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Reviewer #1 (Evidence, reproducibility and clarity (Required)):
The manuscript by Neville et al addresses the link between the localization and the activity of the so-called "Pins complex" or "LGN complex", that has been shown to regulate mitotic spindle orientation in most animal cell types and tissues. In most cell types, the polarized localization of the complex in the mitotic cell (which can vary between apical and basolateral, depending on the context) localizes pulling forces to dictate the orientation.
The authors reexplore the notion that this polarized localization of the complex is sufficient to dictate spindle orientation, and propose that an additional step of "activation" of the complex is necessary to refine positioning of the spindle.
The experiments are performed in the follicular epithelium (FE), an epithelial sheet of cell that surrounds the drosophila developing oocyte and nurse cells in the ovarium. Like in many other epithelia, cell divisions in the FE are planar (the cell divides in the plane of the epithelium). The authors first confirm that planar divisions in this epithelium depends on the function of Pins and its partner mud, and that the interaction between the two partners is necessary, like in many other epithelial structures. Planar divisions are often associated with a lateral/basolateral "ring" of the Pins complex during mitosis. The authors show that in the FE, Pins is essentially apical in interphase and becomes enriched at the lateral cortex during mitosis, however a significant apical component remains, whereas mud is almost entirely absent from the apical cortex. Pins being "upstream" of mud in the complex, this is a first hint that the localization of Pins is not sufficient to dictate the localization of mud and of the pulling forces.
The authors then replace wt Pins, whose cortical anchoring strongly relies on its interaction with Gai subunits, with a constitutively membrane anchored version (via a N-terminal myristylation). They show that the localization of myr-Pins mimics that of wt-Pins, with a lateral enrichment in mitosis, and a significant apical component. Since a Myr-RFP alone shows a similar distribution, they conclude that the restricted localization of Pins in mitosis is a consequence of general membrane characteristics in mitosis, rather than the result of a dedicated mechanism of Pins subcellular restriction. Remarkably, Myr-Pins also rescues Pins loss-of-function spindle orientation defects.
They further show that the cortical localization of Pins does not require its interaction with Dlg (unlike what has been suggested in other epithelia). However, spindle orientation requires Dlg, and in particular it requires the direct Dlg/Pins interaction. The activity of Dlg in the FE appears to be independent from khc73 and Gukholder, two of its partners involved in its activity in microtubule capture and spindle orientation in other cell types.
Based on all these observations, the authors propose that Dlg serves as an activator that controls Pins activity in a subregion of its localization domain (in this case, the lateral cortex of the mitotic FE cell). They propose to test this idea by relocalizing Pins at the apical cortex, using Inscuteable ectopic expression. With the tools that they use to drive Inscuteable expression, they obtain two populations of cells. One population has a stronger apical that basolateral Insc distribution, and the spindle is reoriented along the apical-basal axis; the other population has higher basolateral than apical levels of Insc distribution, and the spindle remains planar. The authors write that Pins localization is unchanged between the two subsets of cells (although I do not entirely agree with them on that point, see below), and that although Mud is modestly recruited to the apical cortex in the first population, it remains essentially basolateral in both. In this situation, the localization of Insc in the cell is therefore a better predictor of spindle orientation than that of Pins or Mud. Remarkably, removing Dlg in an Insc overexpression context leads to a dramatic shift towards apical-basal reorientation of the spindle, suggesting that loss of Dlg-dependent activation of the lateral Pins complex reveals an Insc-dependent apical activation of the complex.
Overall, I find the demonstration convincing and the conclusion appropriate. One of the limitations of the study is the use of different drivers and reporters for the localization of Pins, which makes it hard to compare different situations, but not to the point that it would jeopardize the main conclusions. I do not have major remarks on the paper, only a few minor observations and suggestion of simple experiments that would complete the study.
Minor:
What happens to Pins and Mud in Dlg mutant cells that overexpress Insc and behave as InscA? Are they still essentially lateral, or are they more efficiently recruited to the apical cortex?
This is a terrific question. Of course we would love to know and intend to find out.
One way to do this (consistent with the manuscript) would be to generate flies that are Dlg[1P20], FRT19A/RFP-nls, hsflp, FRT19A; TJ-GAL4/+; Pins-Tom, GFP-Mud/UAS-Insc. (Note that these flies would only allow us to image Mud; we would have to repeat the experiment using GFP FRT19A; hsflp 38 to see Pins. This isn’t ideal given that we’d like to image both together). Generating these flies is a major technical challenge because of the number of transgenes and chromosomes involved.
Our preferred way to do this would be to generate flies that are Dlg[1P20]/Dlg[2]; TJ-GAL4/+; Pins-Tom, GFP-Mud/UAS-Insc. So far, we’ve been unsuccessful. We are now undertaking a modified crossing scheme that we hope will solve the problem, though we aren’t overly optimistic about the outcome. We find that the temperature-sensitive mutation Dlg[2] presents an activation barrier; while we are able to generate flies that are Dlg[2] / FM7 in combination with transgenes and/or mutations on other chromosomes, we do not always recover the Dlg[2] / Y males (which must develop at 18degrees) from these complex genotypes.
In the longer term (outside the scope of revision), we are working to develop more tools for imaging Mud and Pins that we hope will help answer this question.
Regarding the competition between Pins and Insc for dictating the apical versus basolateral localization of Insc, the Insc-expression threshold model could be easily tested in Pins62/62 mutants, where it is expected that only InscA localization should be observed, even at 25{degree sign}C (unless Pins is required for the cortical recruitment of Insc, as it is the case in NBs - see Yu et al 2000 for example).
This is another great experiment and one we’d love to carry out. Again, the genetics are currently challenging, only because both UAS-Inscuteable and FRT82B pinsp62 are on the third chromosome. (Right now we’re trying to hop UAS-Inscuteable to the second).
However, we do have another idea for testing the threshold model, which is to repeat the experiment in which we express UAS-Insc in cells that are DlgIP20/IP20 at 25oC. Because the relevant cells (UAS-Insc OX in Dlg mitotic clones) are relatively rare, we have not yet been able to collect enough examples to make a firm conclusion. However, our preliminary results (only six cells so far!) suggest that more InscB cells are observed at the lower temperature, consistent with the threshold model.
I do not agree with the authors on P.10 and Figure 6A-D, when they claim that the apical enrichment of Pins is equivalent in both InscA and InscB cells. The number of measured cells is very low, and the ratio of apical/lateral Pins differs between the two sets of cells. The number of cells should be increased and the ratios compared with a relevant statistic method.
Totally fair. We are working to add more data to these panels (6B and 6D). The trend observed in 6D may be softening in agreement with the reviewer’s prediction, although we currently don’t yet have enough new data points to be confident in that conclusion. Therefore, we have not yet updated the manuscript, though we expect to do so during the revision period. We will also add a statistical comparison. Importantly, as the reviewer suggested, this does not alter our conclusions.
A lot of the claims on Pins localization rely on overexpression (generally in a Pins null background) of tagged Pins expressed from different promoters or drivers, and fused to different fluorescent tags. Therefore, it is difficult to evaluate to which extent the localization reflects an endogenous expression level, and to compare the different situations. As the cortical localization of Pins relies on interaction with cortical partners (mostly GDP-bound Gai) which are themselves in limiting quantity in the cell, and in the case of Gai-GDP, regulated by Pins GDI activity, this poses a problem when comparing their distribution, because the expression level of Pins may contribute to its cortical/cytoplasmic ratio, but also to its lateral/apical distribution. Although I understand that the authors have been using tools that were already available for this study, I think it would be more convincing if all the Pins localization studies were performed with endogenously tagged Pins, even those with Myr localization sequences. In an age of CRISPR-Cas-dependent homologous recombination, I think the generation of such alleles should have been possible. Although this would probably not change the main claims of the paper, it would have made a more convincing case for the localization studies.
We don’t disagree at all with this point. We did indeed try to stick with the published UAS-Pins-myr-GFP, not only for convenience but because it allows us to make comparisons to other studies using the same tool (Chanet et al Current Biology 2017 and Camuglia et al eLife 2022). Another consideration is that we used only one driver across our experiments (Traffic jam-GAL4). It is quite weak at the developmental stages that we examine, meaning that overexpression is not a major concern. (Indeed we have struggled with the opposite problem).
We certainly take the reviewer’s comment seriously and we therefore described it in the manuscript. We are currently working to develop endogenous tools using CRISPR.
Paragraph added to Discussion – Limitations of our Study:
“Another technical consideration is that our work makes use of transgenes under the control of Traffic jam-GAL4. While this strategy allows us to compare our results with previous work employing the same or similar tools, a drawback is that we cannot guarantee that Traffic jam-GAL4 drives equivalent expression to the endogenous Pins promoter (Chanet et al., 2017, Camuglia et al., 2022). However, given that Traffic jam-GAL4 is fairly weak at the developmental stages examined, we are not especially concerned about overexpression effects.”
The authors should indicate in the figure legends or in the methods that the spindle orientation measurements for controls for Pins62/62 are reused between figures 1, 3, 4, 5, 6 , and between figure 3, 4 and 5, respectively.
Absolutely. Added to the Methods section.
Reviewer #1 (Significance (Required)):
Altogether, this study makes a convincing case that the localization of the core members of the pulling force complex, Pins and Mud, is not entirely sufficient to localize active force generation, and that the complex must be activated locally, at least in the FE.
The notion of activation of the Pins/LGN complex has probably been on many people's mind for years: Pins/LGN works as a closed/open switch depending on the number of Gai subunits it interacts with, it must be phosphorylated, etc... suggesting that not all cortical Pins/LGN was active and involved in force generation. However the study presented here shows an interesting case where localization and activation are clearly disconnected.
The authors show how Dlg plays this role in physiological conditions in the FE, and use ectopic expression of Insc to show that, at least in an artificial context, Insc can have the same "activating activity" (or at least an activating activity that is stronger than its apical recruitment capability and stronger than Dlg's activating activity). It is to my knowledge the first case of such a clear dissociation.
In their discussion, the authors are careful not to generalize the observation to other tissues. Although I did not reexplore all that has been published on the Pins/LGN-NuMA/Mud complex over the last 20 years, my understanding is that despite interesting cases of distribution of the complex like that of Mud in the tricellular junction in the notum, the localization model can still explain most of the phenotypes that have been described without invoking an activation step. If it is the case, then the activation model is another variation (an interesting one!) on the regulation of the core machinery, which are plentiful as the authors indicate in their introduction, and is maybe specific to the FE; if not, then it would be interesting to push the discussion further by reexamining previous results in other systems, and pinpointing those phenotypes that could be better explained with an activation step.
Overall, I find this is an elegant piece of work, which should be of interest to many cell and developmental biologists beyond the community of spindle orientation aficionados.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Summary:
The manuscript by Neville et al. addressed the mechanism how conserved spindle regulators (Pins/Mud/Gai/Dynein) control spindle orientation in the proliferating epithelia by revising "the canonical model", using the Drosophila follicular epithelium (FE). The authors examined the epistatic relationship among Pins, Mud and Dlg in FE and found that Pins controls the cortical localization of Mud by utilizing mutant analyses, and suggested their localization does not fully overlap using the newly generated knock-in allele. They also showed that Pins relocalization during mitosis depends on cortical remodeling, or passive model, where Pins localization changes with other membrane-anchored proteins. Their data further suggest that Pins cortical localization is not influenced by Dlg, but Pins-interacting domain of Dlg does affect spindle orientation. Based on these results, the authors propose that Dlg controls spindle orientation not by redistributing Pins, but by promoting (or "activating" from their definition) Pins-dependent spindle orientation. Interestingly, ectopic expression of Inscuteable (Insc) suggested that Insc localization, either apical or lateral, correlates with spindle orientation, and their localization is a dominant indicator of spindle orientation, compared to the localization of Pins and Mud, implicating potentially distinct roles of activation and localization of the spindle complex. Overall their genetic experiments are well-designed and provide stimulation for future research. However, their evidence is suggestive, but not conclusive for their proposal. I have several concerns about their conclusion and would like to request more detailed information as well as to propose additional experiments.
Major concerns:
1. This report lacks technical and experimental details. As the typical fly paper, the authors need to show the exact genotypes of flies they used for experiments. This needs to be addressed for Figures 1-6, and Supplemental Figures. Especially, which Gal4 drivers were used for UAS-Pins wt or mutant constructs in Figure 4 with pins mutant background, Khc73, GUKH mutant backgrounds. Which exact flies were used for mutant clone experiments for Supplemental Figure 3? (A for typical mosaic, and B for MARCM). Without these details, it is impossible to evaluate results and reproduce by others.
We take this concern very seriously!
- We listed the GAL4 driver (Traffic jam-GAL4) in the first section of the Materials and Methods: Expression was driven by Traffic Jam-GAL4 (Olivieri et al., 2010). The transgene and relevant citation have been added to Table 1.
- We explained the background stock for the MARCM experiment in the Materials and Methods: Mosaic Analysis with a Repressible Cell Marker (after the method of Lee and Luo) was carried out using GFP-mCD8 (under control of an actin promoter) as the marker. The transgene and relevant citation have been added to Table 1.
- In line with other fly studies (eg. Nakajima et al., Nature 2013) and our own Drosophila work (Bergstralh et al Current Biology 2013, Bergstralh*, Lovegrove*, St Johnston NCB 2015, Bergstralh et al Development 2016, Finegan et al EMBO J 2019, Cammarota*, Finegan* et al Current Biology 2020) we were careful to show the relevant genotype components in each figure.
- We included a fully referenced Supplementary Table (Table 1 – Drosophila genetics) listing every mutant allele or transgene with a citation and a note about availability. We have expanded this table in response to the author’s concern (see above).
Related to the comment 1, how did the author perform "clonal expression of Ubi-Pin-YFP" in page 5? As far as I understand, Ubi-Pin-YFP is expressed ubiquitously by the ubiquitin promoter.
The reviewer makes a good point. We regret that we did not make this experiment more clear. Ubi-Pins-YFP was recombined onto an FRT chromosome (FRT82B). We made mitotic clones.
We have clarified this in the Methods section as follows:
“Mitotic clones of Ubi-Pins-YFP were made by recombining the Ubi-Pins-YFP transgene onto the FRT82B chromosome”
- In page 6, if Pins relocalization is passive and is associated with membrane-anchored protein remodeling during mitosis, its relocalization can be suppressed by disrupting the process of mitotic remodeling (mitotic rounding). The authors should test this by either genetic disruption or pharmacological treatment for the actomyosin should cause defects in Pins relocalization, which bolster their conclusion.
We agree that this is a cool experiment and are happy to give it another shot. However, we do note that interpretation could be difficult. We don’t know that mitotic rounding and membrane-anchored protein remodeling during mitosis are inextricably linked. Notably, the remodeling we describe reflects cell polarity; apical components are evidently moved to the lateral cortex. This is contrary to understanding of rounding, which reflects isotropic actomyosin activity (Chanet et al., (2017) Curr.Biol. & Rosa et al., (2015) Dev. Cell.). Therefore we don’t understand what a “negative” result would mean, or for that matter that a “positive” result would be safe to interpret.
We have attempted many strategies to prevent cell rounding in the follicular epithelium, none of which have successfully prevented rounding. 1) We attempted to genetically knockdown Moesin in the FE and did not see an effect on cell rounding. However we couldn’t confirm knockdown and therefore are not confident in this manipulation. 2) It is difficult to interpret the result of genetically disrupting Myosin, because it causes pleiotropic effects, such as inhibition of the cell cycle, and disruption of monolayer architecture. 3) We treated egg chambers with Y-27632 (a Rok-inhibitor) and examined its effect on mitotic cell rounding and on cytokinesis, which are Rok-dependent processes. Our experiments were performed using manually-dissociated ovarioles treated for 45 minutes in Schneider Cell Medium supplemented with insulin. Even at our maximum concentration of 1mM Y-27632, several orders of magnitude above the Ki, we are unable to see any effect on mitotic cell shape or actin accumulation at the mitotic cortex and did not observe any evidence of defective cytokinesis. We also did not observe defects in spindle organization or orientation, as would be expected from failed rounding. We therefore do not believe that the inhibitor works in this tissue. One possible explanation is that the follicle cells are secretory, and likely to pass molecules taken up from the media quickly into the germline. Therefore, we do not anticipate that we can perform this experiment to our satisfaction.
- The critical message in this manuscript is that the core spindle complex mediated by Pins-Mud controls spindle orientation by "activation", but not localization. The findings that Pins and Mud localization is not influenced by Insc and that ecotpic Insc expression and genetic Mud depletion (Figure 6) might support their proposal, but these results just suggest their localization does not matter. I wonder how the authors could conclude and define "activation". What does this activation mean in the context of spindle orientation? Can the authors test activation by enzymatic activity or assess dynamics of spindle alignment?
We intend for the critical message of the manuscript to be that “The spindle orienting machinery requires activation, not just localization.” We absolutely do not make the claim that localization is not important, only that it is not sufficient. The reviewer recognizes this point here and in a subsequent comment: “The authors showed that Pins and Mud localization themselves are not sufficient for the control of spindle orientation with genetic analyses.”
We also do not claim that Pins and/or Mud localization are not impacted by Inscuteable. On the contrary, we plainly see and report that they are; the intensity profiles in Figure 6 are distinct from those in Figure 2, as discussed in the text.
We appreciate the reviewer’s point about activation. Since we do not understand these proteins to be enzymes, we aren’t sure what enzymatic activity would be tested. The dynamics of spindle alignment in this slowly developing system are prohibitively difficult to measure: the mitotic index is very low (~2%) and only a very small fraction of those cells will be in a focal plane that permits accurate live imaging in the apical-basal axis. Alternative modes of activation include conformational change and/or a connection with other important molecules. The simplest possibility would be that Dlg allows Pins to bind Mud, but so far our data do not support it. We have added the following paragraph to our discussion:
“The mechanism of activation remains unclear. While the most straightforward possibility is that Dlg promotes interaction between Pins and Mud, our results show that Mud is recruited to the cortex even when Dlg is disrupted (Figure 4D). Alternatively, Discs large may promote a conformational change in the spindle-orientation complex and/or a change in complex composition. Furthermore, the Inscuteable mechanism is not likely to work in the same way. Dlg binds to a conserved phosphosite in the central linker domain of Pins and should therefore allow for Pins to simultaneously interact with Mud (Johnston et al., 2009). Contrastingly, binding between Pins and Inscuteable is mediated by the TPR domains of Pins, meaning that Mud is excluded (Culurgioni et al., 2011; 2018). While a stable Pins-Inscuteable complex has been suggested to promote localization of a separate Pins-Mud-dynein complex, our work raises the possibility that it might also or instead promote activation.”
- In page 7-8, although Pins-S436D rescue spindle orientation, but Pins-S436A does not in pins null clones background, Pins localization is not influenced by Dlg. This questions how exactly Pins and Dlg can interact, and how Dlg affect Pins function. Related to this observation, in the embryonic Pins:Tom localization in dlg mutant does not provide strong evidence to support their conclusion given the experimental context is different from previous study (Chanet et al., 2017).
We agree with the reviewer. Our data (this paper and previous papers) and the work of others indicate that this interaction is important for spindle orientation (Bergstralh et al., 2013a; Saadaoui et al., 2014; Chanet et al., 2017). However, we show here that Dlg doesn’t obviously impact Pins localization (as proposed in our earlier paper), but does impact the ability of the spindle orientation machinery to work (hence activity).
The reviewer makes a very good point. Our experimental context is different from the previous study concerning Pins and Dlg in embryos: Chanet et al (2017) performed their work in the embryonic head, whereas we look at divisions in the ventral embryonic ectoderm. These are distinct mitotic zones (Foe et al. (1989) Development) and exhibit distinct epithelial morphologies. We show that Pins:Tom localizes at the mitotic cell cortex in Dlg[2]/Dlg[1P20] in cells in the ventral embryonic ectoderm. Our only conclusion from this experiment is that Pins:Tom can localize without the Dlg GUK domain in another cell type (outside the follicular epithelium). In the current preliminary revision we have softened our claim as follows:
“We also examined the relationship between Pins and Dlg in the Drosophila embryo. A previous study showed that cortical localization of Pins in embryonic head epithelial cells is lost when Dlg mRNA is knocked down (Chanet et al., 2017). We find that Pins:Tom localizes to the cortex in the ventral ectoderm of early embryos from Dlg1P20/Dlg2 mothers, indicating that Pins localization in the ventral embryonic ectoderm epithelium does not require direct interaction with Dlg. We therefore speculate that Dlg plays an additional role in that tissue, upstream of Pins (Figure 4G).”
Our intention is to elaborate on our findings with additional data from embryos. To this end we have already acquired preliminary control data investigating the spindle angle with respect to the plane of the epithelium, and are in the process of examining spindle angles in dlg mutant embryonic tissue.
In page 11, the authors state "... that activation of pulling in the FE requires Dlg". I was not convinced by anything related to "pulling". There is no evidence to support "pulling" or such dynamic in this paper, just showing Mud localization, correct?
We appreciate the reviewer’s concern. The original sentence read that “We interpret [our data] to mean that interaction between Pins and Dlg, which is required for pulling, stabilizes the lateral pulling machinery even if Dlg is not a direct anchor.” This statement is based on work across multiple systems, including the C. elegans embryo (Grill et al Nature 2001), the Drosophila pupal notum (Bosveld et al, Nature 2016), and HeLa cells (Okumura et al eLife 2018), which shows that Mud/dynein-mediated pulling (on astral microtubules) orients/positions spindles. This is described in the introduction.
To address the reviewer’s particular concern, we have replaced “pulling” with “spindle-orentation machinery,” so that this sentence now reads …“activation of the spindle-orientation machinery in the FE requires Dlg.”
- Ectopic expression of Insc (Figure 6) provided a new idea and hypothesis, but the conclusion is more complicated given that Insc is not expressed in normal FE. For example, the statement that "Inscuteable and Dlg mediate distinct and competitive mechanism for activation of the spindle-orienting machinery in follicle cells" is probably right, but it does not show anything meaningful since Insc does not exist in normal FE. Is Dlg in a competitive situation during mitosis of FE? If so, which molecules are competitive against Dlg? The important issue is to provide a new interpretation of how spindle orientation is controlled epithelial cells. I strongly recommend to add models in this manuscript for clarity.
We considered the addition of model cartoons very carefully in preparing the original manuscript, and again after review. While we are certainly not going to “dig in” on this point, our concern is that model figures would obscure rather than clarify the message. As the reviewer points out, we do not understand how activation works, and as discussed in the manuscript we don’t think it’s likely to work the same way in follicle cells (Dlg) as it does in neuroblasts (Insc). Therefore model figure(s) are premature.
We do not agree with the statement that "Inscuteable and Dlg mediate distinct and competitive mechanism for activation of the spindle-orienting machinery in follicle cells… does not show anything meaningful.” This is a remarkable finding because it suggests that there is more than one way to activate Pins. Given the critical importance of spindle orientation in the developing nervous system, and the evolutionary history of the Dlg-Pins interaction, we think that this finding supports a model in which the Dlg-Pins interaction evolved in basal organisms, and a second Inscuteable-Pins interaction evolved subsequently to support neural complexity. These ideas are raised in the Discussion.
The reviewer also writes that “The important issue is to provide a new interpretation of how spindle orientation is controlled epithelial cells.” We find this concern perplexing, since the reviewer clearly recognizes that we have provided a new interpretation: Dlg is not a localization factor but rather a licensing factor for Pins-dependent spindle orientation.
Minor comments:
8. Some sections were not written well in the manuscript. "It does not" in page 6. "These predictions are not met". I just couldn't understand what they stand for. Their writing has to be improved.
Again, we are not going to dig in here, but we would prefer to retain the original language, which in our opinion is fairly clear. Our study is hypothesis-driven and based on assumptions made by the current model. We used direct language to help the reviewer understand what happened when we tested those assumptions.
- In page 9, Supplementary Figure 4 should be cited in the paragraph (A potential strategy for..), not Supplemental Figure 1A, and 1B.
Good catch, thank you! We have corrected this.
- In page 10, the authors examine aPKC localization in Insc expressing context of FE. Does aPKC localization correlate with Insc localization (Insc dictates aPKC?)? aPKC is not involved in spindle orientation from the author's report (Bergstralh et al., 2013), so it does not likely provide any supportive evidence.
I’m afraid we don’t entirely understand this comment. The interdependent relationship between aPKC and Inscuteable localization is long-established in the literature and was previously addressed in the follicle epithelium (Bergstralh et al. 2016). We do not make the claim here that aPKC governs spindle orientation. We are emphasizing that the difference between InscA and InscB cells extends to the relocalization of polarity components involved in Insc localization. As described in the manuscript, these data are provided to support our threshold model:
“In agreement with interdependence between Inscuteable and the Par complex, we find that aPKC is stabilized at the apical cortex in InscA cells but enriched at the lateral cortex in InscB cells (Figure 6E). This finding is consistent with an Inscuteable-expression threshold model; below the threshold, Pins dictates lateral localization of Inscuteable and aPKC. Above the threshold, Inscuteable dictates apical localization of Pins and aPKC.”
- In Dicussion page 12, "In addition, we find that while the LGN S408D (Drosophila S436D) variant is reported to act as a phosphomimetic, expression of this variant has no obvious effect on division orientation (Johnston et al., 2012)". Where is the evidence for this? I interpret that this phosphomimetic form can rescue like wt-Pins not like unphospholatable mutant S436A, so it means that have an effect on spindle orientation, correct?
The reviewer makes a good point. We regret the confusion. We mean to point out that the S436D variant is no different from the wild type. We have amended the text to clarify:
“In addition, we find that while the LGN S408D (Drosophila 436D) variant is reported to act as a phosphomimetic, this variant does not cause an obvious mutant phenotype in the follicular epithelium (Johnston et al., 2012). What then is the purpose of this modification? Since the phosphosite is highly conserved through metazoans, one possibility to consider is that the phosphorylation regulates the spindle orientation role of Pins, whereas unphosphorylated Pins plays a different role (Schiller and Bergstralh, 2021).”
Reviewer #2 (Significance (Required)):
The authors showed that Pins and Mud localization themselves are not sufficient for the control of spindle orientation with genetic analyses. While the authors tried to challenge the concept of "canonical model", there is no clear demonstration of "activation" of the spindle complex. I appreciate their genetic evidence and new results, and understand the message that Pins and Mud effects are beyond localization, but there is no alternative mechanism to support their model. At the current stage, their evidence provides more hypothesis, not conclusion. Based on my expertise in Developmental and Cell biology, I suggest that the work has an interest in audience who studies spindle machinery, but for general audience.
We think that the reviewer fundamentally shares our perspective on the study. Our work tests assumptions made by the canonical model and shows that they aren’t always met (meaning that the question of how spindle orientation works in epithelia at least is still unsolved), and that in the FE at least one component (Dlg) has been misunderstood. We reach a major conclusion, which is that localization of Pins is not enough to predict spindle orientation in the FE.
It’s absolutely true that the precise molecular role of Dlg has not been solved by our study. This is a major question for the lab, and we are currently undertaking biochemical work to address it. It’s probably more work than we can (or should) do on our own, which is just one reason to share our current results with colleagues.
One fundamental reason for undertaking this study is that 25 years of spindle orientation studies released into an environment in which “positive” conclusions are the bar for publication success may have burdened the field with claims that are overly-speculative. We appear to have contributed to this problem ourselves in 2013. With that in mind we contend that providing an alternative molecular mechanism at this point is premature and would impair rather than improve the literature.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
Neville et al re-examine the role and regulation of Pins/LGN in Drosophila follicular epithelial cells. They argue that polar or bipolar enrichment of Pins localisation at the plasma membrane is not crucial for spindle orientation, and therefore propose that Pins must be somehow activated to function. These interpretations are not supported by the data. However, the data strongly suggest an alternative interpretation which is of major biological significance.
As an initial point, we disagree with the summary above. We do not argue that enrichment of Pins is not crucial for spindle orientation. We argue that enrichment of Pins is not sufficient. This is why we titled the paper “The spindle orienting machinery requires activation, not just localization” instead of “The spindle orienting machinery requires activation, not localization.”
Although we disagree with reviewer, we appreciate their criticism of our manuscript and are glad for the opportunity to clarify our findings. In our responses to the specific comments (below) we explain why our data contradict the reviewer’s model and what we will do to improve the manuscript.
Comments:
- In the experiments on Dlg mutants (Fig 4D, S3) visualising Pins:Tom, the wild-type needs to be shown next to the Dlg mutant image, otherwise a comparison cannot be made. For example, Pins:Tom looks strongly enriched at the lateral membranes in the wild-type shown in Fig 2B&C, but much more weakly localised at the lateral membranes in Dlg1P20/2 mutants in Fig 4D. Thus, it looks like the Dlg GUK domain is required for full enrichment of Pins:Tom at lateral membranes, even if some low level of Pins can still bind to the plasma membrane in the absence of the Dlg GUK domain. Quantification would likely show a reduction in Pins:Tom lateral enrichment in the Dlg1P20/2 mutants - consistent with the spindle misorientation phenotype in these mutants.
The reviewer raises a reasonable concern about Figure 4D. We noted the difficulty of imaging Pins:Tom, which is exceedingly faint, in our original manuscript. For technical reasons, only one copy of the transgene was imaged in the experiment presented in 4G (two copies were used in Figure 2B), and the lack of signal presented an even greater challenge. In the manuscript we went with the clearest image. To address the reviewer’s concern, we have added signal intensity plots to this figure showing that Pins:Tom and Pins-myr are both laterally enriched at mitosis in Dlg[1P20]/Dlg[2] mutants. These data have been added as a new panel (E) in Figure 4. We were also able to replace the pictures in 4D with new ones generated after review.
- In Fig 4E, the phosphomutant PinsS436A-GFP looks more strongly apical and less strongly lateral than the wildtype Pins-GFP, consistent with the spindle misorientation phenotype in S436A rescued pins mutants.
The reviewer has an eagle eye! We did not detect a difference in localization across the three transgenes, though we were certainly looking for it (that’s why we generated these flies in the first place). Again, the strength of signal was a major challenge in these experiments, and we therefore went with the cleanest image. In response to the reviewer’s concern, we note that the S436A and S436D examples shown have equivalent apical signal, but only the S436A fails to rescue spindle orientation.
Together, Reviewer Comments 1 and 2 suggest a model in which Dlg is required for lateral enrichment of Pins at mitosis. As described in the manuscript, this is the very model proposed in our own previous study (Bergstralh, Lovegrove, and St Johnston; 2013), and reiterated in a subsequent review article (Bergstralh, Dawney, and St Johnston; 2017). We point these publications out because the senior author of the current manuscript is not especially enthusiastic about showing himself to be wrong (twice!) in the literature. He therefore insisted on seeing multiple lines of evidence before making the counterargument:
- The reviewer’s model (the 2013 model) is firstly challenged by work shown in Figure 3. We find that membrane-anchored proteins (even just myristoylated RFP!) demonstrate lateral enrichment at mitosis, regardless of whether or not they interact with the Dlg-GUK domain.
- Even stronger evidence is shown in Figure 4F. Pins-myr-GFP is very plainly enriched at the lateral cortex in Dlg[IP20]/Dlg[2] mutant cells (now demonstrated with signal intensity plots in FIGURE 4E). However, the spindle doesn’t orient correctly (quantified in 4C). Since Dlg is impacting spindle orientation independently of Pins localization, these data support our “claim in the final sentence of the abstract ‘Local enrichment of Pins is not sufficient to determine spindle orientation; an activation step is also necessary’.”
In the InscA examples, Pins:Tom looks apical. In the InscB examples, Pins:Tom looks more laterally localised, consistent with the spindle orientations in these experiments.
These figures (6A-D) don’t only show/quantify Pins:Tom localization. They also show localization of GFP-Mud. Whereas Pins:Tom is certainly apically enriched in the InscA examples, the interesting finding is that GFP-Mud is not. In strong contrast, it instead shows a weak apical localization and a strong lateral enrichment. As described in the manuscript, this pattern of Mud localization predicts normal spindle orientation, which is not observed in these cells.
Thus, these data appear to support the existing model that Pins enrichment at the plasma membrane is a key factor directing mitotic spindle orientation in these cells. The author's claim in the final sentence of the abstract "Local enrichment of Pins is not sufficient to determine spindle orientation; an activation step is also necessary" is not supported by the data.
We are pleased that the reviewer shared this quote; our claim is that Pins localization is not sufficient, not that it is unnecessary (see above). We absolutely do not dispute that “Pins enrichment at the plasma membrane is a key factor directing mitotic spindle orientation.”
The open question posed by the data is why GFP-Mud is excluded apically & basally during mitosis, while Pins:Tom is not. The simple alternative model is that Mud only localises to the plasma membrane where Pins is most strongly concentrated, such that Mud strongly amplifies any Pins asymmetry. Thus, even myr-Pins can still rescue a pins n mutant, because myr-Pins is still enriched laterally compared to apically (or basally).
Thus, I would strongly suggest re-titling the manuscript to: "Mud/NuMA amplifies minor asymmetries in Pins localisation to orient the mitotic spindle".
Well, that is a good-looking title, and we’re therefore sorry to decline the suggestion. However, as described above, Figure 4D shows that Pins enrichment does not always predict spindle orientation. More importantly, Figure 6A (cited by the reviewer in Comment 3) very plainly shows that Mud does not “only locali[ze] to the plasma membrane where Pins is most strongly concentrated.” In this picture – and across multiple InscA cells (Figure 6B) - Pins is strongly concentrated at the apical surface, whereas Mud is not.
Mud/NuMA presumably achieves this amplification by binding to the plasma membrane only where Pins is concentrated above a critical threshold level. This would mean a non-linear model based on cooperativity among Pins monomers that increases the binding avidity to Mud above the threshold concentration of Pins monomers.
This is essentially a minor revision of the standard model, which we expected would hold true in the FE. As described above, it is not supported by our data.
Reviewer #3 (Significance (Required)):
The manuscript is focused on the question of mitotic spindle orientation in epithelial cells, which is a fundamental unsolved problem in biology. The data reported are impressive and important, providing new insights into how the key spindle orientation factors Mud/NuMA and Pins/LGN localise during mitosis in epithelia. I recommend publication after major revisions.
We are delighted that the reviewer finds our data impressive and important, and our experiments insightful. We understand that the “major revisions” requested are meant to bring the paper in line with their model (our own earlier model). Since the data in our original manuscript contradict that model, the revisions are instead focused on clarifying and strengthening our message.
- syntactic transformation tool
- comment : syntax right semantics take care of itself
- ref : Haugland Mind Design
- if you get your syntax right
- semantics can take care of itself
- retort : get your iconcept for your intent right
- software can take care of itself
- symmanthetic manipulation
- combinig symmathetic with semantic
- effectiveness illusion of syntax being right
- integration of knowledge graphs
- for : HyperKnowledge