of "Fig 3H" __

    Done.

    __ SF 1 legend, (C) and (D) are inverted. __

    Done.

    __ SF 4A "Recovered" should be "Full" __

    Done.

    __ SF 5, row 5, under second arrow perhaps missing +PTX __

    Done. We greatly appreciate this close reading of the text and the list of changes making these errors easy to find. We will make these changes in the manuscript.

    __Reviewer #1 (Significance (Required)):____ __

    __Increasing evidence indicates that several MAPKs activated by phosphorylation negatively control cilia length while few studies focus on how MAPK dephosphorylation affects cilia length regulation, largely due to the unknown identity of the phosphatase(s) specifically involved in cilia length regulation. The authors set out to investigate the effect of BCI on cilia length control. BCI specifically inhibits DUSP1 and DUSP6, both of which are known MAPK phosphatase, and therefore may provide a unique opportunity to understand how MAPK pathway is controlled by specific phosphatase(s) activity in cilia length regulation. __

    __Overlooking some inconclusive results and oversimplified interpretations, I find the most striking findings are the BCI's effects including ciliogenesis, kinesin-2 ciliary dynamics and microtubule reorganization. I believe these findings have significant relevance to the stated goal (line 131) and conclusions (line 57) and readers may find them a good starting point for further investigation of the role phosphatases play in cilia length regulation. __

    Cilia length regulation is a complicated mechanism that is affected by many aspects of the cell and functions differently in various systems. My field of expertise may be summarized by cilia biology, cilia length regulation, IFT, kinesin, kinases (MAPKs), microtubules. The membrane trafficking's role in cilia length regulation is somewhat unfamiliar to me. Additionally, the authors used a number of statistical tests and corrections in various assays. The nuance of these choices is not clear to me and neither explained to general readers.

    __Reviewer #2 (Evidence, reproducibility and clarity (Required)): __

    __In their manuscript, "ERK pathway activation inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement," Dougherty et al investigate how BCI, an activator of MAPK signaling, regulates ciliary length. Despite advances in our understanding of the structure and function of cilia, a fundamental question remains as to what are the mechanisms that control ciliary length. This is a critical question because cilia undergo dynamic changes in structure during the cell cycle where they must disassemble as they enter the cell cycle and must rebuild after cell division. This work contributes to a growing body of work to determine mechanisms that regulate cilia length. __

    The authors use a well-established model system, Chlamydomonas, to study cilia dynamics. This work expands on previous findings from these authors that inhibition of MAPK signaling using U0126 lengthens cilia as well as other publications that implicate MAPK signaling in controlling ciliary length. However, the authors only observe a few significant phenotypes with other subtle trends, leaving the conclusion regarding the role of MAPK signaling murky. Furthermore, it is unclear through what mechanism BCI impacts ciliary length. Several issues must be addressed:

    MAJOR ISSUES

    1. __ The basis for this study is the use of the ERK activator BCI, which the authors show activates MAPK signaling. While the authors do use putative DUSP6 ortholog mutants to corroborate some of the phenotypes, the majority of the data (and conclusions) uses BCI. However, there may be off target effects and the authors do not address this limitation of the study. The authors only use 1 pharmacological tool to manipulate MAPK signaling, so it is unclear whether these ciliary disruptions are specifically due to increased MAPK. It is necessary to clarify the following questions about BCI action to interpret the results: __
    • __ ____a.____ What are off target effects of BCI? Does BCI impact proliferation? Why is the BCI phenotype of cilia shortening transient and dose dependent? Why does the phenotype of cilia length and regeneration capacity in Chlamydomonas differ from both ortholog mutants and hTERT-RPE1 cells? __ While we do mention following supplemental figure 1 that other MKPs could be the target for BCI, we also cite Molina et al., 2009 who showed specificity for BCI hydrochloride in zebrafish. BCI targets primarily DUSP6, but also exhibited some activity towards DUSP1. In this study, the authors had also used zebrafish embryos to check expression of 2 other FGF inhibitors, spry 4 and XFD, in the presence of BCI but found that their effects were not reversed. In addition, they checked the ability for BCI to suppress activity of other phosphatases including Cdc25B, PTP1B, or DUSP3/VHR and found that BCI could not suppress these phosphatases. BCI inhibition has previously been found to be more specific to MAPK phosphatases. In addition, we have previously confirmed that U0126 has a slight lengthening effect on Chlamydomonas which further implicates this pathway in cilium length tuning (Avasthi et al. 2012).

    While cell proliferation assays maybe provide more support for MAPK signaling, it does not clarify lack of off target effects that could also contribute to this same phenotype. We do provide a cell proliferation assay for RPE1 cells where we show that higher concentrations of BCI result in cellular senescence as well (Fig 1I).

    The BCI phenotype of cilia shortening is likely transient and dose dependent due to its effect on ciliary protein synthesis demonstrated in Figure 3J. The increase in drug likely increases its substrate binding to exert its effects on the cell faster, even if this includes off target proteins.

    In RPE1 cells, we are likely seeing differences in regeneration capacity potentially due to their different mechanisms of ciliogenesis (RPE1 cells partake in intracellular ciliogenesis where axonemal assembly begins in the cytosol whereas Chlamydomonas cells partake in extracellular ciliogenesis where axonemal assembly begins after basal bodies dock to the apical membrane), or it could be that we’re missing a delay in regeneration in RPE1 cells after waiting 48 hours for ciliogenesis. We do not check this process sooner. There may be a defect that cells overcome. Additionally, among ortholog mutants and RPE1 compared to BCI-treated wild-type Chlamydomonas, there indeed could be off target effects or the drug could be targeting all of these MKPs rather than just one. We will add this to the discussion for clarity.

    __Reviewer #2 (Significance (Required)): __

    __see above __

    __Reviewer #3 (Evidence, reproducibility and clarity (Required)): __

    SUMMARY:

    __In this study, the authors used a pharmacological approach to explore the function of ERK pathway in ciliogenesis. It has been reported that the alteration of FGF signaling causes abnormal ciliogenesis in several animal models including Xenopus, zebrafish, and mice. However, it remains elusive the molecular detail of how ERK pathway is associated with cilia assembling process. The authors found that the ERK1/2 activator/DUSP6 inhibitor, BCI inhibits ciliogenesis, highlighting the importance of ERK during ciliogenesis. Overall, this paper is well written, data are solid and convincing. This paper will be of great interest to many researchers who are interested in understanding ciliogenesis. The following comment is not mandatory requests but suggestions to improve the paper's significance and impact. __

    MAJOR COMMENTS:

    __- Combination of chemical blocker experiments were well controlled and data are solid. The authors are aware of the side effects of BCI, thus they carefully characterized the phenotypes of Mkp2/3/5 in Chlamydomonas. This reviewer wonders if the levels of ERK1/2 phosphorylation are activated in these mutants. Did the authors examine the levels of ERK1/2 phosphorylation in these mutants? __

    While we do not include the data showing ERK activation in these mutants, we have checked pMAPK activation and found that it is not significantly upregulated in these mutants. This could likely be due to compensatory pathways preventing persistent pMAPK activation. For example, constant ERK activation can lead to negative feedback to regulate this signal for cell cycle progression (Fritsche-Guenther et al., 2011). The ERK pathway has not been fully elucidated in Chlamydomonas, but it is possible that these similar mechanisms are in place for MAPKs. We will include this data in the supplement.

    __Reviewer #3 (Significance (Required)): __

    __Accumulated studies suggest that the FGF signaling pathway plays a pivotal role in ciliogenesis. Disruption of either FGF ligands or its FGF receptor results in defective ciliogenesis in Xenopus and zebrafish. On the other hand, FGF signaling negatively controls the length of cilia in chondrocytes that would cause skeletal dysplasias seen in achondroplasia. Therefore, there is strong evidence suggesting that FGF signaling participates in ciliogenesis in cell-type and tissue-context dependent manners. However, the detailed mechanism of the downstream of FGF signaling in ciliogenesis is still unclear. In this regard, this paper is beneficial for the cilia community to expand the knowledge of how ERK1/2 kinase contributes to the regulation of ciliogenesis. __

    __This reviewer therefore suggests that the authors may want to add more discussion to explain how their finding possibly moves the field forward to understand the pathogenesis of multiple ciliopathies. __

    We will add a description of this to the discussion.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

    __Reviewer 1: __

    __Major comment 4____ __

    __A single panel in Fig 4A also can't support the shift in protein density in the TZ in line 317. As line 324 implies protein synthesis defect by BCI, the very minor (in amount and significance) reduction of the NPHP4 fluorescence should not be interpreted as any disruption at all to the transition zone. I suggest checking other TZ proteins such as CEP290 etc or leave this section out. __

    __Also, The additive effect from BFA and BCI treatment in Fig 5A suggests BCI affects cilia length independent of Golgi. The "actin puncta" and arpc4 mutant are not sufficiently introduced. And more importantly, how increase in the actin puncta explains the shorter cilia length caused by BCI while actin puncta are absent in arpc4 mutant with shorter cilia? Also, the Arl6 fluorescence signal "increase" is not significant in either time point. I suggest leaving this section out as well. __

    We agree that one EM image cannot support a protein shift and have removed our observation in the text. However, we do see a statistically significant decrease in NPHP4 fluorescence in BCI treated cells which we consider a disruption in the sense that the structural composition is altered. We will change the word “disruption” to “alteration” for clarity. Though this is a minor defect, we believe it is still worth noting. We believe this data still adds to the model that though the EM-visible structure is unaltered, finer details within the transition zone are indeed altered and we cannot rule out that these smaller changes are not impacting protein entry into cilia. Awata et al. 2014 shows that NPHP4 is important for controlling trafficking of ciliary proteins at the transition zone, and its loss from the transition zone has been found to have effects in ciliary protein composition. Because we see decreased NPHP4 expression, we believe this is a notable finding as we see effects on the abundance of a protein which is known to affect ciliary protein composition and have therefore chosen to leave the data in the manuscript. We will adjust the language to most accurately describe our findings.

    We also agree with the interpretation that the additive effect seen from BFA and BCI treatment could suggest independent pathway collapse separate from the Golgi which we have mentioned in the manuscript.

    We have provided more information to introduce actin puncta and ARPC4 with regards to membrane trafficking. Bigge et al. 2020 shows that ARPC4, a subunit of the ARP2/3 complex which is an actin binding protein important for nucleating actin branches, has a role in ciliary assembly. ARPC4 mutants have repressed ability to regenerate their cilia. One feature they noticed in regenerating cells is the immediate formation of actin puncta which are reminiscent of yeast endocytic pits. This observation in addition to altered membrane uptake pathways in Chlamydomonas suggests that ciliogenesis involves reclaiming plasma membrane for use in ciliogenesis (because of the diffusion barrier preventing a contiguous membrane). Here, we incorporate this assay to assess the ability for the cell to reclaim membrane during BCI treatment and find that there is increased actin puncta. This could indicate that there is increased number of endocytic pits or alternatively that the lifetime of these pits is increased (perhaps due to incomplete endocytosis) such that we are able to detect more of them at a fixed point in time. While we cannot say which is happening here, we have previously found that these actin puncta are likely endocytic and needed to reclaim membrane for early ciliogenesis. An increase in these puncta may suggest dysregulated endocytosis in one way or another. ARPC4 cells cannot form the actin puncta in the first place, whereas we are seeing defects following puncta formation. We have taken out the Arl6 data.

    __Major comment 6____ __

    __Throughout this manuscript, the standard the authors used to interpret statistical significance is erratic. In a few instances, the threshold for p value is clearly indicated such as in Fig 1 legend. Though other times, much higher p values are considered differences. Here are some examples: __

    __SF 1C, p=0.1167 is considered "(mkp5) shorter than wildtype ciliary lengths" (also line 177 "SF 1C" instead of "SF 1D") __

    __Fig 3C, p=0.083 interpreted as "slightly less" in line 262 and possibly as "(KAP-GFP) not being able to enter (cilia)" in line 268 __

    __Fig 3G, p=0.1087 is considered "not decrease after two hours" line 267 __

    __SF 3C, p=0.2929 for mkp2 mutant (misuse of "orthologs" in line 352) is considered "fewer actin puncta compared to wild type cells" (line 352). __

    __SF 6B, p=0.1565for mkp3 mutant (line 421: misuse of "orthologs" and correct use of "ortholog mutants") is considered not be able to "fully reorganize their microtubules" (line 421). __

    __These instances sometimes serve as basis for major conclusions and should be clarified or more carefully characterized. __

    We agree the interpretations are very erratic in places and greatly appreciate this detailed list making it easy to find and correct these interpretations. We have adjusted the text in the mentioned places to reflect these changes, and we have made a statement in the text and under statistical methods that say we consider p __Reviewer 2: __

    __ In multiple instances the conclusions are overstated, and the author must clarify the interpretation of the results to reflect the data presented. Here are some examples: __

    • __ ____a.____ The conclusion that protein synthesis is disrupted is incorrect in two instances (line 258 and 275) as the experiments in figure 3 do not directly examine changes in synthesis (they look at cilia regeneration as a proxy).__ We show that KAP-GFP expression is not normal during regeneration at 120 minutes which suggests, in addition to the inability for cilia to grow in BCI, that synthesis is inhibited because this protein is not replaced. In addition, blocking the proteosome did not rescue this decrease in KAP-GFP expression indicating that this is not a matter of KAP-GFP protein being degraded rapidly. We use regeneration and KAP-GFP readout as a proxy for protein synthesis. We have clarified this in the text.

    • __ ____b.____ The conclusion that BCI disrupts membrane trafficking is too broad when the authors only examined trafficking of one membrane protein, Arl6. __ While we only looked at one membrane protein specifically, we assess other membrane trafficking paths. We looked at BCI vs. BFA to assess Golgi trafficking (Dentler 2010) in addition to formation of actin puncta which is used in Bigge et al. 2020 as an assay for membrane uptake from the plasma membrane for incorporation into cilia.

    • __ ____c.____ The conclusion that the transition zone is disrupted is too broad based on a decrease in the expression of one transition zone protein, NPHP4. __ We have changed the text to be more specific to NPHP4.

    __ Highlighting the overstatement, the conclusion of the header and figure caption on page 10 contradict one another. The manuscript states that "BCI partially disrupts the transition zone" (line 313) and that "The TZ structure is structurally unaltered with BCI treatment" (line 329). __

    In the manuscript, we show that the EM-visible structure is indeed unaltered. Because we see a decrease in NPHP4 fluorescence, we concluded that while the EM-visible structure is unaltered, protein composition within the transition zone is altered which suggests that BCI partially disrupts the transition zone.

    __ Why is kinesin-2 the only target studied for ciliogenesis? Ciliogenesis is a complex process that involves many other critical proteins and investigating kinesin-2 alone is not sufficient to conclude why BCI prevents cilia assembly. __

    We use kinesin-2 because it is the only ciliary anterograde motor in Chlamydomonas which is required for proper ciliogenesis. By assessing kinesin-2, we were able to address whether this protein alone was the cause for inhibited ciliary assembly (and we find that it’s not), whether its ability to enter was impacted (likely owing to defects in other protein entry), and we were able to use this protein to understand how its protein expression was affected. Because KAP-GFP is a cargo adaptor protein and interacts with IFT complexes and other cargoes, defects in this protein can have a wide range of implications. We agree and the data agree that kinesin-2 alone is not sufficient to conclude why BCI prevents cilia assembly. Because of this, we assessed other pathways including membrane trafficking and microtubule stabilization to better understand why we see defects in ciliary assembly. Certainly many other proteins are important in ciliogenesis and we hope that this study sparks further work in this area to identify additional causative explanations for impaired ciliogenesis upon MAPK activation..

    __ Tagged ciliary proteins are sensitive to disruptions in function and expression within cilia. It is important to include proper controls in the study using KAP-GFP Chlamydomonas cells to ensure that KAP-GFP maintains endogenous expression levels and normal function as untagged KAP. Furthermore, if this information is available through the resource where the cells were purchased, then this needs to be discussed. __

    KAP-GFP expressing Chlamydomonas has previously been validated as described in Mueller et al., 2005. We will provide details in the text about validation of this strain.

    __ The authors need to provide clear explanations to a general audience of why this technique is used and how the authors reached the interpretations. There are several instances where the authors use techniques that are cited as fundamental papers in Chlamydomonas. Here are two examples: __

    • __ ____a.____ It is unclear how the authors concluded that decreased frequency and velocity of train size shows that kinesin entry, specifically, is disrupted.__ We have expanded on this in the text. Please see response to reviewer 1, Major comment 2 above.

    • __ ____b.____ It was impossible to follow how the experiment where cells treated with cycloheximide could not regenerate their cilia following BCI treatment shows that BCI inhibits protein synthesis__. We have adapted the text to be more clear regarding this experiment. In this experiment, we deplete the ciliary protein pool by forcing ciliary shedding two times. Following the first shedding, there is enough protein to assemble cilia to half length (Rosenbaum, 1969). We ensure that the protein pool is completely used up by inhibiting further ciliary protein synthesis with cycloheximide. For the second shedding event, completely new ciliary protein must be synthesized for ciliogenesis to occur which is why ciliogenesis takes much longer compared to a single regeneration where half of the ciliary protein pool still remains and can be immediately incorporated into cilia (SF 2C). In the presence of BCI, cilia cannot grow at all as expected; but 4 hours after BCI is washed out, we see ciliogenesis just beginning to occur which indicates that there is protein present for ciliogenesis to begin whereas in cells where BCI is not washed out, we do not see any ciliogenesis.

    __ The impact of BCI treatment on membrane trafficking as presented is confusing. BCI exacerbated the effects of BFA treatment on Golgi, yet the authors do not address that this could be an indirect effect of BCI or an off-target effect of BCI. __

    This is addressed in the discussion (paragraph 4).

    __ The discussion section includes many interpretations of the results, but leaves the reader confused as to what the authors think might be happening. The manuscript would be far clearer if the authors would provide a working model for why BCI impacts cilia length. It is fine for this to be left for future work but, as the experts, the authors must have relevant thoughts to share with the field. __

    Figure 7 provides a model with as much as we can conclude given the data; what we show is that BCI inhibits many different processes in the cell, but we do not necessarily show links between these processes to provide a complete working model of how these are all interconnected; we have provided a summary model that depicts the various, still disconnected processes that are inhibited by BCI. MAP kinases such as ERK have dozens of downstream targets both within and outside the nucleus. Ciliogenesis also is a complex process coordinating many cellular mechanisms. The intersection of these two seem to have a multi-fold effect that results in a dramatic ciliary phenotype through a combination of factors, however not one that fully explains the severity upon initial deciliation in BCI/MAPK activation. Further work is needed to identify the precise cause of completely inhibited cilium growth from zero length.

    MINOR ISSUES

    1. __ The title of the manuscript is inaccurate and overstates the pathway involvement in cilia. The authors do not directly show that ERK pathway activation causes the ciliary phenotypes due to the use of BCI, a drug that modulates ERK. __ We have adjusted the title to “The ERK activator, BCI, causes…”

    __ When discussing results of data that are not statistically significant it creates confusion to state that the results "increased/decreased slightly". __

    We agree that references to statistics are inconsistent or confusing throughout the text and have adjusted these references accordingly.

    __Reviewer 3: __

    __Major comment: __

    - If the authors want to emphasize their finding is associated with MAP kinases, it would be also beneficial to examine other major MAP kinase pathways such as P38/JNK. If not, then this reviewer suggests revising the text as ERK through this manuscript to avoid confusions.

    Because the ERK pathway has not been fully elucidated in Chlamydomonas, we have refrained from using “ERK” as a descriptor because this particular MAPK shares equal identity with multiple MAPKs in Chlamydomonas. Further, BCI may be targeting more than one MAPK phosphatase resulting in the myriad phenotypes we have discovered. At this time, we lack a level of gene-level resolution to map to known MAPK pathways.

    4. Description of analyses that authors prefer not to carry out

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

    __Reviewer 1: __

    __Major comment 2____ __

    __The claim that "BCI treatment decreases kinesin-2 entry into cilia" (line 236) is a misinterpretation of the data presented. The data indicates KAP-GFP have reduced accumulation in cilia, decreased IFT (anterograde) frequency, velocity and injection size associated with BCI treatment. Though as shown in Fig 1D and Fig 2C, cilia length is also shorter due to BCI treatment. Ludington et. al, 2013 showed a negative correlation of cilia length and KAP injection rate in various treatments that affect cilia length. It's essential to rule out that the KAP dynamics reported in the current manuscript is not an outcome of shortened cilia in order to claim as line 236 seems to suggest. One way to demonstrate specific effect by BCI would be to compare KAP dynamic in cilia with equal or similar length, either by only selecting the shorter cilia from wt or use other treatments that are known to decrease cilia length (chemicals, cell cycle, mutants etc.). Given the capability and resource represented in this manuscript, I don't expect a significant cost and time investment for these experiments. __

    Ludington et al., 2013 shows that injection size decreases with increasing length. Our data show that the shorter length cilia have decreased injection size and rate inconsistent with the cause being due to shortened length alone. In other words, in figure 2C and 2G, we see decreased KAP-GFP fluorescence in shorter cilia as opposed to greater fluorescent signal in shorter cilia seen in Ludington et al., 2013. This data, in combination with the decreasing frequency of KAP-GFP entry overtime in figure 2E and decreased velocity in figure 2F support decreased kinesin-2 entry into cilia. If entry was unaltered, we would expect increased KAP-GFP fluorescence in the cilia over time in BCI-treated cells.

    __Reviewer 2: __

    __ The authors state that the decreased length of cilia following BCI treatment could be a result of reduced assembly or increased assembly. Disruptions to cilia assembly and disassembly are not mutually exclusive and both must be evaluated. The authors do not test whether cilia disassembly is disrupted in BCI treatment and therefore, cannot conclude that BCI solely disrupts cilia assembly. __

    While effects on disassembly remains a possibility, the striking inability to increase from zero length upon deciliation and the effects on anterograde IFT through the TIRFM assays suggest an affect on assembly. There may be effects on disassembly and likely many other cilia related processes not investigated but we feel it remains accurate to conclude that assembly is affected by BCI treatment.

    __Reviewer 3: __

    __- If time allows, in addition to examining NPHP4, it would be beneficial to examine other TZ/TF markers such as CEP164 to confirm if BCI partially disrupts the TZ. __

    Given the known outcomes of NPHP4 loss in Chlamydomonas (Awata et al., …) in affecting ciliary protein composition, we suspect the changes in NPHP4 abundance at the transition zone will have a significant impact and agree it would be interesting in a follow up study to see how other transition zone proteins (particularly ones known to interact with NPHP4 or others critical for TZ function) are impacted following BCI treatment.

    __MINOR COMMENTS: __

    __- I suggest moving supplemental figure 1 to the main figure (Fig. 1?) so that the readers appreciate the author's careful examination of BCI through this manuscript. __

    Thank you for your suggestion and kind critique. We have included this data in the supplement for consistency with mutant data in all of the other supplemental figures.

  6. Review Commons

    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:

    In this study, the authors used a pharmacological approach to explore the function of ERK pathway in ciliogenesis. It has been reported that the alteration of FGF signaling causes abnormal ciliogenesis in several animal models including Xenopus, zebrafish, and mice. However, it remains elusive the molecular detail of how ERK pathway is associated with cilia assembling process. The authors found that the ERK1/2 activator/DUSP6 inhibitor, BCI inhibits ciliogenesis, highlighting the importance of ERK during ciliogenesis. Overall, this paper is well written, data are solid and convincing. This paper will be of great interest to many researchers who are interested in understanding ciliogenesis. The following comment is not mandatory requests but suggestions to improve the paper's significance and impact.

    Major comments:

    • Combination of chemical blocker experiments were well controlled and data are solid. The authors are aware of the side effects of BCI, thus they carefully characterized the phenotypes of Mkp2/3/5 in Chlamydomonas. This reviewer wonders if the levels of ERK1/2 phosphorylation are activated in these mutants. Did the authors examine the levels of ERK1/2 phosphorylation in these mutants?

    • If the authors want to emphasize their finding is associated with MAP kinases, it would be also beneficial to examine other major MAP kinase pathways such as P38/JNK. If not, then this reviewer suggests revising the text as ERK through this manuscript to avoid confusions.

    • If time allows, in addition to examining NPHP4, it would be beneficial to examine other TZ/TF markers such as CEP164 to confirm if BCI partially disrupts the TZ.

    Minor comments:

    • I suggest moving supplemental figure 1 to the main figure (Fig. 1?) so that the readers appreciate the author's careful examination of BCI through this manuscript.

    Significance

    Accumulated studies suggest that the FGF signaling pathway plays a pivotal role in ciliogenesis. Disruption of either FGF ligands or its FGF receptor results in defective ciliogenesis in Xenopus and zebrafish. On the other hand, FGF signaling negatively controls the length of cilia in chondrocytes that would cause skeletal dysplasias seen in achondroplasia. Therefore, there is strong evidence suggesting that FGF signaling participates in ciliogenesis in cell-type and tissue-context dependent manners. However, the detailed mechanism of the downstream of FGF signaling in ciliogenesis is still unclear. In this regard, this paper is beneficial for the cilia community to expand the knowledge of how ERK1/2 kinase contributes to the regulation of ciliongenesis.

    This reviewer therefore suggests that the authors may want to add more discussion to explain how their finding possibly moves the field forward to understand the pathogenesis of multiple ciliopathies.

  7. Review Commons

    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

    In their manuscript, "ERK pathway activation inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement," Dougherty et al investigate how BCI, an activator of MAPK signaling, regulates ciliary length. Despite advances in our understanding of the structure and function of cilia, a fundamental question remains as to what are the mechanisms that control ciliary length. This is a critical question because cilia undergo dynamic changes in structure during the cell cycle where they must disassemble as they enter the cell cycle and must rebuild after cell division. This work contributes to a growing body of work to determine mechanisms that regulate cilia length.

    The authors use a well-established model system, Chlamydomonas, to study cilia dynamics. This work expands on previous findings from these authors that inhibition of MAPK signaling using U0126 lengthens cilia as well as other publications that implicate MAPK signaling in controlling ciliary length. However, the authors only observe a few significant phenotypes with other subtle trends, leaving the conclusion regarding the role of MAPK signaling murky. Furthermore, it is unclear through what mechanism BCI impacts ciliary length. Several issues must be addressed:

    MAJOR ISSUES

    1. The basis for this study is the use of the ERK activator BCI, which the authors show activates MAPK signaling. While the authors do use putative DUSP6 ortholog mutants to corroborate some of the phenotypes, the majority of the data (and conclusions) uses BCI. However, there may be off target effects and the authors do not address this limitation of the study. The authors only use 1 pharmacological tool to manipulate MAPK signaling, so it is unclear whether these ciliary disruptions are specifically due to increased MAPK. It is necessary to clarify the following questions about BCI action to interpret the results:

    a) What are off target effects of BCI? Does BCI impact proliferation? Why is the BCI phenotype of cilia shortening transient and dose dependent? Why does the phenotype of cilia length and regeneration capacity in Chlamydomonas differ from both ortholog mutants and hTERT-RPE1 cells?

    1. In multiple instances the conclusions are overstated, and the author must clarify the interpretation of the results to reflect the data presented. Here are some examples:

    a) The conclusion that protein synthesis is disrupted is incorrect in two instances (line 258 and 275) as the experiments in figure 3 do not directly examine changes in synthesis (they look at cilia regeneration as a proxy).

    b) The conclusion that BCI disrupts membrane trafficking is too broad when the authors only examined trafficking of one membrane protein, Arl6.

    c) The conclusion that the transition zone is disrupted is too broad based on a decrease in the expression of one transition zone protein, NPHP4.

    1. Highlighting the overstatement, the conclusion of the header and figure caption on page 10 contradict one another. The manuscript states that "BCI partially disrupts the transition zone" (line 313) and that "The TZ structure is structurally unaltered with BCI treatment" (line 329).

    2. The authors state that the decreased length of cilia following BCI treatment could be a result of reduced assembly or increased assembly. Disruptions to cilia assembly and disassembly are not mutually exclusive and both must be evaluated. The authors do not test whether cilia disassembly is disrupted in BCI treatment and therefore, cannot conclude that BCI solely disrupts cilia assembly.

    3. Why is kinesin-2 the only target studied for ciliogenesis? Ciliogenesis is a complex process that involves many other critical proteins and investigating kinesin-2 alone is not sufficient to conclude why BCI prevents cilia assembly.

    4. Tagged ciliary proteins are sensitive to disruptions in function and expression within cilia. It is important to include proper controls in the study using KAP-GFP Chlamydomonas cells to ensure that KAP-GFP maintains endogenous expression levels and normal function as untagged KAP. Furthermore, if this information is available through the resource where the cells were purchased, then this needs to be discussed.

    5. The authors need to provide clear explanations to a general audience of why this technique is used and how the authors reached the interpretations. There are several instances where the authors use techniques that are cited as fundamental papers in Chlamydomonas. Here are two examples:

    a) It is unclear how the authors concluded that decreased frequency and velocity of train size shows that kinesin entry, specifically, is disrupted.

    b) It was impossible to follow how the experiment where cells treated with cycloheximide could not regenerate their cilia following BCI treatment shows that BCI inhibits protein synthesis.

    1. The impact of BCI treatment on membrane trafficking as presented is confusing. BCI exacerbated the effects of BFA treatment on Golgi, yet the authors do not address that this could be an indirect effect of BCI or an off-target effect of BCI.

    2. The discussion section includes many interpretations of the results, but leaves the reader confused as to what the authors think might be happening. The manuscript would be far clearer if the authors would provide a working model for why BCI impacts cilia length. It is fine for this to be left for future work but, as the experts, the authors must have relevant thoughts to share with the field.

    MINOR ISSUES

    1. The title of the manuscript is inaccurate and overstates the pathway involvement in cilia. The authors do not directly show that ERK pathway activation causes the ciliary phenotypes due to the use of BCI, a drug that modulates ERK.

    2. When discussing results of data that are not statistically significant it creates confusion to state that the results "increased/decreased slightly".

    Significance

    see above

  8. Review Commons

    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

    Summary:

    The authors investigated the effects of an allosteric inhibitor of DUSP (BCI) on cilia length regulation in Chlamydomonas. Among seven conclusions summarized in Fig. 7, BCI is found to severely disrupt cilia regeneration and microtubule reorganization. Additionally, changes in kinesin-II dynamic, ciliary protein synthesis, transition zone composition and membrane trafficking are also explored. All these aspects have been shown to affect cilia length regulation. Findings from this body of work may give insights on how MAPK, a major player in cilia length regulation, functions in various avenues. Additionally, the study of BCI and other specific phosphatase inhibitors may provide a unique addition to the toolset available to uncover this important and complicated mechanism.

    Major comments:

    Major comment 1

    The addition of BCI increases phosphorylated MAPK in Chlamydomonas based on Fig 1B. However, the claim that BCI inhibits Chlamydomonas MKPs is not supported at all. SF1A shows CrMKP2, 3 and 5 are related to each other but distant from HsDUSP6 and DrDUSP6. At the same time, 2 out 3 predicted BCI interacting residues are different from the Hs and Dr DUSP6 in SF1B, contradicting "well conserved" in line 172. Consistently, mutants of these orthologs have little to no ciliary length and regeneration defects compared to BCI treatment (see major comment 6 about statistical significance). I am not convinced that BCI inhibits the identified orthologs or any MKPs in Chlamydomonas. It's possible that BCI inhibits a broad range of phosphatases including the ones listed and/or those for upstream kinases. But such a point is not demonstrated by the presented data.

    Major comment 2

    The claim that "BCI treatment decreases kinesin-2 entry into cilia" (line 236) is a misinterpretation of the data presented. The data indicates KAP-GFP have reduced accumulation in cilia, decreased IFT (anterograde) frequency, velocity and injection size associated with BCI treatment. Though as shown in Fig 1D and Fig 2C, cilia length is also shorter due to BCI treatment. Ludington et. al, 2013 showed a negative correlation of cilia length and KAP injection rate in various treatments that affect cilia length. It's essential to rule out that the KAP dynamics reported in the current manuscript is not an outcome of shortened cilia in order to claim as line 236 seems to suggest. One way to demonstrate specific effect by BCI would be to compare KAP dynamic in cilia with equal or similar length, either by only selecting the shorter cilia from wt or use other treatments that are known to decrease cilia length (chemicals, cell cycle, mutants etc.). Given the capability and resource represented in this manuscript, I don't expect a significant cost and time investment for these experiments.

    Major comment 3

    The claims that "BCI inhibits KAP-GFP protein expression" (line 271) and "BCI inhibits ciliary protein synthesis" (line 286) are not convincingly demonstrated. Overlooking that only KAP is investigated instead of kinesin-II, none of the relative intensity from the WB in 30 or 50 µM BCI and the basal body fluorescence intensity indicates a statistically significant difference. The washout made no difference in any of the assay and it's not explained how phosphatase inhibition by BCI might affect overall ciliary protein synthesis. The claims about protein expression may need a fair amount of effort and time investment to demonstrate, therefore I suggest leaving these out for this manuscript. Though it's very interesting to see that in SF 2C cilia in 20 µM BCI treatment can regeneration slowly. Line 162, the author claimed "In the presence of (30 µM) BCI, cilia could not regenerate at all (Fig 1E)". Since Fig 1E only extends to 2 hours, I think it's important to clarify if in 30 µM BCI cilia indeed can not generate even after 6 or 8 hours.

    Major comment 4

    A single panel in Fig 4A also can't support the shift in protein density in the TZ in line 317. As line 324 implies protein synthesis defect by BCI, the very minor (in amount and significance) reduction of the NPHP4 fluorescence should not be interpreted as any disruption at all to the transition zone. I suggest checking other TZ proteins such as CEP290 etc or leave this section out. Also, The additive effect from BFA and BCI treatment in Fig 5A suggests BCI affects cilia length independent of Golgi. The "actin puncta" and arpc4 mutant are not sufficiently introduced. And more importantly, how increase in the actin puncta explains the shorter cilia length caused by BCI while actin puncta are absent in arpc4 mutant with shorter cilia? Also, the Arl6 fluorescence signal "increase" is not significant in either time point. I suggest leaving this section out as well.

    Major comment 5

    It is very interesting that BCI disrupts microtubule reorganization induced by deciliation and colchicine. Data in Fig 6B and C are presented differently than those in SF 4C. For example, in SF 4C, BCI treatment for 60 min has close to 50 % cells with microtubule partially reorganized while in Fig 6C about 20% cells with microtubule fully (or combined?) reorganized. The nature of the difference is unclear to me without an assay comparing the two directly. Hence the implied claim that BCI affects colchicine induced microtubule reorganization differently than deciliation induced one is hard to interpret (line 398, line 388 vs line 403). The fact that taxol doesn't rescue cilia regeneration defect by BCI is very interesting. Here taxol treatment results in fully regenerated cilia while Junmin Pan's group (Wang et. al., 2013) reported much shorter regenerated cilia. It might be worthwhile to compare the experimental variance as this is a key data point in both instances. The relationship between cilia regeneration and microtubule dynamic is not in one direction. On one side, there's a significant upregulation of tubulin after deciliation. While many microtubule depolymerization factors such as katanin, kinesin-13 positively regulate cilia assembly (though not without exceptions). It is hard to determine that the BCI induced cilia regeneration defect can't be rescued by other forms of microtubule stabilization. Microtubule reorganization is one of the most striking defects related to BCI treatment. I suggest changing the oversimplified claim to a more limited one (such as "PTX stabilized microtubule ...") and an expansion on the discussion about microtubule dynamics and cilia length regulation beyond the use of taxol. Meanwhile, I strongly encourage authors to continue to investigate this aspect and its connection to the cilia regeneration.

    Major comment 6

    Throughout this manuscript, the standard the authors used to interpret statistical significance is erratic. In a few instances, the threshold for p value is clearly indicated such as in Fig 1 legend. Though other times, much higher p values are considered differences. Here are some examples: SF 1C, p=0.1167 is considered "(mkp5) shorter than wildtype ciliary lengths" (also line 177 "SF 1C" instead of "SF 1D") Fig 3C, p=0.083 interpreted as "slightly less" in line 262 and possibly as "(KAP-GFP) not being able to enter (cilia)" in line 268 Fig 3G, p=0.1087 is considered "not decrease after two hours" line 267 SF 3C, p=0.2929 for mkp2 mutant (misuse of "orthologs" in line 352) is considered "fewer actin puncta compared to wild type cells" (line 352). SF 6B, p=0.1565for mkp3 mutant (line 421: misuse of "orthologs" and correct use of "ortholog mutants") is considered not be able to "fully reorganize their microtubules" (line 421). These instances sometimes serve as basis for major conclusions and should be clarified or more carefully characterized.

    Major comment 7

    There are several places where the technical detail or presentation of the data are missing or clearly erroneous.

    • Fig 1B: pMAPK and MAPK antibodies used in the WB are not described in the Material and methods. It's not clear if the same #9101, CST antibody used for RPE1 cell in Fig 1J is used.
    • line 260 and Fig 3A state 20 µM BCI was used while Fig 3 legend repeatedly states 30 µM until (J). Also 30 µM in SF 2A.
    • Fig 6C, the two lines under p value on top mostly likely start from the second column (B) instead of the first (D). Fig 6G, the line is perhaps intended for the second and fourth columns?
    • Fig 6C, legends indicate bars representing each category. But only one bar is shown for each column. Same for 6G?

    Minor comments:

    1. A number of small errors in text were noted above.

    2. "orthologs" is misused in place of "ortholog mutants": line 176, 352, 421 (first), 879, 882, 898, 902, 938 , 939.

    3. Capital names is misused as mutant names (e.g. "MKP2"should be "mkp2"): line 178, SF 1C, 1D and 1E, SF 3C, SF 6A

    4. At several places such statistical analysis lines indicated are chosen confusingly. A simplest example is in Fig 1D, the comparison between 0 to 45 is less important than 0 to 30. Same as in Fig 1H, 1I. The line ends are inconsistent as well. They either end in the middle or the edge of the columns/data points (such as in SF 4B) and some with vertical lines (SF 2B, SF 4A, SF 6B). I suggest adding vertical lines pointing to the middle to indicate the compared datasets clearly.

    5. line 101 remove "the"

    6. line 120 "modulate" to "alter"

    7. line 198 "N=30" should be "N=3"

    8. line 212. The legend for p value is likely for (G)

    9. line 284, "singly" should be "single"

    10. The dataset for "Pre" and "0m" in Fig 6D and 6E are clearly the same. Consider combining the two as in Fig 6C.

    11. Fig 6E, "BCI" on the X-axis should be "DMSO".

    12. line 685, remove "?".

    13. line 894: "Fig 3J" instead of "Fig 3H"

    14. SF 1 legend, (C) and (D) are inverted.

    15. SF 4A "Recovered" should be "Full"

    16. SF 5, row 5, under second arrow perhaps missing +PTX

    Significance

    Increasing evidence indicates that several MAPKs activated by phosphorylation negatively control cilia length while few studies focus on how MAPK dephosphorylation affects cilia length regulation, largely due to the unknown identity of the phosphatase(s) specifically involved in cilia length regulation. The authors set out to investigate the effect of BCI on cilia length control. BCI specifically inhibits DUSP1 and DUSP6, both of which are known MAPK phosphatase, and therefore may provide a unique opportunity to understand how MAPK pathway is controlled by specific phosphatase(s) activity in cilia length regulation.

    Overlooking some inconclusive results and oversimplified interpretations, I find the most striking findings are the BCI's effects including ciliogenesis, kinesin-2 ciliary dynamics and microtubule reorganization. I believe these findings have significant relevance to the stated goal (line 131) and conclusions (line 57) and readers may find them a good starting point for further investigation of the role phosphatases play in cilia length regulation.

    Cilia length regulation is a complicated mechanism that is affected by many aspects of the cell and functions differently in various systems. My field of expertise may be summarized by cilia biology, cilia length regulation, IFT, kinesin, kinases (MAPKs), microtubules. The membrane trafficking's role in cilia length regulation is somewhat unfamiliar to me. Additionally, the authors used a number of statistical tests and corrections in various assays. The nuance of these choices is not clear to me and neither explained to general readers.

  9. ASAPbio crowd review

    Review coordinated via ASAPbio’s crowd preprint review

    This review reflects comments and contributions by Joachim Goedhart, Sónia Gomes Pereira, Ricardo Bruno Carvalho, Anchal Chandra, Akanksha Verma, Claudia Molina, Richa Arya, Rachel Lau, Xianrui Cheng, Ehssan Moglad, Rinalda Proko, Luciana Gallo, Parijat Sil, Yogaspoorthi Subramaniam. Review synthesized by Vasanthanarayan Murugesan.

    The reciprocal regulatory relationship between the cell cycle and ciliogenesis is poorly understood. This study by Dougherty et al. aims to better understand how MAPK signaling pathways control ciliary assembly in Chlamydomonas and RPE1 cells. ERK1/2 is a MAPK protein that is activated predominantly by MEK1/2 and deactivated by DUSP6.

    For this, the study activates ERK, a well-known MAPK pathway, by inhibiting its phosphatase DUSP6 through the compound BIC ((E)-2-benzylidine-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one). The study shows that BIC leads to impaired ciliary assembly and maintenance in Chlamydomonas and impaired ciliary growth in hTERT-RPE1 cells. It further shows, in Chlamydomonas, that BCI inhibits ciliogenesis by disrupting total protein synthesis, microtubule organization, membrane trafficking, and partial kinesin-2 motor dynamics.

    The use of superplots to distinguish between the biological and technical replicates was well received by the community. The discussion is well written and ties together the various experiments conducted in this study. Certain sections could be rephrased to provide more clarity for readers.

    The following items of feedback were raised, to help solidify the claim that BCI affects ciliary assembly only through MAPK signaling:

    The majority of the conclusions about MAP signaling are drawn based on the treatment with the BCI compound whose selectivity is unclear. It is possible that BCI could directly inhibit other phosphatases involved in ciliogenesis such as CDC14, PPP1R35. A reference pointing to the selectivity of BCI towards MKPs or alternatively rescue experiments with the inhibitor U0126 could address this issue.

    It is shown that BCI leads to transient activation of the ERK activity which peaks within 30 minutes and starts fading away after around 60 minutes. However, most of the effects are studied at 2 hours, when the changes in the cilia length are most apparent. But the ERK activity at this time point is unclear. Simultaneous measurements of ERK activity and cilia length would strengthen the correlation between the two processes.

    Specific comments

    Introduction:

    “The cell cycle and ciliogenesis utilize the same structures at different times” can be written as “The cell cycle and ciliogenesis utilize centrioles at different times” as centrioles is the only structure mentioned in the text.

    Small item - “Ciliogenesis occurs when cells the exit cell cycle” to “exit the cell cycle”

    Recommend revising the segment “The ERK pathway controls the cell cycle” as it mostly talks about ERK regulation rather than how the pathway regulates cell cycle.

    Small item – “In C. elegans, mutations to MAPK15 directly regulate...” can be rewritten as “In C. elegans, MAPK15 directly regulates…”

    Results:

    Recommend revising the section “BCI-induced ERK1/2 phosphorylation disrupts ciliary maintenance and assembly in hTERT-RPE1 cells”. The timings of ciliary shortening and its relationship to ERK activation is unclear. In the concluding statement, ciliary assembly was used instead of ciliary shortening despite the data in Figure 1H showing that ciliary assembly is unaffected by BCI.

    “Decreased KAP-GFP at the basal bodies” – This appears to be in contradiction to Figure 2B.

    “These data suggest that BCI inhibits the mechanisms and proteins involved in cytoplasmic microtubule reorganization.” – Recommend adding further clarification about this sentence.

    Figure 1

    Figure 1D – It is unclear in the figure whether the P-value is calculated between concentrations 0 µM and 45 µM, or between 0 and all three other concentrations. A similar comment applies to Figure 1H and Figure 1J.

    Figure 1F – Was any axonemal maker other than acetylated tubulin used to control for tubulin acetylation defects?

    Figure SF 1E – Though the MKP2 mutant does not regenerate to wild type length, it does return to its own original length, can the text be adjusted to reflect this?

    Figure 1G-J – The conclusion in the text that BCI prevents cilia assembly could be clarified, as the data shows growth inhibition rather than assembly inhibition.

    Figure 2

    Figure 2C – The legend is slightly cut off from the image.

    Figure 2C – It is unclear if there is a difference in the fluorescence intensity distribution. A line profile along the cilia would indicate if there is any change in the spatial distribution of KAP.

    Figure SF 2C – Is it possible to elaborate more on what specific conclusion this data suggests.

    Figure 3

    Figure 3B – Is there any reason why the BCI-induced regulation of MAPK signaling affects ciliary protein synthesis in particular? There seems to be no reduction in total protein synthesis.

    Figure 4

    Figure 4A – A clearer description of how BCI “partially” disrupts the transition zone would be beneficial. Cross-sectional imaging of the transition zone with higher concentration of BCI might make changes in the structure more apparent.

    Figure 5

    Figure 5A – 36 µM BFA affects cell morphology and may affect the viability of the cells, can some further clarification be added about this and the concentration used.

    Figure 6

    Figure 6C – The three categories mentioned in the text are not mentioned in the figure.

  10. Version published to 10.1101/2022.05.18.492507v1 on bioRxiv