Lithium-induced ciliary lengthening sparks Arp2/3 complex–dependent endocytosis

  1. Brae M. Bigge
  2. Larissa L. Dougherty
  3. Prachee Avasthi

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Abstract

Lithium is known to lengthen cilia across cell types. Using the organism model organism Chlamydomonas, we confirm that this elongation occurs independent of protein synthesis and show that it is also independent of the Golgi. Additionally, we find that it requires the Arp2/3 complex and endocytosis. Thus, we propose a new mechanism by which lithium sparks endocytosis to reclaim plasma membrane for ciliary elongation.

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  1. Version published to 10.1091/mbc.e22-06-0219
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    Reply to the reviewers

    We thank all reviewers for their very helpful comments. We feel that the comments pointed to a few main issues that we could remedy. First, we found that many comments and concerns could be addressed with work from our previous paper (doi.org/10.1101/2020.11.24.396002). To fix this, we added additional descriptions of experiments done previously and additional citations. We discussed more in depth an experiment that shows that ciliary membrane and membrane proteins can indeed come from the cell body plasma membrane, we talked more about how we determined that the actin puncta are representative of membrane remodeling functions like endocytosis, and we discussed some of the mechanistic insights provided by our previous work that are applicable here. We hope that this helps to answer several of the reviewer questions. Second, there were a few experiments we thought would be useful to add. These are represented in bold in our responses below. Briefly, we added a measure of internalization or endocytosis in the *drp3 *mutant, we added some images of cilia to the phalloidin figure to orient readers’ views of the cell, we added some additional mechanistic insight (supplemental figure 3), and we added an axoneme stain to confirm that the axoneme was extending (supplemental figure 4). Finally, we fixed some of our wording in the paper to represent our findings more accurately. Together, we hope that these revisions will address the reviewer concerns.

    Additionally, we added some data that we collected while waiting on reviews. We investigated the requirement for myosin in this pathway and include this data in the supplement.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    The current manuscript by Bigge et al. demonstrated that the chemical inhibition of GSk3 causes ciliary elongation in Chlamydomonas reinhardtii. They show that lithium induced ciliary lengthening is majorly due to GSK3 inhibition. Consistent with earlier reports, they show that new protein synthesis is not required for lithium induced ciliary elongation. The authors report that targeting endocytosis either by using chemical inhibitors (dynasore and CK-666) or genetic mutants (dpr3 and Arpc4) does not cause lithium induced ciliary elongation. They further reveal enhanced actin dynamics in lithium treated cells and such activity is lost in Arpc4 mutants. Based on these results, the authors concluded that endocytic pathways may be involved in lithium induced ciliary lengthening. The results are interesting, and this work is important in understanding more about ciliary length regulation. However, more experimental evidence addressing the current interpretation that endocytic pathways may be involved in lithium induced ciliary lengthening is required.

    Major comments: 1 The authors use chemical inhibitors as major tools for their study. However, the specificity of these inhibitors is a concern. How specific are these GSK3 inhibitors such as LiCl? Can authors show that LiCl mediated ciliary lengthening is due to inhibition of GSK3? Authors used BFA and Dynasore to show that not the Golgi, but the endocytosis derived membrane is required for ciliary lengthening. Again, here the specificity of these inhibitors is a concern. Especially as Dynasore has been shown to have non-specific effects.

    *We agree that the specificity of chemical inhibitors can be a concern. This is why we used 4 separate inhibitors of GSK3, each showing elongation of cilia and an increase in actin puncta (suggesting an increase in actin dynamics at the membrane). While these different inhibitors may have different off-target effects. Their intended target, GSK3, is the same, suggesting that the shared phenotype from each inhibitor is conserved. The ability of LiCl to affect GSK3 activity in *Chlamydomonas *was also investigated in depth with a kinase assay and a western blot in Wilson, 2004 (doi: 10.1128/EC.3.5.1307-1319.2004). To address the off-target effects of Dynasore, we employed the *drp3 *mutant to confirm genetically what we saw from the chemical inhibition. We also show in our previous paper that Dynasore and PitStop2 have similar effects in *Chlamydomonas, both of them inhibiting the internalization of a dye-labelled membrane, suggesting that they both function to block endocytosis (doi.org/10.1101/2020.11.24.396002). While no mutant or alternative inhibitor is available to look at the effects of BFA, this inhibitor and its effects on cilia have been well-characterized in Dentler, 2013 (doi.org/10.1371/journal.pone.0053366).

    Does inducing/enhancing endocytosis independent of GSK3 by other means has any effect on ciliary length regulation?

    Our concern with the proposed experiment is that even if elongation requires endocytosis, all endocytosis might not lead to ciliary elongation when endocytosis is for other purposes. For example, endocytosis could occur for other purposes, like nutrient uptake, that will have no effect on cilia. The plasma membrane to cilium pathway may be a targeted pathway triggered by specific disruptions. Therefore, we don’t feel that the proposed experiments will add to our model.

    The major claim of this paper is that LiCl mediated ciliary lengthening is due to enhanced endocytosis. Although authors showed that inhibition of endocytosis results in reduced ciliary length, it is important to show if GSK3 inhibition by LiCl (or any other inhibitor) causes any increased cellular endocytosis? Similarly, what is the effect of GSK3 mutants on endocytosis?

    *We show an increase in actin dynamics at the membrane and actin puncta following treatment with LiCl and the other GSK3 inhibitors. We show here and in our previous paper (doi.org/10.1101/2020.11.24.396002), that these puncta are likely endocytic based on the timing of their appearance and the proteins required for puncta formation (including the Arp2/3 complex and Clathrin) (Figure 7, previous paper). We updated our latest version to reflect the data we have already collected and presented as follows: *

    “Further, they rely on proteins typically thought to be involved in endocytosis including the Arp2/3 complex and clathrin, and they form at times when it makes sense for endocytosis to be occurring, like immediately following deciliation when membrane and protein must be recruited to cilia in a timeframe too short for new protein and membrane synthesis, sorting, and trafficking (Bigge et al. 2020). Thus, we stained cells with phalloidin to visualize filamentous actin and these endocytosis-like punctate structures when cells are treated with GSK3 inhibitors.”

    *A phenotypic mutant of GSK3 does not currently exist in *Chlamydomonas, *and methods of reliably introducing mutants in *Chlamydomonas do not currently exist. Thus, we used the array of GSK3 inhibitors.

    Are these endocytic processes enhanced specifically at/or around the cilium during the ciliary lengthening process?

    *Based on our phalloidin staining data, these processes are primarily enhanced near the cilium, but puncta also exist throughout the cell. To more clearly show this and in response to a comment from reviewer 2, we added a set of images with brightfield to demonstrate where the dots are in relation to cilia. We also added arrows to the images in the figure to point out the apex of the cell as determined by the filamentous actin structures in the cells. *

    Authors claim that drp3 is a target of GSK3 and, similar to the canonical dynamin, functions in endocytosis. While, it is an important observation, experiments are required to show the role of drp3 in endocytosis and also to show that it is indeed a target of GSK3.

    *To address this comment, we are employing an experiment that was designed in our previous paper (doi.org/10.1101/2020.11.24.396002, Figure 5B-E). This experiment uses a lipophilic membrane dye, FM4-46FX. The dye binds to the membrane but is unable to enter the cell alone. It is quickly endocytosed and results in vesicular-like structures within the cell. We added a panel to Figure 3 where we do this experiment in wild-type and *____drp3 mutant cells. This shows that endocytosis is affected by the mutation in DRP3. The discussion of this new data is summarized in the text as follows:

    “Additionally, we showed that this DRP is required for internalization of a lipophilic membrane dye, FM4-46FX through endocytosis. This dye binds to the membrane but is unable to enter the cells on its own and must be endocytosed. In wild-type cells it is quickly endocytosed and visible as puncta within the cell (Figure 3F, H) (Bigge et al. 2020). However, in drp3 mutants the amount of dye endocytosed is significantly lower (Figure 3G-H), suggesting that DRP3 is required for optimal endocytosis in these cells.”

    Mechanistic insights into how endocytosis/actin dynamics regulate ciliary lengthening would be interesting to see. Further, it is interesting to see if the ciliary signaling defects caused by abnormal ciliary length can be rescued by inhibition of endocytosis.

    *In our previous paper (doi.org/10.1101/2020.11.24.396002), we dive into the mechanisms tying together actin dynamics, endocytosis, and cilia. We find that Arp2/3 complex-nucleated actin networks are required for endocytosis to reclaim ciliary membrane and membrane proteins from a pool in the plasma membrane for the rapid early stages of ciliary assembly. We believe that this is a similar mechanism to what is occurring when cells elongate following lithium treatment. This is because there are several parallels in phenotypes: *

    -The Arp2/3 complex is required for both ciliary assembly (Figure 1, previous paper) and ciliary elongation resulting from lithium treatment. In the case of ciliary assembly, treating with cycloheximide to block the synthesis of new protein fully eliminates regrowth in the absence of the Arp2/3 complex, suggesting this Arp2/3 complex dependent mechanism in early ciliary assembly does not involve new protein synthesis (Figure 2, previous paper). Similarly, the process of ciliary elongation in response to lithium does not require new protein synthesis.

    *-A burst in actin dynamics/actin puncta occurs immediately following deciliation during early regrowth and during growth initiated by lithium treatment. We know these puncta are Arp2/3 complex and clathrin dependent (Figures 4 and 7, previous paper). *

    *-Both initial ciliary assembly or ciliary maintenance and elongation of cilia due to lithium treatment require endocytosis (Figures 5, 7-8, previous paper) but not require Golgi-derived membrane (Figure 3, previous paper). *

    *-Also in the previous paper, we find that this mechanism is required for the internalization and relocalization of a ciliary membrane protein for mating (Figure 6, previous paper). We also find that ciliary membrane proteins move from the plasma membrane to the cilia during ciliary assembly (Figure 7-8, previous paper). *

    *This is summarized in the text as follows: *

    *In the introduction we added: *

    “Previous data from our lab suggest that the Arp2/3 complex and actin are involved in reclaiming material from the cell body plasma membrane that is required for normal ciliary assembly (Bigge et al. 2020). We show that the Arp2/3 complex is required for the normal assembly of cilia and for endocytosis of both plasma membrane and plasma membrane proteins in various contexts. Further, we find that deciliation triggers Arp2/3 complex-dependent endocytosis by observing an increase in actin puncta immediately following deciliation (Bigge et al. 2020).”

    And in the discussion we added:

    “Previous work has shown that while the Golgi is required for ciliary maintenance and assembly (Dentler 2013), it is not the only source of membrane. Instead, we found that membrane reclaimed through actin and Arp2/3-complex dependent endocytosis is required for ciliary assembly or growth from zero length (Bigge et al. 2020). More specifically, we found that the Arp2/3 complex is required for normal ciliary maintenance and ciliary assembly, especially in the early stages when membrane and protein are needed quickly. The Arp2/3 complex is also required for the internalization of membrane and a specific ciliary membrane protein required for mating. Further, we show that endocytosis-like actin puncta form immediately following deciliation in an Arp2/3 complex and clathrin-dependent manner, and that membrane from the cell body plasma membrane can be reclaimed and incorporated into cilia (Bigge et al. 2020). This led us to question whether that same mechanism might be required for ciliary elongation from steady state length induced by lithium treatment.”

    Minor comments:

    1. The paper needs a thorough proof reading as it harbors many spelling mistakes, grammatical errors, and poor sentence formation in multiple instances.

    *The paper was thoroughly read, and spelling mistakes and grammar were fixed. *

    Supplemental Figure S2A and S2B should be quoted separately from S2C and S2D.

    *This was updated in the latest version of the paper. *

    In Page 6 paragraph 2 - "authors wrote "To determine if GSK3 could be a potential kinase for this protein, we employed ScanSite4.0, which confirmed that of the 9 DRPs of Chlamydomonas, the only one with a traditional GSK3 target sequence was DRPs (Supplemental Figure 2)." No data is shown in S2 with regard to this. Either data needs to be shown or change the text in a way to avoid confusion.

    *The text was changed in a way to avoid confusion. *

    It would be nice to see if GSK3 can actually phosphorylate DRP3.

    *This would be interesting, however there is not currently a simple way to test this. There is not an antibody for DRP3 that shares enough of its immunogen sequence with the Chlamydomonas DRP3 sequence to use for a western blot. *

    The authors observe that arpc4 mutants do not form actin puncta upon LiCl treatment. Could this phenotype be rescued by complementing with WT ARPC4.

    *We showed in our previous paper (doi.org/10.1101/2020.11.24.396002) that the actin puncta could be rescued by re-expression of wild-type ARPC4 (Figure 4). *

    The concentration of inhibitors is described differently in the text and figure legends (for example Fig. 4A)

    *In the figure legend of figure 4, the concentration of 6-BIO was accidentally reported as 100 µM instead of the correct value (100 nM) as it was throughout the rest of the paper. This was addressed in the latest version. *

    The p values are not significant in some of the figures. (Fig. 4D &Fig. 5C)

    P values were provided for all comparisons in an effort to be transparent and so that readers could draw their own conclusions about the data.

    Reviewer #1 (Significance (Required)):

    The current manuscript by Bigge et al. demonstrates that endocytosis is required for GSK3 inhibition mediated ciliary lengthening. Maintenance of proper length of cilia is crucial and its dysregulation results in pathogenesis. This work takes the field forward and helps in our understanding of how ciliary length is regulated. This work is of interest to researchers working in the field of ciliary biology as well as to those working on endocytosis.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Summary: The authors show in this study that Lithium and other GSK3-beta inhibitors induce cilia elongation in Chlamydomonas. They further demonstrate that inhibition of endocytosis by Dynasore prevents the induced elongation of cilia. They speculate that a Dynamin-related protein might be involved in this process, and determine 9 Dynamin related proteins (DRPs) in Chlamydomonas of which DRP3 shows the highest sequence similarity. Lithium-induced ciliary elongation is prevented in DRP3 mutants supporting the author's hypothesis and indicating that DRP3 might be a GSK3-beta target, similar to some animal Dynamins. Since Dynamins interact with the F-actin regulator ARP3/3-complex, and because F-actin reorganization is observed in cells after GSK3-beta inhibition, they test the induction of ciliary elongation in arpc4 mutants and after blocking the ARP-complex by CK-666. Indeed, F-actin remodeling and cilia elongation were prevented after loss of ARP-complex function. The induction of ciliary elongation and F-actin remodeling also correlates with the emergence of strong F-actin punctae in cells, and the authors interpret that as induction of Dynamin-dependent endocytosis (also addressed in a current preprint from the group). From that, the conclude that endocytosis is required for delivering membrane to the growing cilium and that this is required for the observed effects. While this claim is somewhat supported by a lack of cilia elongation inhibition after treatment to prevent protein synthesis or Golgi function, direct evidence for membrane delivery to the cilium, the need for membrane delivery for ciliary elongation, and presence of bona fide endocytotic vesicles is sadly missing. Therefore, this study sheds new light on an important process in ciliary functional regulation and also furthers our understanding on why GSK3-beta inhibition induces elongated cilia in many cell systems, but I am not convinced that the conclusions are actually supported by the data, as the two key points in question were not experimentally addressed at this point.

    Main points:

    1. The authors need to demonstrate that new membrane is delivered in the process to the growing cilium. E.g. this could be done by membrane stains (pulse) and static or live-cell imaging analysis in untreated, GSK3-beta inhibitor treated and in mutants.

    *In our previous paper (doi.org/10.1101/2020.11.24.396002), we do an experiment similar to the one described here (Figure 8, previous paper). We biotinylated all surface proteins, then removed the cilia (and therefore all labelled ciliary surface proteins) and allowed them to regrow. We then isolated the new cilia and probed for biotinylated proteins because any biotinylated proteins must have come from the surface of the cell. We found that the cilia did contain membrane proteins from the surface of the cell. This experiment shows that membrane and membrane proteins derived from the plasma membrane are entering growing cilia during regeneration. We added a description of this experiment to the text as follows: *

    “Conversely, when treated with Dynasore to inhibit endocytosis, cilia could not elongate to the same degree as untreated cells (Figure 3A-B), implying endocytosis is required for lithium-induced elongation and that endocytosis requires dynamin. This is consistent with results from our previous studies which show that ciliary membrane and membrane proteins are delivered from the cell body plasma membrane to the cilia. In an experiment first performed in Dentler 2013 and then later in Bigge et al. 2020, we biotinylated all cell surface proteins. Then, deciliated cells and allowed cilia to regrow. We then isolated cilia and probed for biotinylated proteins. Any biotinylated proteins present must have come from the cell body plasma membrane, and we found that indeed biotinylated proteins exist in the newly grown cilia, suggesting that ciliary membrane and membrane proteins can be recruited from the cell body plasma membrane (Dentler 2013; Bigge et al. 2020).”

    However, this experiment cannot be done in the case of lithium because cilia are not removed meaning they already will contain labelled surface proteins. Additionally, cells do not regrow cilia in the presence of lithium, meaning that we cannot add a regeneration. Regardless, work from our previous paper described above does establish that ciliary membrane and membrane proteins are able to come from the cell body plasma membrane as the reviewer requested.

    Along the same line, the authors need to demonstrate that the punctae are truly endocytotic vesicles. For that uptake assays/stains could be used and additional markers. Furthermore, there are multiple modes of endocytosis (e.g. Clathrin) besides Dynamin. The authors should determine if blocking other modes of endocytosis has similar or divergent effects on cilia elongation.

    *In our previous paper (doi.org/10.1101/2020.11.24.396002) we supplement the actin puncta data with membrane labelling to show that the puncta are likely endocytic pits (doi.org/10.1101/2020.11.24.396002, Figure 5). We also show that the puncta require both the Arp2/3 complex and active clathrin to form, further suggesting that they are endocytic (Figure 7, previous paper). We added this to the paper as follows: *

    “Further, they rely on proteins typically thought to be involved in endocytosis including the Arp2/3 complex and clathrin, and they form at times when it makes sense for endocytosis to be occurring, like immediately following deciliation when membrane and protein must be recruited to cilia in a timeframe too short for new protein and membrane synthesis, sorting, and trafficking (Bigge et al. 2020). To provide additional evidence that these are endocytic puncta, we also showed that a corresponding increase in membrane internalization occurs during this same timeframe using a fluorescent membrane dye that is endocytosed in wild-type cells (Bigge et al. 2020).”

    *Additionally, Dynamin is required for most forms of endocytosis, including clathrin mediated endocytosis. In the previous paper (doi.org/10.1101/2020.11.24.396002), which we cite here, we do a deep dive into which endocytic proteins are present in *Chlamydomonas. We found that clathrin mediated endocytosis is the most highly conserved on the endocytic processes we looked at (Figure 5, previous paper).

    *We did add a new figure to this paper (Figure 4) using a dye that labels membrane in lithium treated cells. This dye binds to the plasma membrane but is unable to enter cells by itself and must be endocytosed. We found that during the first 30 minutes of lithium treatment there is increased membrane dye internalization. *

    No cilia are actually shown in the study. I personally, would like to see how these cilia look like, especially in relation to the sites of F-actin remodeling and punctae formation. What comes first? Please also provide a axoneme staining to confirm elongation of the ciliary core and what happens to the tubulin pool when cilia cannot elongate any more? Is it accumulating at the ciliary base?

    We added a panel demonstrating where the puncta are in relation to cilia in Figure 4 with a brightfield overlay.* We also look at the appearance and timing of these puncta more in depth in our previous paper (doi.org/10.1101/2020.11.24.396002, Figure 7). We find that puncta form immediately following deciliation and start to return to normal following about 10 minutes of regrowth. We think that this mechanism of ciliary elongation in lithium is similar to what occurs during those early steps of ciliary assembly suggesting that the dots likely form very early on. *

    We also included axoneme staining in Supplemental figure 4*. We show that the axoneme does continue to elongate with the cilia. After about 90 minutes, the cilia actually stop growing and detach from the cells (doi: 10.1128/EC.3.5.1307-1319.2004, doi: doi.org/10.1247/csf.12.369). However, we are interested in the more acute mechanisms that result in ciliary elongation. *

    The authors also claim that the method of GSK3 inhibition is not important. It would be more correct to say that the mode/drug of GSK3 inhibition is not important, but discuss how some of the minor variance between treatments could be explained (incl. the timeline and temporal dynamics of the diverging effects; and the dose-dependency as low concentrations of BIO seem to induce shortening but high doses induce elongation of cilia).

    *We further discussed this in the text as follows: *

    “The minor variances between the drugs could be explained by the timeline in which we tested cilia (90 minutes) or the exact dosages we used. An example of this is 6-BIO where treatment with a low dose of 100 nM caused ciliary lengthening, but treatment with a higher concentration of 2 µM reportedly caused ciliary shortening (Kong et al. 2015). Together, the data suggest that the mode of inhibition by chemical targets of GSK3 is not important for ciliary lengthening. Whether GSK3 was inhibited via competition for ATP binding or phosphorylation, cilia were able to elongate.”

    They propose here a positive effect of F-actin build up in cilia length regulation, while most studies to date report ciliary shortening to correlate with increased F-actin at the ciliary base. I believe that this is not highlighted and discussed enough, which I find reduces the overall quality of the paper (but is easy to improve). It might be also interesting to test if other F-actin inducers/stabiliziers have the same effect?

    *This is addressed in the discussion in the latest version in depth as follows: *

    “One important detail to point out is that Chlamydomonas differ from mammalian cells in that they have a cell wall. The stability awarded by the cell wall means that Chlamydomonas does not require a cortical actin network as mammalian cells do. Thus, in Chlamydomonas, we are able to investigate actin dynamics and functions without the interference of the cortical actin network. This also means that some of the effects we see might be masked in mammalian cells by the presence of the cortical actin network and the effect that it has on ciliary assembly and maintenance.”

    *We also added a section to the introduction to address this concern early on so that readers will have this difference in mind as they read the paper: *

    “Additionally, unlike mammalian cells, Chlamydomonas lacks a cortical actin network which simplifies the relationship between cilia and actin and makes this an ideal model to study such interactions.”

    Also, F-actin inducers/stabilizers do not typically have the same effect because the filamentous actin needed for these processes must be dynamic, or able to undergo rapid depolymerization and repolymerization as needed during this fairly quick timeframe. This is demonstrated in Avasthi, 2014 (*doi.org/10.1016/j.cub.2014.07.038). Cells were treated with several actin targeting inhibitors including LatB which results in depolymerization of filaments and Jasplakinolide which results in stabilization of filaments. In both cases, ciliary regeneration is impaired suggesting that actin must be dynamic for its functions related to cilia. *

    Minor points:

    1. In many Figures, the x-axis is labeled "Number of values", but I think that maybe number of observations might be more appropriate.

    We discussed this point and decided to change the axis titles to “Number of cilia”.

    The author often use the word "normally" elongating, but in all cases the elongation is induced = abnormal situation. Maybe the authors could use a different term.

    We originally used “normally” because there are times when we get defective elongation but not no elongation. In the latest version we changed this to “elongation consistent with untreated wild-type cells” or something along those lines.

    It is puzzling as to why DRP3 was chosen, while DRP2 actually is most similar in terms of domain composition. Maybe they could discuss that. They also could explain a bit better how the mutants were generated in which a "cassette was inserted early in the gene". What kind of disruption is expected?

    DRP3 was chosen because it has the highest sequence identity (and similarity). DRP2 while containing all domains, has low overall sequence conservation. DRP3 is also the only DRP that showed a potential GSK3 target site when investigated with ScanSite4.0. This was all made clearer in the text as follows:

    “Chlamydomonas contains 9 DRPs with similarity to a canonical dynamin (DRP1-9). Despite lacking 2 of the canonical dynamin domains, the DRP with the highest sequence similarity and identity to canonical dynamin is DRP3 (Supplemental Figure 2C-D). To determine if GSK3 could be a potential kinase for this protein, we employed ScanSite4.0, which confirmed that of the 9 DRPs of Chlamydomonas, the only one with a traditional GSK3 target sequence was DRP3.”

    The representative images in Figure 4A do not really seem to match the quantifications.

    *The quantitative data suggest that these different treatments have increased dots, which we believe the representative images do show. LiCl and CHIR99021 have the most dots, while 6-BIO and Tideglusib have more dots, but less than LiCl and CHIR99021. *

    line 109: "of-targets" should be off-targets

    Fixed in the latest version, thanks for pointing this out.

    line 141: "delivery form the Golgi" should be FROM the Golgi

    Fixed in the latest version, thanks for pointing this out.

    line 160: "was DRPs" should be was DRP3

    Fixed in the latest version, thanks for pointing this out.

    line 204/205: the sentence starting "Thus, we phalloidin..." should be rephrased. It sounds not quite correct

    Fixed in the latest version, thanks for pointing this out.

    line 209: Figure 4A should refer to Figure 4B

    Fixed in the latest version, thanks for pointing this out.

    line 211: "times or rapid ciliary" should be of rapid ciliary...

    Fixed in the latest version, thanks for pointing this out.

    line 257: "in lithium." Should be in lithium treated cells Fixed in the latest version, thanks for pointing this out.

    Reviewer #2 (Significance (Required)):

    This study sheds new light on an important process in ciliary functional regulation and also furthers our understanding on why GSK3-beta inhibition induces elongated cilia in many cell systems, but I am not convinced that the conclusions are actually supported by the data, as the two key points in question were not experimentally addressed at this point.

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

    Chlamydomonas maintains relatively regular length of cilia (flagella). However, when the cell is exposed to high concentration of lithium ions, it elongates cilia further. In this work, Bigge and Avasthi made experiments to build a potential hypothesis of molecular mechanism of this unusual cilia elongation. Their hypothesis is (1) cilia elongation is triggered, depending on supply of extra membrane (not proteins), (2) membrane is supplied from plasma membrane by clathrin-dependent endocytosis (not from Golgi), (3) this endocytosis contains Arp2/3 complex, (4) GSK3 downregulates Arp2/3 dependent endocytosis and (5) GSK3 is suppressed by lithium. They conducted well-organized experiments to prove each step. While some of them are indirect, their hypotheses were supported experimentally in outline.

    (1) is undoubted, since the authors demonstrated that inhibition of protein production by cycloheximide did not influence cilia elongation.

    (2) The authors clearly demonstrated that source of ciliary membrane for elongation is plasma membrane and not Golgi by examining specific inhibitors' effect. They also showed protein transfer from plasma membrane to cilia, by biotinylaing surface proteins in the cell, deciliating and growing cilia and detecting biotinylated proteins in cilia. This part rather characterizes initial growth of cilia, not elongation. Therefore this result must be properly described in the context of this work (which is elongation of cilia).

    This comment was particularly helpful as it also helps us address some of the comments from the other reviewers. We updated the description of this experiment in the context of this work in the latest version as follows:

    “Further, they rely on proteins typically thought to be involved in endocytosis including the Arp2/3 complex and clathrin, and they form at times when it makes sense for endocytosis to be occurring, like immediately following deciliation when membrane and protein must be recruited to cilia in a timeframe too short for new protein and membrane synthesis, sorting, and trafficking (Bigge et al. 2020). To provide additional evidence that these are endocytic puncta, we also showed that a corresponding increase in membrane internalization occurs during this same timeframe using a fluorescent membrane dye that is endocytosed in wild-type cells (Bigge et al. 2020).”

    For (3)-(4), they visualized Arp2/3 localization, showing highly condensed Arp2/3. They interpreted these particles as sign of clathrin endocytosis. Since so far such an endocytosis particle has not been reported in Chlamydomonas, the authors confirmed that DRPs are target of GSK3 to indirectly show GSK3 influences formation of endocytosis. This reviewer thinks the author should be able to directly confirm endocytosis for example by electron microscopy (of traditional epon-embedded and stained cells).

    *We visualized Arp2/3 complex-dependent filamentous actin localization. We provide DRP3 as a potential target of GSK3, but do not report that it is the target that results in increased endocytosis or increased ciliary length. We agree that electron microscopy would be ideal to visualize endocytosis in these cells. However, we feel this is outside the scope of this current work. But, we do have plans to look at endocytosis in *Chlamydomonas *using electron microscopy in the future and hope that the increased context from the previous data are sufficient at this time. *

    (5) was elegantly proved by multiple drugs (all known as inhibitor of GSK3), including lithium.

    After fixing these points, this manuscript will be ready for publication.

    Minor points: Line188-191: not clear. What are *** and ****?

    Fixed in the latest version, thanks for pointing this out.

    Line262-264: It would be helpful how the initial cilia growth of the arpc4 cell.

    We agree that this would be helpful information, and included more of a description of how ciliary growth is affected by loss of Arp2/3 complex function in the latest version: “Specifically, we found that the Arp2/3 complex is required for reclamation of membrane from a pool in the plasma membrane during the rapid growth that occurs during early ciliary assembly”.

    Line321: it should read as follows. Cang 2014; carlsson and Bayly 2014). While we...

    Fixed in the latest version, thanks for pointing this out.

    Line329: were -> where

    Fixed in the latest version, thanks for pointing this out.

    Line365-366: Lithium-treated cells are not motile. Any thought why? Maybe protein production is not necessary for apparent cilia elongation, but necessary for elongation of functional cilia.

    *This is an interesting idea. However, even when protein production is allowed to proceed, Lithium-treated cells are not motile. This is a ciliary dysfunction, and in fact, after about 90 minutes incubation with lithium, the cilia of these cells start to crash out or fall off, demonstrating that these are not healthy cells or healthy cilia. *

    Reviewer #3 (Significance (Required)):

    This work is an important step toward the understanding of cilia elongation and thus growth mechanism. It will attract wide audience who have interest in cell biology and motility. My expertise is about motile cilia and their 3D structure.

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    Referee #3

    Evidence, reproducibility and clarity

    Chlamydomonas maintains relatively regular length of cilia (flagella). However, when the cell is exposed to high concentration of lithium ions, it elongates cilia further. In this work, Bigge and Avasthi made experiments to build a potential hypothesis of molecular mechanism of this unusual cilia elongation. Their hypothesis is (1) cilia elongation is triggered, depending on supply of extra membrane (not proteins), (2) membrane is supplied from plasma membrane by clathrin-dependent endocytosis (not from Golgi), (3) this endocytosis contains Arp2/3 complex, (4) GSK3 downregulates Arp2/3 dependent endocytosis and (5) GSK3 is suppressed by lithium. They conducted well-organized experiments to prove each step. While some of them are indirect, their hypotheses were supported experimentally in outline.

    (1) is undoubted, since the authors demonstrated that inhibition of protein production by cycloheximide did not influence cilia elongation.

    (2) The authors clearly demonstrated that source of ciliary membrane for elongation is plasma membrane and not Golgi by examining specific inhibitors' effect. They also showed protein transfer from plasma membrane to cilia, by biotinylaing surface proteins in the cell, deciliating and growing cilia and detecting biotinylated proteins in cilia. This part rather characterizes initial growth of cilia, not elongation. Therefore this result must be properly described in the context of this work (which is elongation of cilia).

    For (3)-(4), they visualized Arp2/3 localization, showing highly condensed Arp2/3. They interpreted these particles as sign of clathrin endocytosis. Since so far such an endocytosis particle has not been reported in Chlamydomonas, the authors confirmed that DRPs are target of GSK3 to indirectly show GSK3 influences formation of endocytosis. This reviewer thinks the author should be able to directly confirm endocytosis for example by electron microscopy (of traditional epon-embedded and stained cells). (5) was elegantly proved by multiple drugs (all known as inhibitor of GSK3), including lithium. After fixing these points, this manuscript will be ready for publication.

    Minor points:

    Line188-191: not clear. What are *** and ****?

    Line262-264: It would be helpful how the initial cilia growth of the arpc4 cell.

    Line321: it should read as follows.

    Cang 2014; carlsson and Bayly 2014). While we...

    Line329: were -> where

    Line365-366: Lithium-treated cells are not motile. Any thought why? Maybe protein production is not necessary for apparent cilia elongation, but necessary for elongation of functional cilia.

    Significance

    This work is an important step toward the understanding of cilia elongation and thus growth mechanism. It will attract wide audience who have interest in cell biology and motility. My expertise is about motile cilia and their 3D structure.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The authors show in this study that Lithium and other GSK3-beta inhibitors induce cilia elongation in Chlamydomonas. They further demonstrate that inhibition of endocytosis by Dynasore prevents the induced elongation of cilia. They speculate that a Dynamin-related protein might be involved in this process, and determine 9 Dynamin related proteins (DRPs) in Chlamydomonas of which DRP3 shows the highest sequence similarity. Lithium-induced ciliary elongation is prevented in DRP3 mutants supporting the author's hypothesis and indicating that DRP3 might be a GSK3-beta target, similar to some animal Dynamins. Since Dynamins interact with the F-actin regulator ARP3/3-complex, and because F-actin reorganization is observed in cells after GSK3-beta inhibition, they test the induction of ciliary elongation in arpc4 mutants and after blocking the ARP-complex by CK-666. Indeed, F-actin remodeling and cilia elongation were prevented after loss of ARP-complex function. The induction of ciliary elongation and F-actin remodeling also correlates with the emergence of strong F-actin punctae in cells, and the authors interpret that as induction of Dynamin-dependent endocytosis (also addressed in a current preprint from the group). From that, the conclude that endocytosis is required for delivering membrane to the growing cilium and that this is required for the observed effects. While this claim is somewhat supported by a lack of cilia elongation inhibition after treatment to prevent protein synthesis or Golgi function, direct evidence for membrane delivery to the cilium, the need for membrane delivery for ciliary elongation, and presence of bona fide endocytotic vesicles is sadly missing. Therefore, this study sheds new light on an important process in ciliary functional regulation and also furthers our understanding on why GSK3-beta inhibition induces elongated cilia in many cell systems, but I am not convinced that the conclusions are actually supported by the data, as the two key points in question were not experimentally addressed at this point.

    Main points:

    1. The authors need to demonstrate that new membrane is delivered in the process to the growing cilium. E.g. this could be done by membrane stains (pulse) and static or live-cell imaging analysis in untreated, GSK3-beta inhibitor treated and in mutants.
    2. Along the same line, the authors need to demonstrate that the punctae are truly endocytotic vesicles. For that uptake assays/stains could be used and additional markers. Furthermore, there are multiple modes of endocytosis (e.g. Clathrin) besides Dynamin. The authors should determine if blocking other modes of endocytosis has similar or divergent effects on cilia elongation.
    3. No cilia are actually shown in the study. I personally, would like to see how these cilia look like, especially in relation to the sites of F-actin remodeling and punctae formation. What comes first? Please also provide a axoneme staining to confirm elongation of the ciliary core and what happens to the tubulin pool when cilia cannot elongate any more? Is it accumulating at the ciliary base?
    4. The authors also claim that the method of GSK3 inhibition is not important. It would be more correct to say that the mode/drug of GSK3 inhibition is not important, but discuss how some of the minor variance between treatments could be explained (incl. the timeline and temporal dynamics of the diverging effects; and the dose-dependency as low concentrations of BIO seem to induce shortening but high doses induce elongation of cilia).
    5. They propose here a positive effect of F-actin build up in cilia length regulation, while most studies to date report ciliary shortening to correlate with increased F-actin at the ciliary base. I believe that this is not highlighted and discussed enough, which I find reduces the overall quality of the paper (but is easy to improve). It might be also interesting to test if other F-actin inducers/stabiliziers have the same effect?

    Minor points:

    1. In many Figures, the x-axis is labeled "Number of values", but I think that maybe number of observations might be more appropriate.
    2. The author often use the word "normally" elongating, but in all cases the elongation is induced = abnormal situation. Maybe the authors could use a different term.
    3. It is puzzling as to why DRP3 was chosen, while DRP2 actually is most similar in terms of domain composition. Maybe they could discuss that. They also could explain a bit better how the mutats were generated in which a "cassette was inserted early in the gene". What kind of disruption is expected?
    4. The representative images in Figure 4A do not really seem to match the quantifications.
    5. line 109: "of-targets" should be off-targets
    6. line 141: "delivery form the Golgi" should be FROM the Golgi
    7. line 160: "was DRPs" should be was DRP3
    8. line 204/205: the sentence starting "Thus, we phalloidin..." should be rephrased. It sounds not quite correct
    9. line 209: Figure 4A should refer to Figure 4B
    10. line 211: "times or rapid ciliary" should be of rapid ciliary...
    11. line 257: "in lithium." Should be in lithium treated cells

    Significance

    This study sheds new light on an important process in ciliary functional regulation and also furthers our understanding on why GSK3-beta inhibition induces elongated cilia in many cell systems, but I am not convinced that the conclusions are actually supported by the data, as the two key points in question were not experimentally addressed at this point.

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    Referee #1

    Evidence, reproducibility and clarity

    The current manuscript by Bigge et al. demonstrated that the chemical inhibition of GSk3 causes ciliary elongation in Chlamydomonas reinhardtii. They show that lithium induced ciliary lengthening is majorly due to GSK3 inhibition. Consistent with earlier reports, they show that new protein synthesis is not required for lithium induced ciliary elongation. The authors report that targeting endocytosis either by using chemical inhibitors (dynasore and CK-666) or genetic mutants (dpr3 and Arpc4) does not cause lithium induced ciliary elongation. They further reveal enhanced actin dynamics in lithium treated cells and such activity is lost in Arpc4 mutants. Based on these results, the authors concluded that endocytic pathways may be involved in lithium induced ciliary lengthening. The results are interesting, and this work is important in understanding more about ciliary length regulation. However, more experimental evidence addressing the current interpretation that endocytic pathways may be involved in lithium induced ciliary lengthening is required.

    Major comments:

    1. The authors use chemical inhibitors as major tools for their study. However, the specificity of these inhibitors is a concern. How specific are these GSK3 inhibitors such as LiCl? Can authors show that LiCl mediated ciliary lengthening is due to inhibition of GSK3? Authors used BFA and Dynasore to show that not the Golgi, but the endocytosis derived membrane is required for ciliary lengthening. Again, here the specificity of these inhibitors is a concern. Especially as Dynasore has been shown to have non-specific effects.
    2. Does inducing/enhancing endocytosis independent of GSK3 by other means has any effect on ciliary length regulation?
    3. The major claim of this paper is that LiCl mediated ciliary lengthening is due to enhanced endocytosis. Although authors showed that inhibition of endocytosis results in reduced ciliary length, it is important to show if GSK3 inhibition by LiCl (or any other inhibitor) causes any increased cellular endocytosis? Similarly, what is the effect of GSK3 mutants on endocytosis?
    4. Are these endocytic processes enhanced specifically at/or around the cilium during the ciliary lengthening process?
    5. Authors claim that drp3 is a target of GSK3 and, similar to the canonical dynamin, functions in endocytosis. While, it is an important observation, experiments are required to show the role of drp3 in endocytosis and also to show that it is indeed a target of GSK3.
    6. Mechanistic insights into how endocytosis/actin dynamics regulate ciliary lengthening would be interesting to see. Further, it is interesting to see if the ciliary signaling defects caused by abnormal ciliary length can be rescued by inhibition of endocytosis.

    Minor comments:

    1. The paper needs a thorough proof reading as it harbors many spelling mistakes, grammatical errors, and poor sentence formation in multiple instances.
    2. Supplemental Figure S2A and S2B should be quoted separately from S2C and S2D.
    3. In Page 6 paragraph 2 - "authors wrote "To determine if GSK3 could be a potential kinase for this protein, we employed ScanSite4.0, which confirmed that of the 9 DRPs of Chlamydomonas, the only one with a traditional GSK3 target sequence was DRPs (Supplemental Figure 2)." No data is shown in S2 with regard to this. Either data needs to be shown or change the text in a way to avoid confusion.
    4. It would be nice to see if GSK3 can actually phosphorylate DRP3.
    5. The authors observe that arpc4 mutants do not form actin puncta upon LiCl treatment. Could this phenotype be rescued by complementing with WT ARPC4.
    6. The concentration of inhibitors is described differently in the text and figure legends (for example Fig. 4A)
    7. The p values are not significant in some of the figures. (Fig. 4D &Fig. 5C)

    Significance

    The current manuscript by Bigge et al. demonstrates that endocytosis is required for GSK3 inhibition mediated ciliary lengthening. Maintenance of proper length of cilia is crucial and its dysregulation results in pathogenesis. This work takes the field forward and helps in our understanding of how ciliary length is regulated. This work is of interest to researchers working in the field of ciliary biology as well as to those working on endocytosis.

  6. Version published to 10.1101/2022.04.18.488674v1 on bioRxiv