MAST4 promotes primary ciliary resorption through phosphorylation of Tctex-1

The mechanism that underlies cilium resorption is not well understood. This study reveals the microtubule-associated serine/threonine kinase family member 4 (MAST4) is a novel kinase that promotes ciliary resorption by localizing ciliary base-phosphorylation of Tctex-1 at Thr94 and its downstream effectors.

There's a puncta for P-(T94)Tctex-1 in the KD & point mutants so presumably some other kinase is phosphorating this substrate.Please add to the discussion that MAST4 is not solely responsible (unless you have additional supportive data to include) & anything that's known about other kinases.

Additional specific issues:
General: some wording throughout like "expression of X protein at ciliary TZ"-I think you mean localization?Surely not implying gene expression, right?I'm also unclear on how a transient interaction like that of a kinase & substrate can be stable enough for a pulldown.Some discussion might help here.
Figure 1 B: It doesn't say here, but I'm assuming these measurements were made off ac-tub signal.That's a bit problematic since deacetylation happens before resorption as the authors cite in Pugacheva et al.E: That's a lot of background for MAST4-mC.The fluorescence intensity line plot should be segmented to cover the length of the cilium as defined by ac-tub staining.As displayed, it looks like mC signal ends at tip, but as drawn, it looks like it's due to the straight line.If not, fix the line in the first panel to reflect appropriately.Honestly, not sure if this adds much though will all the background.What's the true signal here?Would like to see larger whole cell pic to get a better idea of signal-to-noise.F: need scale bar & asterisks definition Figure 2 A & B: These blots leave much to be desired.With signals like these, this reviewer requests full panel of these membranes.Tctex-1 "signal" is especially problematic as it doesn't even really look like a band, but rather a smudge from signal above.Shouldn't these weights be different for MAST4-mC & endogenous MAST4? Looks nearly identical.D: Signal for frag 1 too in anti-MBP blot?Why aren't we seeing a clear 56 kDA band in lane 2 indicative of MBP-Tctex-1? Figure 3 A: for consistency, please label the figure with MAST4-sh1 B: missing scale bar Figure 4 C: Of all the puncta in these little panels, you're quantifying those that overlap with ac-tub signal?
Figure 5 C-F: Need a better explanation of how you're measuring active Cdc42 (I had to google it but it should be in the paper.If you're including densitometry of blots & running stats, this reviewer wants to see full panels in the supplement of all blots used to generate D & F. It's also unclear how you're measuring relative levels: is the active & total Cdc42 on the same membrane?I should hope so.Need more in M & M than lns 466-467.EV3 What's going on with K506A signal here?Need to show clearly that it is expressed for other experiments.
EV4 Way too much mC background here to say where this signal is coming from!One could generate line intensity plots anywhere in these small panels.This is not acceptable data to support subcellular localization.The figure isn't labeled with mutants.No scale bar definition in figure legend.
Ln 111: These are not isoforms of one protein but rather a family of kinases as you said in the intro.This sentence leads one to believe these are isoforms of MAST (even a thing)?Consider "MAST4 is the predominant kinase of the MAST family" Ln 140: is this ciliary pocket staining?I'd like to see a secondary pocket marker to be convinced Ln 140-142: how do these data support that MAST4 plays an impt role in only the early phasic resportion 0-2hr when resorption is also perturbed at 24 hours?Ln 144: seems like an illogical jump here if you read results first.Consider adding a similar statement about the interaction identified in a previous paper here Ln 175: Haven't shown MAST4 phosphorylates Tctex-1-either provide direct data or adjust to "likely phosphorylates" or the like Ln 181: AGC? Define Ln 201-202: cannot say from these images that anything moved from basal body to axoneme Ln 203: expression→ localization?"Variants resulted in reduced signal from" Ln 442→ TIRF?Ln 704: scale bar in F also 1?
Ln 724-725: could put kDa in the figure easier for the reader to interpret the blot below-to scale with C Ln 776: PAK-PBD beads?Please explain these beads so readers don't have to google to figure out your experiments.
Ln 812: major member?Consider "predominately expressed" Reviewer #2 (Comments to the Authors (Required)): MAST4 promotes primary ciliary resorption by phosphorylating Tctex-1 K.Sakaji., S. Ebrahimiazar, Y. Harigae, T. Sato, T. Yoshikawa, C. Sung, M. Saito 1.A short summary of the paper, including description of the advance offered to the field.In this work Sakaji et al. study the phenomenon of primary cilia resorption in a cell line in culture.In particular, they address the function of MAST4 (a Ser/Thr kinase) in the process, paying special attention to its interaction with Tctex-1 as a potential substrate.Tctex-1 is a protein that was previously identified as a participant in cilia resorption.The phosphorylation of Thr94 in Tctex-1 (T94) is necessary for the activation of downstream effectors involved in ciliary resorption.In this work the authors determine that MAST4 is involved in ciliary resorption, interacts with Tctex-1 by its kinase domain and that the downstream mechanisms involved in ciliary resorption are the ones triggered by Tctex-1 phosphorylation, thus proposing Tctex-1 a substrate of MAST4.The data obtained in this work provides new information about ciliary resorption, a process that is still not completely understood.It is clear from their work that MAST4 is another player in ciliary resorption, and although they provide evidence of interaction between this kinase and Tctex-1, I think some other simple experiments would further support the claim that MAST4 activity in ciliary resorption is due to Tctex-1 phosphorylation.2. For each main point of the paper, please indicate if the data are strongly supportive.If not, explicitly state the additional experiments essential to support the claims made and the time frame that these would require.a) MAST4 accelerates ciliary resorption The participation of MAST4 in ciliary resorption is clearly supported by knockdown (KD) and overexpression (OE) experiments and the evaluation of the cilia length and % of ciliated cells in transfected cell lines.The conclusion that MAST4 is localized at the ciliary pocket needs to be further confirmed using a marker of the ciliary pocket in the immunofluorescence (IF).This marker was used in Figure 6 and thus should be included in the IF.b) The kinase domain of MAST4 directly binds to Tctex-1 The authors observed the interaction between MAST4 and Tctex-1 when they overexpressed both proteins, and also with the endogenous proteins.The WB showing the immunoprecipitation of endogenous proteins, which reflects physiological interaction, is not of very good quality, it would be great if it could be improved.Moreover, the authors mentioned in the introduction of the present manuscript that the interaction of MAST4 and Tctex-1 was observed in a previous paper (Saito et al. 2017), in the proteomic analysis of material immunoprecipitated with an anti-Tctex-1 antibody.I could not find this result in the cited reference.It would be nice, if possible, that the authors could include the proteomic data (the corresponding author of the present manuscript is the first author of the one involving proteomics), as another evidence of the interaction between the two proteins.The evidence that the kinase domain of MAST4 is the one involved in interacting with Tctex-1 comes from pull-down experiments where Tctex-1 and different fragments of MAST4 were expressed in bacteria.The results are convincing.However, there is a contradiction between what is described in Materials and Methods and in Legend of Figure 2D.Whereas in M&M it is said that crude extracts from bacteria expressing MAST-fragments was incubated with extracts from bacteria expressing Tctex-1, in Legend of Figure 2D says that purified MAST4 fragments were incubated with purified Tctex-1.It is important to clarify which of the two procedures was used.In case the authors have used crude extracts, they cannot assure that the interaction between the MAST4 kinase domain and Tctex-1 is direct.Please check.I think Figure 2D could improve very much if the pull down of the different MAST4 fragments is visualized by Western Blotting with an anti-GST antibody instead of Coomassie staining.c) MAST4 enhances the ciliary transition zone expression of phospho-(T94) Tctex-1 First of all, I do not like the use of the word "expression" when referring to localization of a protein.Expression maybe confused with translation, that does not occur at the cilium.Please change "expression" for localization.The effect of MAST4 KD is reversed by the overexpression of Tctex-1T94E but not the WT or the Tctex-1T94A proteins, suggesting that Tctex-1 could be a substrate of the kinase.I think the results support the idea but other evidences are needed in order to establish that MAST4 phosphorylates Tctex-1.As the authors mentioned in the Discussion section, MAST4 is a big protein, difficult to produce recombinantly so as to test its activity on Tctex-1 directly.However, as this point is very relevant for the work, I suggest that an effort has to be made in order to have more evidence of MAST4 phosphorylating Tctex-1.I am proposing this because there are at least two experiments, that look relatively simple, that could shed light on this point: i) it would be very informative to see what happens with Tctex-1 phosphorylation levels in MAST4 KD cells.This could be studied by WB using the anti-phospho-Tctx-1 antibody as previously done (Saito et al, 2017).The results could further support the idea that MAST4 phosphorylates Tctex-1.ii) Does Tctex-1T94A suppress the acceleration of ciliary resorption caused by overexpression of MAST4?MAST4 KD also decrease the serum-induced localization of phsopho-Tctx-1at the transition zone (TZ).This is clearly supported by the IF images.
There is an issue regarding Tctex-1 localization that is not clear.I did not find in the previous papers (Li et al 2011 and Saito et al 2017) which is the localization of un-phosphorylated Tctex-1.Is it also present at the TZ and then phosphorylated at this place?Or is it present somewhere else and moved to the TZ upon phosphorylation?Would you be able to answer these questions?
d) The kinase activity of MAST4 is required for Tctex-1-mediated ciliary resorption.
The results supporting that the catalytic domain of the kinase is important for ciliary resorption and phospho-Tctex-1 TZ localization are convincing.e) MAST4 regulates ciliary resorption by activating the downstream effectors of phospho-(T94) Tctex-1 I consider that this conclusion is well supported by the experiments and the results.However, I found an inconsistency with the previous paper (Saito et 2017).In Figure 6B there is no effect of the overexpression of the Ccdc42 CA protein, while it was expected to accelerate ciliary resorption.Please comment on that.Once the authors showed that MAST4 mediates Cdc42 activation and Arp2/3 activity, the rest of the experiments involving endocytosis of the ciliary pocket membrane are a bit redundant, as the link between Cdc42-Arp2/3-endocytosis of the ciliary pocket membrane and ciliary resorption was previously established (Saito et al 2017).

f) Discussion
Line 259-262: Our mechanical studies showed that MAST4 regulates ciliary resorption by promoting the expression localization of phospho-(T94)Tctex-1 in the ciliary transition zone and its downstream activation of Cdc42-ARPC2-mediated ciliary pocket membrane endocytosis.
"Endosomes from the ciliary pocket membrane do not simply remove the ciliary components (e.g., depolymerized tubulin, ciliary membrane) after axonemal microtubule disassembly; they would actively prime ciliary resorption".I do not understand how depolymerized tubulin, a soluble and intracellular protein would be eliminated by endocytosis.
"This idea is supported by the fact that phospho-Smad2/3 accumulates on the endosome membrane, which is generated from the ciliary pocket membrane (Saito et al., 2017)".Please expand on the link between activation of Smad signaling and ciliary resorption as it is not clear from the reference."MAST4 certainly controls corticogenesis by phosphorylating (T94)Tctex-1 in the cells".This idea has not been tested in this work, so it should be expressed in a conditional way.Pg 8, line 175: change "expression" for localization Pg 11, line 231: change "extracellularly fed" by extracellular Materials and Methods Ciliary resorption assay, GFP-F imaging assay, and immunostaining Please provide details of the temperature of incubation of the antibodies.Co-immunoprecipitation assay and western blotting Please provide details of the time and temperature of incubation of the antibodies Pg 30, line707-708: Please re-write the following sentence "A representative immunoblot of HEK293 cells overexpressing MAST4WT-mCherry and FLAG-Tctex-1WT was subjected to a co-immunoprecipitation assay" as A representative immunoblot of an immunoprecipitation assay using HEK293 cells overexpressing MAST4WT-mCherry and FLAG-Tctex-1WT.

Reviewer summary
This study follows up on previous findings that Tctex-1-in its T94 phophorylated state and dynein independent function-regulates cilia resorption by activating cdc42 and regulating the branching dynamics of actin that lead to ciliary resorption.In order to further study the mechanisms underlying Tctex-1 function, this study builds upon the identification of MAST4 as a ligand for Tctex-1 using a proteomics approach.In this work, the authors analyze MAST4 role in ciliary dynamics as well as the physical and functional association between Tctex-1 and MAST4.The data provided support the conclusions of a physical and functional interactions.The study also aims to study the involvement of MAST4 kinase activity and does succeed in providing evidence that MAST4 kinase domains residues are important for Tctex-1 function and Tctex-1 effectors.However more evidence is needed to demonstrate that MAST4 kinase activity is involved in these processes.
Overall, the work is relevant to the field of cilia dynamics, ciliopathies and contributes original information to the understanding of primary cilium regulatory mechanisms.The manuscript is well written and is clear except for the parts where I asked for clarification (mostly in methods description) The length of the paper is appropriate.Data and literature are succinctly but sufficiently discussed.
The major claims made by this report are that: 1.MAST4 is expressed in cilia 2.MAST4 and Tctex-1 interact physically and functionally 3.The catalytic loop of MAST4 is important for Tctex-1 phosphorylation 4.Phosphorylated TcTex-1 is localized to the transition zone of the cilium during resorption 5.MAST4 Kinase activity is required for Tctex-mediated cilia resorption Claims 1-3 are sufficiently supported by the data in this manuscript.
For claims 4 and 5 additional experiments or rephrasing would be necessary to accurately reflect the conclusions allowed by the data.The statement referred to the kinase activity of MAST4 being required for the observed that needs to be consistent throughout the manuscript.My major concern is related to the section 4 of results (the kinase activity of MAST4 is required for Tctex-1mediated ciliary resorption).In order to fully support this statement, some direct readout for the kinase activity of MAST4 Wt and the point mutations should be provided, since a phosphoTctex1 antibody is available and is actually used in microscopy in this work a western blotting approach would add evidence, provided that the antibody works in western blot.The phrasing that describes the data currently available seems more accurate both in the figure legend and in the abstract.Similarly, the conclusion about the localization of pTctex-1, should reflect that it is somewhat speculative (although reasonable) but colocalization with a transition zone markers would strengthen this conclusion.
Reviewer comments: Methods Cdc42 activity assay.Please provide more details not referring to previous paper.Al least a rationale basis for the assay would be appreciated.
Statistics in Material and methods and figure legends: It has been discussed that presenting data with SEM does not add much meaningful information to the graphical representation.Rather, providing a visual representation of 95% Confidence interval-in addition to the statistical significance test results (p values)-would be more informative RT-PCR: I am not sure about why "cDNA was PCR amplified with specific primers" and was this followed by real time PCR?How do you keep this quantitative?Perhaps this is 2 different experiments?This is somewhat confusing and should be made more clear.

MAST4 accelerates ciliary resorption
Minor comment on Fig 1A-C: control cell resorption in biphasic manner.What does biphasic mean in this context?Could this be explained or rephrased?I am not sure that he dynamics seen Both in A and B is biphasic.It looks more linear.The rest of these panels does support the description in the text.Line 132; the sentence "These cilia underwent further shortly after 1 h up to 24 h time points" is perhaps mistyped.A word seems to be missing after "further" Fig 1E .Since there is not any good antibody available to test endogenous distribution of MAST4 , a different MAST4 fusion protein with a different tag could be used to verify the observed localization.Also, are these cells stably expressing MAST4-mCherry or are they transiently transfected?

MAST4 enhances the ciliary transition zone expression of phospho-(T94)Tctex-1
The title of this section is a more accurate description of the conclusion supported by the data than the title of Figure 3  Claiming that phosphoTctex-1 localizes at the transition zone might require a maker for the transition zone (perhaps CEP290?) .Otherwise the description of the data would be more accurately described by analyzing Tctex-1 localization at the pericentriolar region or cilia base.Some of these concerns are somewhat addressed in figure EV4.As similar approach could be performed for MAST4WT-mCherry.Also, these data do not fully support the conclusion that MAST4 phosphorylates TcTex1 as stated on Figure 3 legend.The title of the section is more accurate.

The kinase activity of MAST4 is required for Tctex-1-mediated ciliary resorption
The prediction of the catalytic motifs of MAST4 kinase domain is based on data about MAST2 kinase domain and it is correct in my opinion.Since alphafold2 tool is available it would be interesting to see how these predictions map on a predicted structure of MAST4.This would probably have only a visualization value since the functional data shown in 4A is sufficient for the purpose of suggesting that the catalytic activity of MAST4 kinase domain is required for promoting the resorption of cilia.Is there any other known substrate for MAST4 kinase activity that could be used to corroborate that the mutants have a different activity?This could be performed by a western blot of transfected cells using a phospho-specific antibody (eg phosphoTcTex-1, phosphoERM or overall levels of p-Ser) Otherwise since no direct evidence for kinase activity is shown the title of this section and the conclusion might need to reflect that the mutated residues are required for ciliary resorption, rather than the kinase activity.

Discussion
Line 260.Mechanical might no be the best word here Line 263 where it says were it should be was Line 284.Explanation for lack of direct evidence for phosphorylation of Tctex-1 by MAST4.However some experiment could be attempted.Precisely a list of potential readouts for MAST4 kinase activity is shown next.Line 303."MAST4 certainly controls corticogenesis by phosphorylating (T94)Tctex-1 in the cells" No reference is given so this is a speculation, albeit reasonable, based on data from this paper so it should be stated as such.
The conclusions are supported partially by data.MAST4 is a regulator of cilia dynamics is supported by the data in this study.However, there is only indirect evidence showing that MAST4 kinase activity is required and that MAST4 phosphorylates Tctex-1.Since there is an anti-phosphoTctex antibody available that was used in IF, it might be possible to use it in a western blot testing the levels of pTctex-1 in the presence of each of the point mutations.
Issues to be addressed: I explained the details in previous sections but I summarize main concerns here: Please provide direct evidence of kinase activity of MAST4 wild type and mutants preferably on Tctex-1 or on any of its known substrates.Alternatively, adjust text (mostly title and title section 4 of results and figure 3 title legend) to reflect the data available.The conclusions are more accurately described in the title of the legend for figure 4 and result section 3.
Please provide evidence of colocalization of Phospho-Tctex-1 with a marker for the transition zone of the cilium.Alternatively, adjust text to reflect the data available.

Referee cross comments:
In addition to my previous comments, I agree with the important points raised by both the other reviewers about changing the text of section 3 of results using Localization instead of expression activation is perturbed, providing some rationale on how MAST4 fits into the larger known mechanisms underlying ciliary resorption.
Overall the manuscript was well written, provides new information about ciliary resorption, and will be interest to Life Science Alliance readers.All comments offered herein are in good spirit to both help the authors and future readers reproduce these experiments.
2) Main points: •MAST4 accelerates ciliary resorption Yes, the data is supportive.It would be enhanced with better MAST4-mC images as those presented in Figure 1 E-F have much background making it difficult to distinguish signal from noise (especially in E).
Reply: As suggested, we replaced the original Fig. 1E and F with a better-quality image in revised Fig. 1E and Fig. EV3, respectively, to depict the ciliary distribution of MAST4-mCherry.
According to this reviewer's suggestion below, we also added a low-power view of a transfected cell in both figures.As a result of these addition, we removed the original Reply: We agreed with the reviewer retrospectively.The original Fig. 2B showed that the amount of the endogenous Tctex-1 pulled down by the MAST4 antibody was low, and, hence not a strong evidence supporting the endogenous protein-protein interaction.We, therefore, deleted the original Fig. 2B.
•MAST4 enhances the ciliary transition zone expression of phospho-(T94)Tctex-1 Without details on how these experiments were performed, I cannot evaluate if the data support the claims or not.
Reply: We apologize for our oversight.We extensively rewrote the Materials & Methods section and added greater detail for the procedures for cell staining, imaging, and quantification of the ciliary base signal of phospho-(T94)Tctex-1.
•The kinase activity of MAST4 is required for Tctex-1-mediated ciliary resorption The data supports that the kinase activity of MAST4 is required for ciliary resorption.
Again, need details on qIF to interpret properly.
Reply: As suggested, we added additional detail for qIF (also see above).
Retrospectively, we agreed with the reviewers that our available data are not adequate to directly support the original claim calling "the kinase activity of MAST4 is required for ciliary resorption".We deleted that statement from the manuscript.Overall, we significantly softened the original claim.Instead of calling MAST4 is the kinase that phosphorylates Tctex-1, we concluded MAST4 is a novel kinase that regulates ciliary resorption by modulating the ciliary base activation of phospho-(T94)Tctex-1.
•MAST4 regulates ciliary resorption by activating the downstream effectors of phospho- The data supports indirect activation of downstream effectors & upstream involvement of MAST4 with Tctex-1.I cannot interpret how the GFP-F experiment was quantified due to lack of details.
Reply: We agreed with this reviewer more detailed description of these experiments will help data interpretation.However, a holistic consideration of this reviewer's comment and the comments from Reviewer 2, who called the GFP-F studies are redundant, we decided to delete the original Fig 6C , D. We believed that the removal of these figures did not impact on our conclusions.Reply: We appreciate this valuable suggestion.We discussed this possibility in the revised Discussion .
Additional specific issues: General: some wording throughout like "expression of X protein at ciliary TZ"-I think you mean localization?Surely not implying gene expression, right?I'm also unclear on how a transient interaction like that of a kinase & substrate can be stable enough for a pulldown.Some discussion might help here.
Reply: We made the editorial changes as suggested, we used either "localization" or "distribution" instead of "expression".
The reviewer is right; stable complexes are more likely to be co-immunoprecipitated.
However, precedents showed certain kinases and their substrates could be identified through pull-down assays especially when these proteins are overexpressed (e.g., Dart et a., J. Biol. Chem., 2001).MAST4 was initially identified in the proteomics of the GFP immunoprecipitants of Tctex-1-GFP stably expressed in the RPE-1 cells (Saito et al., EMBO Rep., 2017).The current paper showed that MAST4-mCherry pulled down the co-expressed FLAG-Tctex-1 in RPE-1 cells (Fig. 2A).The rest pull-down assays were carried out using recombinant bacterial fusion proteins in vitro (Fig. 2C).Would like to see larger whole cell pic to get a better idea of signal-to-noise.
Reply: As suggested, in the revised Fig. 1E, we modified the fluorescent intensity line plot so that the Ac-Tub staining spanning the entire cilium was segmented and presented.We also better labeled the orientation (basal body at left and distal cilium at right) in the y-axis.Also, we added a whole cell view, and, understandably, MAST4-mCherry is not exclusively expressed in the cilium.The cilium-associated MAST4-mCherry signals are likely to be true signals because no similar ciliary association was found in RPE-1 cells transfected with mCherry alone (data not shown).

F: need scale bar & asterisks definition
Reply: We added the scale bar and asterisk definition in the original Fig. 1F (now revised Fig. EV3).
Figure 2 A & B: These blots leave much to be desired.With signals like these, this reviewer requests full panel of these membranes.Tctex-1 "signal" is especially problematic as it doesn't even really look like a band, but rather a smudge from signal above.Shouldn't these weights be different for MAST4-mC & endogenous MAST4? Looks nearly identical.
Reply: As suggested, the revised Fig. 2A shows a full panel of the original Fig. 2A.As shown, a clear Tctex-1 (Flag-Tctex-1) band was detected in the immunoprecipitant pulled down by mCherry antibody but not control anti-GFP antibody.

The original Fig. 2B was aimed to pull down the endogenous Tctex-1 using anti-MAST4 antibody from RPE-1 cells. Understandably, co-immunoprecipitation of endogenous (vs. overexpressed) proteins is generally more challenging due to the trace amount of expression. Two factors further increased the practical difficulty of this experiment. (I)
Transferring a very big protein (MAST4) and a very small protein (Tctex-1) simultaneously in a same protein blot, and (2) Tctex-1 and immunoglobulin light chain (used for IP) have similar molecular weights.We agreed with this reviewer that this blot leaves much to be desired.As a result, we decided to delete the original Fig. 2B considering this removal will not impact the conclusion of the present paper.
D: Signal for frag 1 too in anti-MBP blot?Why aren't we seeing a clear 56 kDA band in lane 2 indicative of MBP-Tctex-1?
Reply: We agreed with this reviewer about the first comment.In the revised manuscript, we mentioned weak signal of MBP-Tctex-1 was also pulled down by GST-MAST4 fragment 1 (Lines 181-182).
We respectfully disagreed with the second comment.We speculated this misunderstanding was due to the poor figure labeling.Therefore, we revised the labeling of the original Fig. 2D (revised Fig. 2C).We added the molecular weights of all recombinant proteins tested and marked their position on protein gels.The active form of Cdc42 and the total Cdc42 ran on the same protein gels and were detected by the anti-Cdc42 antibody on immunoblots.The full panels of Figs.5D and E were presented in the revised Fig. EV7.EV2 Why isn't there an endogenous MAST4 signal in B while you can see it in A?
GAPDH loading is similar so one should see it.
Reply: In Fig. EV2B, the high level of transfected MAST4 is likely to obscure the relatively lower level of endogenous MAST4 due to the former sequesters the majority of the primary and secondary antibodies.Regardless, the most important message here is that the transfected MAST4 and endogenous MAST4 migrated to the similar position on protein gels.
EV3 What's going on with K506A signal here?Need to show clearly that it is expressed for other experiments.
Reply: For unclear reasons, the transfection rate of MAST4 K506A was lower compared to that of other variants, this was consistent with its weaker protein signal on immunoblots (Fig. EV5).Since we only counted the cilium expression in GFP + transfected cells, the lower transfection rate of MAST4 K506A would not affect the cilium counting results.
Nevertheless, for the subsequent studies (Figs. 4C,D,EV6), we primarily focused on testing MAST4 R503A and MAST4 D504A mutants.These comments were added in revised Results .
EV4 Way too much mC background here to say where this signal is coming from!One could generate line intensity plots anywhere in these small panels.This is not acceptable data to support subcellular localization.The figure isn't labeled with mutants.No scale bar definition in figure legend.
Reply: We replaced the original Fig. EV4 (revised EV6) with a better-quality representative image with lower background.We also added a whole cell view, the label of the mutants, and scale bars.
Ln 111: These are not isoforms of one protein but rather a family of kinases as you said in the intro.This sentence leads one to believe these are isoforms of MAST (even a thing)?Consider "MAST4 is the predominant kinase of the MAST family" Reply: Thanks for the suggestion.We described this point in the Introduction .Ln 140-142: how do these data support that MAST4 plays an impt role in only the early phasic resportion 0-2hr when resorption is also perturbed at 24 hours?
Reply: Thanks for the comment.We removed the "early phase" from this context in the revised Results .
Ln 144: seems like an illogical jump here if you read results first.Consider adding a similar statement about the interaction identified in a previous paper here Reply: As suggested, we added an introduction sentence in the first paragraph of Result.
Ln 175: Haven't shown MAST4 phosphorylates Tctex-1-either provide direct data or adjust to "likely phosphorylates" or the like Reply: We significantly softened the claim throughout the entire revised manuscript.We Reviewer #2 1.
In this work Sakaji et al. study the phenomenon of primary cilia resorption in a cell line in culture.In particular, they address the function of MAST4 (a Ser/Thr kinase) in the process, paying special attention to its interaction with Tctex-1 as a potential substrate.
Tctex-1 is a protein that was previously identified as a participant in cilia resorption.The phosphorylation of Thr94 in Tctex-1 (T94) is necessary for the activation of downstream effectors involved in ciliary resorption.In this work the authors determine that MAST4 is involved in ciliary resorption, interacts with Tctex-1 by its kinase domain and that the downstream mechanisms involved in ciliary resorption are the ones triggered by Tctex-1 phosphorylation, thus proposing Tctex-1 a substrate of MAST4.The data obtained in this work provides new information about ciliary resorption, a process that is still not completely understood.It is clear from their work that MAST4 is another player in ciliary resorption, and although they provide evidence of interaction between this kinase and Tctex-1, I think some other simple experiments would further support the claim that MAST4 activity in ciliary resorption is due to Tctex-1 phosphorylation.

a) MAST4 accelerates ciliary resorption
The participation of MAST4 in ciliary resorption is clearly supported by knockdown (KD) and overexpression (OE) experiments and the evaluation of the cilia length and % of ciliated cells in transfected cell lines.The conclusion that MAST4 is localized at the ciliary pocket needs to be further confirmed using a marker of the ciliary pocket in the immunofluorescence (IF).This marker was used in Figure 6 and thus should be included in the IF.

Reply: Claiming the localization of MAST4 at ciliary transition zone and ciliary pocket
localization is not pertinent to the conclusion of this report.As a result, we modified the claim by stating "MAST4-mCherry is broadly distributed in the cilium, including the ciliary base, the ciliary axoneme and the region juxtaposing to the ciliary axoneme, possibly ciliary pocket".
The co-staining of the most commonly used ciliary transition marker CEP290 is not feasible because that both CEP290 and MAST4 antibodies were made in rabbits.The colocalization study of ciliary pocket marker GFP-F (GFP fusion of farnesylated motif) of a protein.Expression maybe confused with translation, that does not occur at the cilium.Please change "expression" for localization.
Reply: As suggested, we avoided using the word "expression".We used either localization or distribution instead.
The effect of MAST4 KD is reversed by the overexpression of Tctex-1T94E but not the WT or the Tctex-1T94A proteins, suggesting that Tctex-1 could be a substrate of the kinase.I think the results support the idea but other evidences are needed in order to establish that MAST4 phosphorylates Tctex-1.As the authors mentioned in the Discussion section, MAST4 is a big protein, difficult to produce recombinantly so as to test its activity on Tctex-1 directly.However, as this point is very relevant for the work, I suggest that an effort has to be made in order to have more evidence of MAST4 phosphorylating Tctex-1.I am proposing this because there are at least two experiments, that look relatively simple, that could shed light on this point: i) it would be very informative to see what happens with Tctex-1 phosphorylation levels in MAST4 KD cells.This could be studied by WB using the anti-phospho-Tctx-1 antibody as previously done (Saito et al, 2017).The results could further support the idea that MAST4 phosphorylates Tctex-1.
ii) Does Tctex-1T94A suppress the acceleration of ciliary resorption caused by overexpression of MAST4?
Reply.As suggested in (ii), we added a new figure showing FLAG-Tctex-1 T94A suppressed the acceleration of ciliary resorption caused by co-transfected MAST4 (revised Fig. 3B).
Regarding to the (i), the currently available phospho-Tctex-1 antibody is not sensitive enough to detect the protein expressed in RPE-1 cells.As a result, we softened our claim.We no longer claimed MAST4 is a kinase that phosphorylates Tctex-1.Rather, we described MAST4 is required for the ciliary base activation of phospho-Tctex-1.
We did previously showed a WB blot of phospho-(T94)Tctex-1 in serum-stimulated RPE-1 cells (Fig. 2 in Li et al., Nat. Cell Biol., 2011).However, due to the very low level of phospho-Tctex-1, in these experiments, immunoprecipitation was first carried out using a saturated amount of anti-(pan)Tctex-1 Ab, and the immunoprecipitates were electrophoresed and immunoblotted with phospho-(T94)Tctex-1 Ab (see Fig.2 legends in Li et al., Nat. Cell Biol., 2011).We felt strongly that this IP/IB approach is not reliable for a quantitative comparison between specimens.
MAST4 KD also decrease the serum-induced localization of phsopho-Tctx-1at the transition zone (TZ).This is clearly supported by the IF images.
There is an issue regarding Tctex-1 localization that is not clear.I did not find in the previous papers (Li et al 2011 and Saito et al 2017) which is the localization of unphosphorylated Tctex-1.Is it also present at the TZ and then phosphorylated at this place?Or is it present somewhere else and moved to the TZ upon phosphorylation?
Would you be able to answer these questions?
Reply: The ciliary localization of Tctex-1 has not been reported using the (pan)Tctex-1 antibody.Nonetheless, proteomics of primary cilia identified the presence of Tctex-1 (aka DYNLT1) along with other dynein components (Fig 2, Mick et al., Dev. Cell, 2015, PMID 26585297).Given the broad ciliary distribution of MAST4, we do not know the exact location where Tctex-1 is phosphorylated.Related discussion is not included in the revised Discussion.
d) The kinase activity of MAST4 is required for Tctex-1-mediated ciliary resorption.
The results supporting that the catalytic domain of the kinase is important for ciliary resorption and phospho-Tctex-1 TZ localization are convincing.
Reply: Thank you.
e) MAST4 regulates ciliary resorption by activating the downstream effectors of phospho-(T94) Tctex-1 I consider that this conclusion is well supported by the experiments and the results.
Reply: Thank you.
However, I found an inconsistency with the previous paper (Saito et 2017).In Figure 6B there is no effect of the overexpression of the Ccdc42 CA protein, while it was expected to accelerate ciliary resorption.Please comment on that.
Reply: Fig. 5D in Saito et al. (EMBO rep., 2017) showed overexpression of Cdc42-CA accelerated ciliary resorption.Instead beginning at 2 h time point, cilia began to resorb 0.5 h and 1 h post-serum readdtion in these cells.Nonetheless, the percentage of ciliated cells is not significantly different between 2 and 24 h time points in Cdc42-CA transfected cells.In the current manuscript, Fig. 5B, we only measure the 2 and 24 h time points in Cdc42-CA transfected cells.So that the acceleration of ciliary resorption is not observed, as expected .
Once the authors showed that MAST4 mediates Cdc42 activation and Arp2/3 activity, the rest of the experiments involving endocytosis of the ciliary pocket membrane are a bit redundant, as the link between Cdc42-Arp2/3-endocytosis of the ciliary pocket membrane and ciliary resorption was previously established (Saito et al 2017).
Reply: We appreciate the viewpoint.As suggested by this reviewer stating "the rest of the experiments" (after Fig. 6A &B) are redundant, we decided to delete the original

Reviewer summary
This study follows up on previous findings that Tctex-1-in its T94 phophorylated state and dynein independent function-regulates cilia resorption by activating cdc42 and regulating the branching dynamics of actin that lead to ciliary resorption.In order to further study the mechanisms underlying Tctex-1 function, this study builds upon the identification of MAST4 as a ligand for Tctex-1 using a proteomics approach.In this work, the authors analyze MAST4 role in ciliary dynamics as well as the physical and functional association between Tctex-1 and MAST4.The data provided support the conclusions of a physical and functional interactions.The study also aims to study the involvement of MAST4 kinase activity and does succeed in providing evidence that MAST4 kinase domains residues are important for Tctex-1 function and Tctex-1 effectors.However more evidence is needed to demonstrate that MAST4 kinase activity is involved in these processes.
Overall, the work is relevant to the field of cilia dynamics, ciliopathies and contributes original information to the understanding of primary cilium regulatory mechanisms.The manuscript is well written and is clear except for the parts where I asked for clarification (mostly in methods description) The length of the paper is appropriate.Data and literature are succinctly but sufficiently discussed.
The major claims made by this report are that: 1.MAST4 is expressed in cilia 2.MAST4 and Tctex-1 interact physically and functionally 3.The catalytic loop of MAST4 is important for Tctex-1 phosphorylation 4.Phosphorylated TcTex-1 is localized to the transition zone of the cilium during resorption 5.MAST4 Kinase activity is required for Tctex-mediated cilia resorption Claims 1-3 are sufficiently supported by the data in this manuscript.
For claims 4 and 5 additional experiments or rephrasing would be necessary to accurately reflect the conclusions allowed by the data.
The statement referred to the kinase activity of MAST4 being required for the observed that needs to be consistent throughout the manuscript.My major concern is related to the section 4 of results (the kinase activity of MAST4 is required for Tctex-1-mediated ciliary resorption).In order to fully support this statement, some direct readout for the kinase activity of MAST4 Wt and the point mutations should be provided, since a phosphoTctex1 antibody is available and is actually used in microscopy in this work a western blotting approach would add evidence, provided that the antibody works in western blot.The phrasing that describes the data currently available seems more accurate both in the figure legend and in the abstract.
Similarly, the conclusion about the localization of pTctex-1, should reflect that it is somewhat speculative (although reasonable) but colocalization with a transition zone markers would strengthen this conclusion.
Reviewer comments: Methods Cdc42 activity assay.Please provide more details not referring to previous paper.Al least a rationale basis for the assay would be appreciated.
Reply: We added the rationale of Cdc42 activity assay in revised Results and procedures details in M&M section.Reply: In the revised Introduction, we described the serum induced biphasic cilium resorption.Briefly, this concept was first described by Golemis and her colleagues (Pugacheva et al, 2007) who used a systematic kinetic studies that showed the disassembly of the primary cilium occurs in a biphasic manner, with the first wave occurring at 2 h and a second wave at 24 h post-serum-readdition.This observation was reproduced by several other labs, including ours.We do acknowledge that the biphasic pattern is not as apparent when only two time points were tested.

Statistics in
Line 132; the sentence "These cilia underwent further shortly after 1 h up to 24 h time points" is perhaps mistyped.A word seems to be missing after "further" Reply: Thank you.We revised and completed the sentence.Overall data do support the conclusion that MAST4 plays a role in ciliary resorption.
The main weakness lies in the interpretation of the localization data.
Reply: This comment is well received.Taken together with the comments raised by other reviewers, we made two major editorial changes (1) we used "localization" or "distribution" instead of "expression".(ii) Because we do not have the colocalization data using a ciliary transition marker or ciliary pocket marker, we modified our description.i.e., we used "ciliary base" instead of "ciliary pocket or ciliary transition zone".Reply: As suggested, we added this comment in the revised Result section.

MAST4 enhances the ciliary transition zone expression of phospho-(T94)Tctex-1
The title of this section is a more accurate description of the conclusion supported by the data than the title of Figure 3 legend.Is there any other known substrate for MAST4 kinase activity that could be used to corroborate that the mutants have a different activity?This could be performed by a western blot of transfected cells using a phospho-specific antibody (eg phosphoTcTex-1, phosphoERM or overall levels of p-Ser) Otherwise since no direct evidence for kinase activity is shown the title of this section and the conclusion might need to reflect that the mutated residues are required for ciliary resorption, rather than the kinase activity.
Reply: We appreciated the suggestions.Also, see the replies above and to other reviewers, we removed the conclusion that "MAST4 is a kinase that phosphorylates Tctex-1" throughout the entire manuscript.Also, we added a section in the revised  An explanation of the cdc42 activity assay would be helpful for the interpretation of this article as a stand-alone set of results.Thank you for submitting your revised manuscript entitled "MAST4 promotes primary ciliary resorption through phosphorylation of Tctex-1".We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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Reviewer #2 (Comments to the Authors (Required)): The authors had answered all the comments I made in the first round of revision.Answers are satisfactory, and when suggested experiments could not be carried out, they gave reasonable explanations.Modifications in the text were introduced to make it clearer and to soften some assertions that were not fully supported by the results obtained.I think that the revised version is clearly better than the first one and should be accepted for publication after a thorough revision of the English.

Figure 6 B
Figure 6 B: How are you differentiating/identifying internalized Tf-594.From this data, I cannot believe those specific puncta are internalized vs all the others.C: This experiment seems shaky from what's presented.How are you claiming this signal is from the ciliary pocket?? See major points above regarding quantification of this experiment.D: Figure legend says this is in %-please indicate likewise on the figure EV2 Why isn't there an endogenous MAST4 signal in B while you can see it in A? GAPDH loading is similar so one should see it.
Pg 30, line 712: idem Pg 30, line C: Diagram showing MAST4 domains Pg 31, line 738: Values (C)...is the same for Values in A? Please include.Legend of Figure 5. "The histogram shows the percentage of GFP+ cells expressing Ac-Tub-labeled primary cilia at the indicated time points".Did you count GFP+ cells or GFP+-HA+ cells?The same question applies for Figure 6, is GFP+ cells or GFP+-FLAG+ cells?Legend of Figure 6C: please specify the type of microscopy used (confocal, airyscan, SR-SIM) Reviewer #3 (Comments to the Authors (Required)): Fig 1F.Asterisks.If the picture shows a single cilium then, the bottom asterisks seem to point to the ciliary pocket but it is unclear what is signaled by the top asterisk Overall data do support the conclusion that MAST4 plays a role in ciliary resorption.The main weakness lies in the interpretation of the localization data.2. The kinase domain of MAST4 directly binds to Tctex-1 Fig 2A and 2B suggestion: adding IP mCherry and IP MAST4 labels to the figures would help the reading.Fig 2D.Possible discussion point: the faint band detected for the pull down of MBP-Tctex-1 with Fr.1 might suggest that other regions of the protein contribute to the interaction.
Fig 3Band C. Claiming that phosphoTctex-1 localizes at the transition zone might require a maker for the transition zone (perhaps CEP290?) .Otherwise the description of the data would be more accurately described by analyzing Tctex-1 localization at the pericentriolar region or cilia base.Some of these concerns are somewhat addressed in figureEV4.As similar approach could be performed for MAST4WT-mCherry.Also, these data do not fully support the conclusion that MAST4 phosphorylates TcTex1 as stated on Figure3legend.The title of the section is more accurate.
activating the downstream effectors of phospho (T94)Tctex-1 Fig 5A supports the claims in the text.An explanation of the cdc42 activity assay would be helpful for the interpretation of this article as a stand-alone set of results.Fig 6 supports the conclusion that MAST4 acts upstream of known effectors of Tctex-1 figure/panel

Fig
Fig. 1F to the Supplementary figure EV3 due to space constraints in the main figure.

3 )
Fig.3, 4, and 6 in detail (note Fig. 6C and D were deleted).It now reads "For quantitative immunofluorescence analysis, the confocal fluorescence intensity of MAST4-mCherry across the entire cilium and ciliary base was plotted using Zen software (Zeiss).The representative line scans from the images of 10-16 cilia were shown.To quantify the ciliary basal signal of phospho-(T94)Tctex-1, the total fluorescence of the region of interest (ROI), i.e., the proximal end of Ac-Tub labeled cilium, was quantified in the projection confocal images of 1.25 μm-thick Z-series sections of cilium-containing regions (x = 1 μm, y = 1 μm) in randomly selected cells using NIH ImageJ 64 software.The numbers of analyzed cilia were indicated in the figure legend.All experiments were repeated at least three times.The ciliary length was measured using the acquired confocal Ac-Tub images on NIH ImageJ 64 software.For presentation purposes, the brightness and the background of the images were adjusted by Photoshop Elements 12 software (Adobe Systems)."

Figures 3 &
Figures 3 & 4:  There's a puncta for P-(T94)Tctex-1 in the KD & point mutants so presumably some other kinase is phosphorating this substrate.Please add to the discussion that MAST4 is not solely responsible (unless you have additional supportive

Figure 1 B
Figure 1 B: It doesn't say here, but I'm assuming these measurements were made off ac-tub signal.That's a bit problematic since deacetylation happens before resorption as the authors cite in Pugacheva et al.

Figure 3 A
Figure 3 A: for consistency, please label the figure with MAST4-sh1 B: missing scale bar

Figure 4 C:
Figure4C: Of all the puncta in these little panels, you're quantifying those that overlap with actub signal?

Figure 5 C
Figure 5 C-F: Need a better explanation of how you're measuring active Cdc42 (I had to google

Figure 6 B:
Figure 6 B: How are you differentiating/identifying internalized Tf-594.From this data, I cannot believe those specific puncta are internalized vs all the others.

Ln 140 :
is this ciliary pocket staining?I'd like to see a secondary pocket marker to be convinced Reply: We agreed with the reviewer calling the ciliary pocket localization of MAST4-mCherry requires the co-labeling with a marker.We avoid performing the colocalization study using the transfected ciliary pocket reporter (GFP fusion of farnesylated motif), which might cause unnecessary complications.Since the ciliary pocket localization of MAST4 is not pertinent to any key conclusion of the paper, we simply avoid calling this out.In the revised Results and figure legends (original Fig 1F/revised Fig. EV3), we described "MAST4 WT -mCherry signals were detected in the ciliary base (arrow) and ciliary axoneme (arrowhead).The asterisk marks the MAST4 WT -mCherry signal juxtaposing to the ciliary axoneme, possibly representing the ciliary pocket.." (Lines[956][957][958]

Fig
Fig. 6C, D (i.e., the GFP-F ciliary pocket membrane assays).Please also see the related comments raised by Reviewer 1.

Fig 1E .
Fig 1E.Since there is not any good antibody available to test endogenous distribution of MAST4 , a different MAST4 fusion protein with a different tag could be used to verify the observed localization.Also, are these cells stably expressing MAST4-mCherry or are they transiently transfected?

Fig
Fig 1F.Asterisks.If the picture shows a single cilium then, the bottom asterisks seem to point to the ciliary pocket but it is unclear what is signaled by the top asterisk

Tctex- 1 Fig
Fig 2A and 2B suggestion: adding IP mCherry and IP MAST4 labels to the figures would help the reading.

Fig
Fig 3A strong data of the functional association and the proposal or MAST4 upstream of phospho-Tctex-1 Fig 3B and C. Claiming that phosphoTctex-1 localizes at the transition zone might Fig 5A supports the claims in the text.

Fig 6
Fig 6 supports the conclusion that MAST4 acts upstream of known effectors of Tctex-1

figure 4 and result section 3 .
figure 4 and result section 3.Please provide evidence of colocalization of Phospho-Tctex-1 with a marker for the transition zone of the cilium.Alternatively, adjust text to reflect the data available.Referee cross comments: In addition to my previous comments, I agree with the important points raised by both the other reviewers about changing the text of section 3 of results using Localization instead of expression.
please update your callouts for the Supplementary Figures and tables in the manuscript Fig EV1A = Fig S1A, Table This is somewhat confusing and should be made more clear.Could this be explained or rephrased?I am not sure that the dynamics seen Both in A and B is biphasic.It looks more linear.The rest of these panels does support the description in the text.]