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  1. Author Response:

    Thank you to the reviewers and editors at eLife for the comments on our manuscript. We believe we can address or rebut all of the reviewer comments, and our responses are included below.

    Reviewer #1 (Public Review):

    In this manuscript Bigge et al. use chemical inhibitors and a mutant in ARPC4arpc4 mutant to investigate the role of the Arp2/3 complex in regulating cilia length and assembly in Chlamydomonas. The authors have previously shown that the actin cytoskeleton is required for ciliary assembly and maintenance in this organism, but the precise mechanism(s) involved were unclear. Furthermore, while previous studies targeted the actin cytoskeleton in general, the current study focuses on branched actin networks regulated by the Arp2/3 complex. The authors first demonstrate that chemical inhibition of the Arp2/3 complex leads to shortening of existing cilia, a phenotype that is recapitulated in the arpc4 mutant and which can be rescued be reintroducing V5-tagged ARPC4 in the latter mutant. Next, using similar approaches they show that initial stages of cilium biogenesis are also impaired upon Arp2/3 complex inhibition. They next use a variety of approaches, mostly involving chemical inhibitors and F-actin- or membrane dyes, to assess the mechanism by which Arp2/3 complex affects ciliary biogenesis. They provide evidence indicating that Arp2/3 complex specifically promotes endocytosis at the plasma membrane to support lipid and protein for the growing ciliary membrane. This is an interesting discovery that advances our understanding of how ciliary membrane biogenesis is regulated, especially in Chlamydomonas.

    Thank you for these comments and the accurate summary of our findings.

    Reviewer #2 (Public Review):

    Previous studies have demonstrated that interfering with actin polymerization leads to the shortening of flagella in Chlamydomonas cells, indicating that F-actin is important for ciliary/flagellar elongation. However, the precise roles of F-actin, and especially of branched actin networks nucleated by the Arp2/3 complex in cilia formation have not been elucidated. Here, the authors aimed to examine one of the mechanisms that may be involved in F-actin-dependent cilia elongation/formation, namely, actin- and clathrin-dependent endocytosis of membrane proteins.

    The authors used both pharmacological inhibition and genetic disruption of the Arp2/3 complex to demonstrate that interfering with the activity of the Arp2/3 complex reduces the ability of Chlamydomonas to form or elongate cilia. The authors then showed that incorporation of existing (not newly synthesized) proteins into cilia is perturbed by the genetic or pharmacological inhibition of the Arp2/3 complex, and that new membrane for building cilia may be derived via an Arp2/3-mediated membrane retrieval pathway.

    These experiments are carefully performed and convincing. A description of the Arp2/3 complex components and clathrin-containing structures in Chlamydomonas is novel and will be of interest to the cell biologists working on this model organism.

    The authors propose that the Arp2/3-mediated actin assembly promotes cilia elongation/formation due to the contribution of the branched actin to clathrin-dependent endocytosis. This is an intriguing idea that ties together previous findings showing that F-actin is needed for cilia elongation and that some ciliary proteins are internalized from the plasma membrane and then redistributed to cilia. One concern regarding this portion of the manuscript in that the experiments addressing the role of endocytosis rely solely on the use of a pharmacological inhibitor of clathrin-dependent endocytosis, PitStop2. PitStop2 was previously shown to have non-specific effects on endocytosis, suggesting that its mechanism of action may not be directly related to disrupting clathrin heavy chain interactions (see, for example, Willox et al., 2014).

    We thank this reviewer for their helpful comments regarding the work presented in our manuscript. We understand the concerns regarding the off-target effects of PitStop2. However, the PitStop2 data is only one of multiple lines of evidence that we provide supporting endocytosis occurring in these cells (internalization of membrane dye in Figure 5, internalization and relocalization of a ciliary membrane protein in Figure 6, actin patches in Figures 4 & 7, the presence of plasma membrane proteins in the cilia in Figure 8). Further, the requirement for Arp2/3 in the phenotypes listed points to an endocytic mechanism as the Arp2/3 complex has repeatedly been shown to be involved in actin dynamics in endocytosis in other systems. To determine the mechanism of endocytosis most likely occurring in these cells, we searched the Chlamydomonas genome for proteins commonly thought to be involved in endocytic pathways. We propose clathrin mediated endocytosis is occurring in these cells because this is the only pathway where the most important components of the mechanism are present in Chlamydomonas. This is opposed to every other form of endocytosis which is lacking some major component. For example, Chlamydomonas do not contain endophilin for endophilin-mediated endocytosis, flotillin for flotillin-mediated endocytosis, or caveolin for caveolin-mediated endocytosis. Therefore, our conclusions regarding clathrin mediated endocytosis do not rely entirely on PitStop2 data. The PitStop2 data is merely present to support the findings of our comparative analysis and other endocytosis assays. Further, mutants of clathrin heavy or light chain does not exist and cannot be reliably generated in this organism.

    We now have further evidence that endocytosis is occurring in these cells using dynamin inhibitors. Specifically, using the dynamin inhibitor, Dynasore, reduces membrane uptake in a dye internalization assay. We plan to include this data in the next version of the paper.

    Intriguingly, a previous paper by Kim et al., 2010 demonstrated that interfering with the Arp2/3-dependent actin assembly resulted in cilia elongation in mammalian cells, suggesting that branched actin assembly was counteracting growth of cilia. Similarly, actin depolymerization is known to promote ciliogenesis in mammalian cells. The difference between mammalian actin organization/cilia growth regulation vs. the new observations in the Chlamydomonas system should be discussed by the authors to help the readers understand whether the findings can be generalized to the diverse ciliated cell types or are unique to algae.

    While Chlamydomonas is an excellent model for ciliary studies due to the structural and mechanistic conservation of the cilia in relation to mammalian cilia, there are several important differences between mammalian cells and Chlamydomonas cells that might influence ciliary dynamics. These differences have been reported on by others in the field (Jack et al. 2019). Briefly, one difference is that Chlamydomonas cells have a cell wall, which means they have no need for a cortical actin network as mammalian cells do. This cortical actin network in mammalian cells is thought to potentially block access of basal bodies and ciliary proteins from the cortex of the cell. This in turn blocks ciliary formation and assembly.

    We now have data that a mechanism of ciliary elongation mediated by lithium, which is conserved between mammalian cells and Chlamydomonas, is blocked by loss of the Arp2/3 complex, suggesting that ciliary/membrane growth not just from zero length but also from steady state requires the Arp2/3 complex. This data will be reported in a subsequent publication. While we could speculate on the conservation of ciliary growth between mammalian cells and Chlamydomonas in this paper, a more well-supported speculation would be in the subsequent paper where we directly discuss the role of the Arp2/3 complex in a conserved ciliary growth process.

    Reviewer #3 (Public Review):

    Wild-type Chlamydomonas cells possess a pool of ciliary precursors that, in conjunction with newly synthesized precursors, are used to build a new cilium after de-ciliation. The cellular and molecular mechanisms the underly retrieval of the pool are unknown. Given the role of the Arp2/3 complex in endocytosis in other systems, it was reasonable to test whether it also functions in reclaiming of the pool of precursors. The central finding is that cells bearing a mutation in an essential Arp2/3 component, ARPC4, fail to assemble cilia in a timely fashion after de-ciliation. Although the failure of assembly is well-documented here, the manuscript lacks evidence that cells missing ARPC4 actually establish a pool of ciliary precursors in the first place. Without such information it is not possible to determine the cellular function that fails to occur after de-ciliation in the mutant: Retrieval of a pool of ciliary precursors? Establishing the pool during the ciliary assembly that occurs after cell division? Synthesis of the precursors as new cilia are formed? Sensing loss of cilia and activating the events needed for re-ciliation?

    Upon discovering that there was almost no initial ciliary assembly in mutants of the Arp2/3 complex caused by loss of the functional Arp2/3 complex, we immediately suspected that either it lacked a precursor pool, as the reviewer suggests, or that mutants were unable to incorporate it. To discriminate between these possibilities, you typically need to be able to regenerate cilia in cycloheximide. However, the inability of genetic mutants to regenerate in cycloheximide prevents us from being able to do the typical studies testing new protein synthesis, precursor pool size, and new protein incorporation as they all require regeneration in cycloheximide. Therefore, to get around this roadblock, we used an acute perturbation through chemical inhibition on wild-type cells that have a normal ciliary precursor pool (as evidenced by their ability to grow to half-length in cycloheximide). These cells were deciliated and then the Arp2/3 complex inhibitor CK-666 (in addition to cycloheximide) was added only for the regrowth, and thus it was not able to affect the size of the precursor pool. Cells treated with CK-666 and cycloheximide could not incorporate the precursor pool we know exists in these wild-type cells (Figure 2B). We referred to these results in the text saying: “This can be further dissected because we know in the case of cells treated with cycloheximide and CK-666, the protein pool is available but is still not incorporated, suggesting a problem with membrane incorporation or delivery as opposed to a problem with protein availability” (Lines 195-200). We understand that this piece of data may be confusing the way it is displayed (only the final growth length after 2 hours), so we plan to provide the full regeneration graphs in the supplement.

    While we are not able to concretely rule out a problem with signal transduction alerting cells that they have lost their cilia or with protein synthesis, we believe these are lesser effects because some arpc4 mutant cells do grow immediately following deciliation and because eventually cells reach near full length in the absence of cycloheximide (Figure 1C). However, we plan to test protein synthesis immediately following deciliation using semiquantitative PCR for the next iteration of this manuscript.

    Finally, we provide several pieces of orthogonal data to suggest that membrane incorporation is a problem in these cells. First, we show that blocking membrane delivery from the Golgi causes increased ciliary shortening in arpc4 mutant cells compared to wild-type cells. This suggests there is an alternate source of membrane that is defective in the arpc4 mutant cells (Figure 3). We go on to show that actin structures that form near the membrane at the base of the cilia are absent in the arpc4 mutant but present in wild-type cells, and that these structures increase following deciliation (Figures 4 and 7). We show that aprc4 mutants are less capable of internalizing membrane than wild-type cells (Figure 5) and less capable of internalizing a ciliary membrane protein for mating (Figure 6). Lastly, we show that the pattern of ciliary membrane proteins that came from the cell body plasma membrane is altered in arpc4 mutants suggesting defects in at least one pathway (Figure 8). Based on this entire body of data together, in addition to our CK-666 data on wild-type cells with an existing precursor pool, we feel that the best-supported model is one in which a defect in membrane delivery may be responsible for the impact we see on ciliary assembly in cells lacking a functional Arp2/3 complex.

    Other conclusions also were not sufficiently supported by the experimental results, including the following: Clathrin function in endocytosis: Pitstop2 was described as specifically blocking clathrin-mediated endocytosis, but reports in the literature indicate that Pitstop2 also blocks nuclear functions and clathrin-independent endocytosis. Experiments with an anti-clathrin antibody were interpreted as showing mislocalization of clathrin in the arpc4 mutants. The antibody was raised against a peptide near the N-terminus of human clathrin, but the antibody was not validated, and it was not reported whether Chlamydomonas clathrin even has that peptide.

    We understand the concerns regarding the off-target effects of PitStop2, and as this concern was shared by reviewer 2 we have addressed this above.

    If further evidence is needed of endocytosis occurring in these cells, we also have new data suggesting that dynamin is also involved in these processes.

    We thank the reviewers for this comment, and plan to add a western blot confirming the clathrin light chain antibody works in Chlamydomonas in the next version. It is true that mammalian antibodies do not always work in Chlamydomonas. This particular antibody was selected based on the immunogen sequence. The immunogen of this antibody was the most similar we could find to the Chlamydomonas sequence (61.5% similar). Our intent with the included data was to highlight the similarities between both membrane internalization and clathrin behavior whether endocytosis or the Arp2/3 complex are inhibited.

    Internalization of a protein from the plasma membrane: In the experiments to use protease-sensitivity to examine SAG1-HA relocalization induced by db-cAMP, the authors assumed that all of the SAG1-HA was on the cell surface in untreated cells and the 2 chemically treated cells, but they never experimentally documented this assumption.

    We respectfully disagree that our assumption is that all of the SAG1-HA is on the surface prior to induction. In fact, our data support the opposite, that there is indeed some percentage of SAG1-HA on the surface and some percentage of SAG1-HA already inside the cell even in uninduced cells given that some portion is protected from trypsin treatment. Regardless of whether the full population of SAG1-HA is on the surface, we can still draw conclusions about the amount of SAG1-HA internalized under the conditions through quantification of the changes in band intensity. This specific assay can only report on whether there is an internalization defect for a plasma membrane protein destined for the cilium.

    Arp2/3 relation to actin dots: Structures termed actin dots that stained with Phalloidin were reported to undergo changes after de-ciliation of wild-type cells and were missing in the arpc4 mutant. The conclusion that the properties of the dots in the wild-type cells changed with de-ciliation were not supported by statistical analysis. Also, without experiments showing localization of Arp2/3-V5 at the dots, it was not possible to assess whether, as the text asserts, Arp2/3 functioned at the dots.

    Statistics were not done for Figure 4 where we show dots are present in wild-type cells and absent in the arpc4 mutant cells because it is clear even without statistics. We do not feel that including an infinitesimally small p value added any substantive information to this piece of data. However, statistics can and should be included in the next version for Figure 7 which shows an increase in dots (both percentage of cells with dots and number of dots per cell) following deciliation. To fully convince readers, we also plan to include images with a larger field of view showing a population of cells, which will make it clear that dots increase following deciliation.

    We attempted to look at ARPC4-V5 localization in Figure 1 Supplemental Figure 2, but saw mostly diffuse localization with high noise. The ARPC4 component of the Arp2/3 complex may be diffusely localized with only some proportion being incorporated into the complex at sites where it is active, or the diffuse nature could be due to overexpression, or both. While we do not definitively show that overexpressed ARPC4 is at the dots in addition to its diffuse localization, we do not believe that this prevents us from drawing conclusions about Arp2/3 complex function in the dots. We do show that the Arp2/3 complex is required for dot formation as cells lacking a functional Arp2/3 complex do not have dots while cells that contain the Arp2/3 complex do have dots. Further, expression of the ARPC4-V5 construct rescues the presence of the dots.

    Cilia resorption induced by BFA in arpc4 mutants: In the experiments with the Golgi-active agent BFA, the percent ciliation in the arpc4 mutants, but not wild type, fell rapidly after drug addition. The authors concluded that BFA induced ciliary resorption, but they did not determine whether the lack of cilia on cells was a consequence of cilia resorption or cilia detachment.

    As the reviewer points out, the percent ciliation of the arpc4 mutant cells treated with BFA fell rapidly after drug addition. We believe this is due to resorption and not ciliary detachment because we see ciliary length decrease over time (not all at once). Additionally, in Figure 3B, we only measure cells that have cilia, and we know that arpc4 mutant cells cannot regrow their cilia to half-length in 1 hour (Figure 1C). Therefore, we know that the cilia we are measuring are in fact shortening. Further, there was no increase in free cilia in the samples. Images can be provided in the supplement for the next version.

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  2. Evaluation Summary:

    This manuscript addresses the role of the Arp2/3 complex in endocytic retrieval of ciliary precursors from the plasma membrane for use in assembly of Chlamydomonas cilia, a topic of broad interest to cell biologists. The manuscript can serve as basis for future research addressing whether Arp2/3 affects cilium biogenesis solely via endocytic retrieval or through additional mechanisms, and whether these findings apply to species other than Chlamydomonas.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

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  3. Reviewer #1 (Public Review):

    In this manuscript Bigge et al. use chemical inhibitors and a mutant in ARPC4arpc4 mutant to investigate the role of the Arp2/3 complex in regulating cilia length and assembly in Chlamydomonas. The authors have previously shown that the actin cytoskeleton is required for ciliary assembly and maintenance in this organism, but the precise mechanism(s) involved were unclear. Furthermore, while previous studies targeted the actin cytoskeleton in general, the current study focuses on branched actin networks regulated by the Arp2/3 complex. The authors first demonstrate that chemical inhibition of the Arp2/3 complex leads to shortening of existing cilia, a phenotype that is recapitulated in the arpc4 mutant and which can be rescued be reintroducing V5-tagged ARPC4 in the latter mutant. Next, using similar approaches they show that initial stages of cilium biogenesis are also impaired upon Arp2/3 complex inhibition. They next use a variety of approaches, mostly involving chemical inhibitors and F-actin- or membrane dyes, to assess the mechanism by which Arp2/3 complex affects ciliary biogenesis. They provide evidence indicating that Arp2/3 complex specifically promotes endocytosis at the plasma membrane to support lipid and protein for the growing ciliary membrane. This is an interesting discovery that advances our understanding of how ciliary membrane biogenesis is regulated, especially in Chlamydomonas.

    Read the original source
    Was this evaluation helpful?
  4. Reviewer #2 (Public Review):

    Previous studies have demonstrated that interfering with actin polymerization leads to the shortening of flagella in Chlamydomonas cells, indicating that F-actin is important for ciliary/flagellar elongation. However, the precise roles of F-actin, and especially of branched actin networks nucleated by the Arp2/3 complex in cilia formation have not been elucidated. Here, the authors aimed to examine one of the mechanisms that may be involved in F-actin-dependent cilia elongation/formation, namely, actin- and clathrin-dependent endocytosis of membrane proteins.

    The authors used both pharmacological inhibition and genetic disruption of the Arp2/3 complex to demonstrate that interfering with the activity of the Arp2/3 complex reduces the ability of Chlamydomonas to form or elongate cilia. The authors then showed that incorporation of existing (not newly synthesized) proteins into cilia is perturbed by the genetic or pharmacological inhibition of the Arp2/3 complex, and that new membrane for building cilia may be derived via an Arp2/3-mediated membrane retrieval pathway.

    These experiments are carefully performed and convincing. A description of the Arp2/3 complex components and clathrin-containing structures in Chlamydomonas is novel and will be of interest to the cell biologists working on this model organism.

    The authors propose that the Arp2/3-mediated actin assembly promotes cilia elongation/formation due to the contribution of the branched actin to clathrin-dependent endocytosis. This is an intriguing idea that ties together previous findings showing that F-actin is needed for cilia elongation and that some ciliary proteins are internalized from the plasma membrane and then redistributed to cilia. One concern regarding this portion of the manuscript in that the experiments addressing the role of endocytosis rely solely on the use of a pharmacological inhibitor of clathrin-dependent endocytosis, PitStop2. PitStop2 was previously shown to have non-specific effects on endocytosis, suggesting that its mechanism of action may not be directly related to disrupting clathrin heavy chain interactions (see, for example, Willox et al., 2014).

    Intriguingly, a previous paper by Kim et al., 2010 demonstrated that interfering with the Arp2/3-dependent actin assembly resulted in cilia elongation in mammalian cells, suggesting that branched actin assembly was counteracting growth of cilia. Similarly, actin depolymerization is known to promote ciliogenesis in mammalian cells. The difference between mammalian actin organization/cilia growth regulation vs. the new observations in the Chlamydomonas system should be discussed by the authors to help the readers understand whether the findings can be generalized to the diverse ciliated cell types or are unique to algae.

    Read the original source
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  5. Reviewer #3 (Public Review):

    Wild-type Chlamydomonas cells possess a pool of ciliary precursors that, in conjunction with newly synthesized precursors, are used to build a new cilium after de-ciliation. The cellular and molecular mechanisms the underly retrieval of the pool are unknown. Given the role of the Arp2/3 complex in endocytosis in other systems, it was reasonable to test whether it also functions in reclaiming of the pool of precursors. The central finding is that cells bearing a mutation in an essential Arp2/3 component, ARPC4, fail to assemble cilia in a timely fashion after de-ciliation. Although the failure of assembly is well-documented here, the manuscript lacks evidence that cells missing ARPC4 actually establish a pool of ciliary precursors in the first place. Without such information it is not possible to determine the cellular function that fails to occur after de-ciliation in the mutant: Retrieval of a pool of ciliary precursors? Establishing the pool during the ciliary assembly that occurs after cell division? Synthesis of the precursors as new cilia are formed? Sensing loss of cilia and activating the events needed for re-ciliation?

    Other conclusions also were not sufficiently supported by the experimental results, including the following: Clathrin function in endocytosis: Pitstop2 was described as specifically blocking clathrin-mediated endocytosis, but reports in the literature indicate that Pitstop2 also blocks nuclear functions and clathrin-independent endocytosis. Experiments with an anti-clathrin antibody were interpreted as showing mislocalization of clathrin in the arpc4 mutants. The antibody was raised against a peptide near the N-terminus of human clathrin, but the antibody was not validated, and it was not reported whether Chlamydomonas clathrin even has that peptide.

    Internalization of a protein from the plasma membrane: In the experiments to use protease-sensitivity to examine SAG1-HA relocalization induced by db-cAMP, the authors assumed that all of the SAG1-HA was on the cell surface in untreated cells and the 2 chemically treated cells, but they never experimentally documented this assumption.

    Arp2/3 relation to actin dots: Structures termed actin dots that stained with Phalloidin were reported to undergo changes after de-ciliation of wild-type cells and were missing in the arpc4 mutant. The conclusion that the properties of the dots in the wild-type cells changed with de-ciliation were not supported by statistical analysis. Also, without experiments showing localization of Arp2/3-V5 at the dots, it was not possible to assess whether, as the text asserts, Arp2/3 functioned at the dots.

    Cilia resorption induced by BFA in arpc4 mutants: In the experiments with the Golgi-active agent BFA, the percent ciliation in the arpc4 mutants, but not wild type, fell rapidly after drug addition. The authors concluded that BFA induced ciliary resorption, but they did not determine whether the lack of cilia on cells was a consequence of cilia resorption or cilia detachment.

    Read the original source
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