Adenomatous polyposis coli (APC) regulates internalization and signaling of the chemorepellent receptor, Roundabout (ROBO) 1

  1. Yi-Wei Huang
  2. Jonathan St-Germain
  3. Bo Wen Pang
  4. Richard F Collins
  5. Etienne Coyaud
  6. Wenjuan Li
  7. Amir Mohamed
  8. Brian Raught
  9. Sergio Grinstein
  10. Lisa A Robinson

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Abstract

The SLIT-ROBO signaling pathway regulates axon guidance and cell migration, and ROBO1 is a receptor for SLIT ligands. ROBO1 undergoes constitutive endocytosis which is enhanced upon SLIT2 binding, but the molecular mechanisms and functional consequences of this process are not well understood. Using pharmacologic inhibitors and molecular techniques, we found that clathrin-mediated endocytosis is necessary for SLIT2-induced inhibition of cell spreading. To explore the underlying mechanisms, we performed BioID to identify ROBO1-interacting proteins whose association with the cytoplasmic domain of ROBO1 is differentially regulated by SLIT2. We discovered that adenomatous polyposis coli (APC), a multifunctional tumor suppressor, constitutively interacts with ROBO1 but dissociates upon binding of SLIT2 and that this dissociation is necessary for clathrin-mediated endocytosis of ROBO1 and subsequent effects on cell morphology. These findings provide new insights into the functional mechanisms by which SLIT2 binding to ROBO1 effects changes in actin cytoskeletal architecture.

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    Reply to the reviewers

    Reply to the Reviewers

    We sincerely thank the reviewers for their comprehensive and constructive feedback.

    Reviewer #1

    Major comments:

    The data and key conclusions of the paper are convincing. However, the reliability of the findings in terms of the new interaction could be improved by not relying solely on proximity ligation approaches (BioID, PLA), but employing a complementary biochemical strategy. The authors state that an immunoprecipitation (IP) was not possible due to a lack of antibodies for IP. This does not seem convincing since in the paper Saito-Diaz et al which they cite commercial antibodies were used to immunoprecipitate APC. Alternatively, the cell line expressing tagged ROBO1 could be used together with endogenous or tagged APC for an biochemical interaction experiment.

    Response

    We thank the reviewer for this important suggestion. In our initial studies, we attempted co-immunoprecipitation (co-IP) experiments using several different antibodies directed to APC. The signal detected was very low, possibly reflecting relatively low endogenous expression of ROBO1 in COS-7 cells, technical challenges associated with co-IP of APC and ROBO1, which are both large proteins (>200 kDa), and/or transient interactions between the two proteins. As part of the revision plan we will carry out co-IP experiments using HEK293A cells stably expressing full length ROBO1 (5H9 cells).

    Regarding the PLA experiment, I was very surprised by the very strong labeling for Clathrin+ROBO1 shown in the representative image. It is hard to believe that this image is representative when the average number of dots in the quantification is about 100. From the image it is also hard to see how it would be possible to quantify individual dots. For this, a zoom would be helpful.

    Response

    We thank the reviewer for this helpful comment. In the revised manuscript, we have added a magnified panel to Figure 4E.

    Clathrin and ROBO1 are likely not even direct interactors but come together by their common interaction with AP2. Therefore, to back this surprisingly strong result up, I would recommend to include one more control such as another rabbit antibody recognizing a protein that does not associate with clathrin or use e.g. the ROBO1 wildtype vs the ROBO1 mutant, that does not bind AP2 and therefore should also not associate with clathrin, for the experiment. Even better, the authors could confirm the PLA results by the mentioned complementary biochemical experiments to bolster the findings by an independent approach.

    Response

    We thank the reviewer for this suggestion. As recommended, we will use the complementary biochemical approaches suggested, and will perform immunoprecipitation experiments to examine interactions with clathrin in cells that express wildtype ROBO1 vs. cells that express mutant ROBO1 that does not bind AP2. As recommended, we will further perform experiments using control antibody directed to a protein that does not associate with clathrin.

    Minor comments:

    In general, data and methods are presented in a manner that should make them reproducible by others. Some small things to improve are:

    In the paragraph on antibodies the used concentrations for the different applications should be provided.

    Response

    We thank the reviewer for this suggestion and apologize for the omission. In the revised manuscript, we have added a supplementary table to clarify the concentrations of antibodies used for different experimental applications. Please see Table s1.

    It should be described how the poly-D-lysine coating was exactly performed.

    Response

    We thank the reviewer for this comment. In the revised manuscript, we have added the procedure for poly-D-lysine coating in the "Materials and Methods" section. Please see page 7 line 143-144.

    The statistical analysis looks adequate. There are just some minor things that should be specified:- Just to make sure: Is it really always SD which is provided and not SEM? Sometimes the error bars look so small that I was wondering about this.

    Response

    We appreciate the opportunity to clarify that we used SD consistently in the manuscript.

    • It should be specified for each experiment which post-hoc test is used or stated that one is always used for the One-Way ANOVA and the other for the Two-Way ANOVA resp. a rationale should be provided why two different post-hoc tests are used.

    Response

    We have added the post hoc tests used for each assay in the figure legend. The rationale for the different post hoc tests used has also been added in the "Materials and Methods" section as "Two-tailed paired Student's t-test was used for two-group comparisons. One-way ANOVA followed by Tukey's post hoc multiple comparison test was used for multiple-group comparisons with a single independent variable, and two-way ANOVA followed by Sidak's post hoc multiple comparison test was used for multiple-group comparisons with two independent variables". Please see page 12 line 275-279, page 20 line 519-520, page 21 line 524-525, 533-534, 537-538, 540-541, 543-544, page 22 line 551-552, 555-556, 561-562, 576-577, page 23 line 582, 584-585, 587, 589-590, 595, 599, page 24 line 624-625, 629, 635-636, page 25 638-639.

    • When using the t-test, it should be stated whether it is paired or unpaired and one- or two-tailed.

    Response

    Two-tailed paired Student's t-test was used in Fig. 5C. We have added in the "Materials and Methods" section and figure legend in the revised manuscript. Please see page 12 line 275-276, page 23 line 587.

    • It should be stated whether it was tested that the data fulfill the requirements for parametric tests (normal distribution).

    Response

    We have added "The data fulfilled the requirements for normal distribution using the Shapiro-Wilk test" in the "Materials and Methods" section in the revised manuscript. Please see page 12 line 274-275 in the revised manuscript.

    Text and figures are mostly clear, apart from some small things:

    • I was wondering about figure 1B. If I understand the methods description right, all cells were permeabilized prior to secondary antibody application. Why then is so little fluorescence for Flag visible in the first PBS row at 30 min? That would only make sense for me if the cell was not permeabilized and the protein internalized. So where did the majority of the protein end up after 30 min since you should see the entire population in a permeabilized cell? Could you please comment on this?

    Response

    We thank the reviewer for this comment. The cells were permeabilized prior to secondary antibody application. Since NSLIT2 binding to ROBO1 can facilitate ADAM10-mediated ROBO1 cleavage to release the extracellular domain of ROBO1 (Coleman et al., 2010), this may have caused little fluorescence for Flag to be visible in the first PBS row at 30 min. In the revised manuscript we have added a comment about the finding described. Please see page 13 line 293-296.

    • Fig. 2A the upper left image (0 min PBS) should be very similar to the upper left image in Fig. 1B, shouldn´t it? But it looks quite different to me in terms of surface amount of ROBO1-Flag. Could you please comment on this?

    Response

    We apologize for the confusing images included in the original version of the manuscript. As noted, the upper left image (0 min PBS) in Fig. 2A should be very similar to the upper left image in Fig. 1B. We have now instead included an image for Fig. 2A that is more representative of the data from the experiments we performed.

    • Please explain what the molecular difference between bio-active NSLIT2 and bio-inactive CSLIT2 is. Please provide a rationale why you sometimes use CSLIT2 as negative control and sometimes DD2SLIT2. In Fig. 3G you are using DD2SLIT2. Even though there is no significance reached with the analyzed n, it is very striking that the bars are consistently higher upon DD2SLIT2 application. Can you comment on this effect? Or am I misunderstanding the labeling of the figure?

    Response

    Bio-active NSLIT2 consists of the N-terminal fragment of SLIT2 and contains the second leucine-rich repeat (LRR) domain (D2), which binds to the first two Ig domains of the ROBO1 receptor (Ig1-2). Bio-inactive CSLIT2 consists of the C-terminal fragment of SLIT2, which does not bind ROBO1. DD2SLIT2 consists of the N-terminal fragment of SLIT2 but lacks D2 LRR domain that is essential for ROBO1 binding. Neither CSLIT2 nor DD2SLIT2 can bind the ROBO1 receptor (Bhosle et al., 2020; Mukovozov et al., 2015; Patel et al., 2012). In Fig. 3G, DD2SLIT2 was used as negative control and did not affect cell spreading, so the bars are consistently higher upon D2SLIT2 application. The use of CSLIT2 or DD2SLIT2 in different experiments was due to the availability of these reagents. In Fig. 3F and 3G, we have made modifications to the X axis to clarify.

    • On page 3 it states "...endocytosis of ROBO1...requires...APC": I found this confusing since it is the dissociation of APC that is required for promoting endocytosis. Therefore, it would be good to rephrase this sentence.

    Response

    We apologize for the confusing language. In the revised manuscript, we have changed "endocytosis of ROBO1 from the cell surface requires the tumor suppressor protein, APC" to "endocytosis of ROBO1 from the cell surface requires the dissociation of the tumor suppressor protein, APC". Please see page 4 line 35-36.

    • On page 8 is written "...cells surface ROBO1 [is] removed". Please be more accurate since the acid wash does not remove ROBO1, but only the antibody bound to the extracellular epitope.

    Response

    We apologize for the confusing language. In the revised manuscript, we have changed "cell surface ROBO1 removed" to "anti-Flag antibody binding ROBO1 removed from the cell surface". Please see page 8 line 153-154.

    • On page 8 provide an explanation for the abbreviation HAC.

    Response

    To enhance clarity, in the revised manuscript we have used the full name "acetic acid" instead of using the abbreviation "HAC". Please see page 8 line 155.

    • On page 15 you speak of "mutant AP2". Please be more accurate since there is no mutant AP2 involved, but you are refering to ROBO1 with mutations in its AP2 binding motifs.
    • On page 14 you speak of "cells expressing the mutant alleles of AP2". As above, please be more accurate and replace with "cells expressing ROBO1 harboring mutations in both AP2 binding sites".

    Response

    We thank the reviewer for this suggestion and apologize for the confusion. For the sake of accuracy, we have made the changes as suggested by the reviewer. Please see page 15 line 351 and page 14 line 331-332.

    • On page 19 you write: "Using proximity ligation assays, we observed that ROBO1, APC and clathrin interact with one another". I am maybe a bit picky here, but in my eyes with these assays you only show that they are very close together and might be in a complex, but you do not show (direct) interaction in a strict sense. Therefore, I would downtone this a bit.

    Response

    We thank the reviewer for this important comment. As suggested, in the revised manuscript, we replaced "Using proximity ligation assays, we observed that ROBO1, APC and clathrin interact with one another" with "Using proximity ligation assays, we observed that ROBO1, APC and clathrin are in close proximity to one another". Please see page 18 line 458. We have similarly amended the language throughout the manuscript. Please see page 3 line 11-12, page 4 line 37, page 16 line 391, 394, 396, 398, page 22 line 564.

    • In Fig. 5B I would find it easier for the reader if siRNA and control were shown side by side for the different conditions.

    Response

    In the revised manuscript, we have made the changes suggested by the reviewer to enhance clarity.

    • Between the internalization assays and the spreading assays, you switch from HEK293 cells to COS7 cells. Please provide a rationale for this for the reader.

    Response

    Because the endogenous expression of ROBO1 is relatively low in COS-7 cells, we generated a HEK293A cell line that stably expresses ROBO1, and used these cells to examine subcellular traffic of ROBO1 and explore interactors of ROBO1. We next sought to explore the functional consequences of internalization of ROBO1 and the functional role of APC. As we and others previously showed that SLIT2-ROBO1 signaling inhibits cell spreading (Bhosle et al., 2020; Patel et al., 2012; Tole et al., 2009), we elected to use this measure as a biologic read-out. Because HEK293A cells do not spread as much as COS-7 cells, we instead used COS-7 cells for the spreading assays.

    • You provide a table with putative interactors within the paper and as supplementary table. Could you please explain better to the reader what your criteria were for including hits into the "short-list" presented in Table1.

    Response

    We chose proteins based on two criteria. The first was association with full-length ROBO1, but not with ROBO1 lacking the intracellular domain. The second was association with full-length ROBO1 under basal conditions, but loss of association with full-length ROBO1 after exposure of cells to NSLIT2. In the revised manuscript, we have added the criteria in the manuscript. Please see page 15 line 363-366.

    Typos

    • p. 6: CO2 instead of CO2
    • p21 last line: Immunoblotting should not be capitzalized.
    • Figure s1 legend: full-lenth is missing a g

    Response

    We apologize for the oversight. In the revised manuscript, we have corrected these typos. Please see page 6 line 97, page 21 line 535 and page 24 line 611.

    Significance

    It was already known from Drosophila and for mammalian cells that SLIT2 induces the endocytosis of ROBO1 and that this is necessary for its repulsive function in axon guidance as the authors point out. The key advance of the study is the identification of APC as an interactor of ROBO1 which decreases its endocytosis until it dissociates upon SLIT2 binding to ROBO1. This is an interesting aspect which opens up parallels to the regulation of Wnt signaling by APC as the authors discuss. The significance of this finding would be even greater if it would have been shown that this mechanism actually operates in axon guidance. That not being the case, the authours might want to discuss in more detail if APC has previously been implicated to affect axon guidance.

    Researchers working on endocytosis, adhesion, cellular signaling and the development of the nervous system will be interested in these findings.

    Response

    We thank the reviewer for the positive comments regarding the significance of our findings. As recommended, in the discussion section of the revised manuscript we will discuss in more detail what is known about the role of APC in axon guidance.

    Reviewer #2

    Major comments:

    As the authors emphasize the role of NSlit2 in Robo1 internalization throughout their manuscript, I suggest authors include "NSlit" in their title. Something like this "Adenomatous polyposis coli (APC) regulates the NSlit2-induced internalization and signaling of the chemo repellent receptor, hRoundabout (ROBO) 1" or maybe a better title.

    Response

    As suggested, we have changed the title of the revised manuscript to "Adenomatous polyposis coli (APC) regulates the NSLIT2-induced internalization and signaling of the chemorepellent receptor, Roundabout (ROBO) 1".

    In addition to transferrin as the control for their internalization studies, have the authors tested the specificity of NSlit-2-induced internalization with other Robo receptors such as Robo2? Does the APC bind to Robo2 also?

    Response

    We thank the reviewer for this comment. Due to significant cost constraints, we focused our BioID experiments on identifying proteins that interact with ROBO1. In the revised manuscript, we will expand the discussion to consider the questions raised here by the reviewer.

    The N-Slit group at 0' in Figure 1 b and Figure 2a, the Flag-Robo staining looks very different. Is it because the authors did not use ADAM protease inhibitor in Figure 2a that's why they are seeing more internalized Flag-Robo at 0'? It is not very clear either in the Results or the legend.

    Response

    We apologize for the confusing images. We used ADAM protease inhibitor for all endocytosis assays, as mentioned in the "Materials and Methods" section. The upper left image (0 min PBS) in Fig. 2A should be very similar to the upper left image in Fig. 1B. We have now replaced the image in 2A with one that is more representative of the overall results.

    Have the authors tested the Surface Robo1 pool in siAPC cells induced with or without N-Slit2?

    Response

    We added NSLIT2 to cells as we started endocytosis assay. At the time point of 0 min, the surface ROBO1 pool was not affeacted by NSLIT2.

    Does the Robo1 mutated with AP2 binding motifs interact with APC? Have authors performed a Proximity ligation assay with AP2-binding motifs mutated Robo1 and APC?

    Response

    We thank the reviewer for this suggestion. As recommended, we will perform proximity ligation assays to examine interactions between APC and ROBO1 which lacks AP2-binding motifs.

    The resolution of PLA dots in the current version is very low. Authors should include higher magnification pictures for these interactions and also PLA dots channel should be separately represented in addition to the DAPI merged images for better clarity and interpretation.

    Response

    We thank the reviewer for these suggestions. In the revised manuscript, we have included figures with the recommended modifications to enhance clarity. Please see figure 4A, 4C and 4E.

    Do the Slit2 treated cells affect APC mRNA expression? Or does Slit2 only inhibit the interaction between APC and Robo1? Have the authors tested the mRNA expression of APC in slit2-treated and untreated cells?

    Response

    We thank the reviewer for this question. We will perform the experiments suggested and include the results in the revised manuscript.

    The authors have tested the effect of Slit2-induced inhibition of cell spreading under different experimental conditions however it is also important to test the cell migration/proliferation rates under control and siAPC conditions with or without Slit2 treatment.

    Response

    We thank the reviewer for this comment. In order to test the effect of APC on SLIT2-induced cell migration, a migratory cell type would be required. This would involve introducing a third cell type in addition to the HEK293 and COS-7 cells we have already used, and first validating our key experimental findings in the new cell type. Please see our response to the 10th sub-comment in Minor Comment 4) of Reviewer 1.

    Do authors see the inhibition of Robo1 and Cyfip interactions also in the presence of Slit2 by PLA assay?

    Response

    We thank the reviewer for this interesting question. As this was beyond the scope of the current study, we did not examine whether SLIT2 inhibits interactions between ROBO1 and CYFIP. In the Discussion section of the revised manuscript, we will address this question as a potential line of future investigation.

    Studying the endogenous Robo1 and APC interaction by PLA is good but I suggest authors do standard co-IP assays to visualize these interactions since authors have already generated a variety of general epitope- tagged constructs for both Robo1 and APC. These epitope-specific antibodies that are best suitable for IP are easily available with many antibody companies. This is the first study to suggest that the interaction between Robo1 and APC so the strong biochemistry would have a good impact on the findings.

    Response

    We appreciate this important suggestion. We will perform the recommended studies and include the results in the revised manuscript. Please also see our response to Reviewer 1, Major Comment 1).

    Minor comments:

    I suggest the authors show the single-channel images of Flag-robo (green) in Figure 2B for a clear visualization of internalized Robo in a cell. With DAPI-merged images, it is hard to specifically visualize Robo in these cells.

    Response

    We assume the reviewer was referring Figure 2A instead of 2B. To enhance clarity, in the revised manuscript we have made the changes suggested by the reviewer.

    In Figure 1C, the Y axis should have a clear indication. Instead of "% internalized" it should be mentioned as "% Internalized Robo1".

    Response

    We thank the reviewer for this suggestion and apologize for the oversight. In the revised manuscript, we have made the suggested change in Figure 1C, 2B, 2D, 2E, 5B and 5D.

    I suggest authors to include the simple schematic of the mechanism they are proposing in the manuscript.

    Response

    We thank the reviewer for the suggestion. To enhance clarity, in the revised manuscript we will include a simple schematic of the mechanism our findings suggest.

    The authors should mention the rationale or the function of using the acid wash method for their experimental conditions for a better understanding of the reader.

    Response

    We thank the reviewer for this suggestion and apologize for the oversight. We performed acid wash experiments to remove the anti-Flag antibody that binds ROBO1 from the cell surface for the endocytosis assay. To increase the clarity, in the "Materials and Methods" section of the revised manuscript we have included the rationale for using acid wash. Please see page 8 line 153-154.

    siRNA-mediated knockdown of specific genes should be correctly denoted in the figure. For example, instead of "CLTC", it should be "siCLTC" for easy understanding. The same correction has to be done in all the figures with siRNA data.

    Response

    We thank the reviewer for this helpful comment and apologize for the oversight. As suggested, we have made the suggested changes throughout the revised manuscript and in Figure 2C, 2D, 5A, 5B, 6A, 6B, 6C, s2C and s2D.

    Reference

    Bhosle, V.K., Mukherjee, T., Huang, Y.W., Patel, S., Pang, B.W.F., Liu, G.Y., Glogauer, M., Wu, J.Y., Philpott, D.J., Grinstein, S., et al. (2020). SLIT2/ROBO1-signaling inhibits macropinocytosis by opposing cortical cytoskeletal remodeling. Nat Commun* 11*, 4112.

    Coleman, H.A., Labrador, J.P., Chance, R.K., and Bashaw, G.J. (2010). The Adam family metalloprotease Kuzbanian regulates the cleavage of the roundabout receptor to control axon repulsion at the midline. Development (Cambridge, England)* 137*, 2417-2426.

    Mukovozov, I., Huang, Y.W., Zhang, Q., Liu, G.Y., Siu, A., Sokolskyy, Y., Patel, S., Hyduk, S.J., Kutryk, M.J., Cybulsky, M.I., et al. (2015). The Neurorepellent Slit2 Inhibits Postadhesion Stabilization of Monocytes Tethered to Vascular Endothelial Cells. J Immunol* 195*, 3334-3344.

    Patel, S., Huang, Y.W., Reheman, A., Pluthero, F.G., Chaturvedi, S., Mukovozov, I.M., Tole, S., Liu, G.Y., Li, L., Durocher, Y., et al. (2012). The cell motility modulator Slit2 is a potent inhibitor of platelet function. Circulation* 126*, 1385-1395.

    Tole, S., Mukovozov, I.M., Huang, Y.W., Magalhaes, M.A., Yan, M., Crow, M.R., Liu, G.Y., Sun, C.X., Durocher, Y., Glogauer, M., et al. (2009). The axonal repellent, Slit2, inhibits directional migration of circulating neutrophils. Journal of leukocyte biology* 86*, 1403-1415.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Comments on Huang et al.

    In their manuscript, Robinson and colleagues explore the role of the APC protein in the regulation of Slit-Robo signaling in mammalian cell culture. In particular, the authors investigate the importance of APC in controlling the endocytosis of Robo and its subsequent signaling. The authors begin by demonstrating that as previously reported in a Drosophila model that Slit induces the internalization of Robo through Clathrin dependent endocytosis and that this effect depends on two AP binding motifs in the Robo c-terminal tail. In a cellular readout for Slit- signaling the authors demonstrate that Slit inhibition of cell spreading in COS7 cells depends on the receptor endocytosis. A BioID screen for proteins that bind Robo in a slit-dependent manner identified a number of candidate proteins and the authors chose to focus on the APC protein whose association with Robo is down-regulated by Slit. Using a series of in vitro and cell based experiments the authors propose a model in which APC negatively regulates Robo internalization and subsequently impacts receptor signaling. Overall the findings are interesting and many of the experiments are well controlled; however, significant technical and conceptual concerns limit enthusiasm for the manuscript in its current form.

    General- throughout the manuscript the magnification of the cells in the micrographs are too low. I would recommend including insets to more clearly show the observed effects (especially for the localization figure).

    Specific comments

    Figure 1: The effect of Slit on Robo internalization appears to be robust and the transferrin control is welcome. The authors need to do a better job of explaining how exactly they are quantifying the internalized pool, especially given the likely cell to cell variability in the amount of expression of the transfected constructs. Labeling the figure more clearly to indicate what is Scarlet tagged would help and the purpose of the acid-washing procedure should be more clearly explained.

    Figure 2: There seems to be a mismatch in the representative images for the Dyn treatments- both concentrations appear to reduce internalization of Robo R ~ 2 fold, but the images show essentially 0 internalized receptor. (2A and B)

    Figure 3: The analysis of the Robo c-terminal AP binding motifs is a bit confusing. The authors refer to these manipulations as Robo alleles; however, my understanding is that these are over-expressed constructs in stably transfected lines. They are not 'alleles.' It is puzzling how the effects of Slit appear to be restricted to the transfected cells, especially given that all of these cells should be expressing endogenous Robo. Some clarification would be welcome.

    Figure 4: This figure presents the 'validation' of the Robo APC interaction. There are major problems here. First, PLA is not an adequate substitute for biochemical interaction and it does not allow for clear documentation of ligand-dependent effects. To suggest as the authors do that this analysis provides evidence for a multi-protein complex between Robo, APC and clathrin is not legitimate. Furthermore, the nature of the PLA assay and what they are counting is unlear and misleading. In the methods the author's state

    'Interactions were quantified by counting the number of dots per nucleus as well as the intensity of the signal per dot. An increase in intensity is the consequence of a concentration of interactions in the same cellular dots (Gauthier et al., 2015)'

    Surely, we are not to believe that nuclear PLA signals would exist between this transmembrane protein and endocytic machinery.

    The authors should perform co-IP experiments +/- Slit to validate their findings from BioID.

    Figures 5 and 6: These data represent the functional analysis of APC's role in Robo signaling. There are several observations that do not it with the model which states that APC associates with Robo to prevent endocytosis until Slit arrives. For example, this model would predict that loss of APC should lead to an increase in Robo internalization and an increase in Robo dependent inhibition of cell spreading- neither of these predictions match their data.

    Significance

    In their manuscript, Robinson and colleagues explore the role of the APC protein in the regulation of Slit-Robo signaling in mammalian cell culture. In particular, the authors investigate the importance of APC in controlling the endocytosis of Robo and its subsequent signaling. The authors begin by demonstrating that as previously reported in a Drosophila model that Slit induces the internalization of Robo through Clathrin dependent endocytosis and that this effect depends on two AP binding motifs in the Robo c-terminal tail. In a cellular readout for Slit- signaling the authors demonstrate that Slit inhibition of cell spreading in COS7 cells depends on the receptor endocytosis. A BioID screen for proteins that bind Robo in a slit-dependent manner identified a number of candidate proteins and the authors chose to focus on the APC protein whose association with Robo is down-regulated by Slit. Using a series of in vitro and cell based experiments the authors propose a model in which APC negatively regulates Robo internalization and subsequently impacts receptor signaling. Overall the findings are interesting and many of the experiments are well controlled; however, significant technical and conceptual concerns limit enthusiasm for the manuscript in its current form.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In this paper, the authors sought to identify the mechanism of Slit2-induced hRobo1 internalization and its signaling. They demonstrated that Slit2-induced hRobo1 internalization is regulated by one of the Robo1 C-terminal binding partners, adenomatous polyposis coli (APC). By using various in vitro experiments, the authors have concluded that APC constitutively interacts with hRobo1, and this interaction is disrupted upon the binding of Slit2 to the extracellular domain of hRobo1. They also showed that the dissociation of interaction between APC and hRobo1 is important for clathrin-mediated endocytosis of hRobo1 and subsequent cell morphology. In conclusion, while this study presents intriguing findings, there are notable experimental concerns. In several instances, the authors fail to sufficiently elucidate the experimental setup or provide specific conditions for certain experiments, which may pose challenges for readers in understanding the methodology thoroughly. Also, the labels for the figures can be more accurate and clearly stated.

    Major comments:

    1. As the authors emphasize the role of NSlit2 in Robo1 internalization throughout their manuscript, I suggest authors include "NSlit" in their title. Something like this "Adenomatous polyposis coli (APC) regulates the NSlit2- induced internalization and signaling of the chemo repellent receptor, hRoundabout (ROBO) 1" or maybe a better title.
    2. In addition to transferrin as the control for their internalization studies, have the authors tested the specificity of NSlit-2-induced internalization with other Robo receptors such as Robo2? Does the APC bind to Robo2 also?
    3. The N-Slit group at 0' in Figure 1 b and Figure 2a, the Flag-Robo staining looks very different. Is it because the authors did not use ADAM protease inhibitor in Figure 2a that's why they are seeing more internalized Flag-Robo at 0'? It is not very clear either in the Results or the legend.
    4. Have the authors tested the Surface Robo1 pool in siAPC cells induced with or without N-Slit2?
    5. Does the Robo1 mutated with AP2 binding motifs interact with APC? Have authors performed a Proximity ligation assay with AP2-binding motifs mutated Robo1 and APC?
    6. The resolution of PLA dots in the current version is very low. Authors should include higher magnification pictures for these interactions and also PLA dots channel should be separately represented in addition to the DAPI merged images for better clarity and interpretation.
    7. Do the Slit2 treated cells affect APC mRNA expression? Or does Slit2 only inhibit the interaction between APC and Robo1? Have the authors tested the mRNA expression of APC in slit2-treated and untreated cells?
    8. The authors have tested the effect of Slit2-induced inhibition of cell spreading under different experimental conditions however it is also important to test the cell migration/proliferation rates under control and siAPC conditions with or without Slit2 treatment.
    9. Do authors see the inhibition of Robo1 and Cyfip interactions also in the presence of Slit2 by PLA assay?
    10. Studying the endogenous Robo1 and APC interaction by PLA is good but I suggest authors do standard co-IP assays to visualize these interactions since authors have already generated a variety of general epitope- tagged constructs for both Robo1 and APC. These epitope-specific antibodies that are best suitable for IP are easily available with many antibody companies. This is the first study to suggest that the interaction between Robo1 and APC so the strong biochemistry would have a good impact on the findings.

    Minor comments:

    1. I suggest the authors show the single-channel images of Flag-robo (green) in Figure 2B for a clear visualization of internalized Robo in a cell. With DAPI-merged images, it is hard to specifically visualize Robo in these cells.
    2. In Figure 1C, the Y axis should have a clear indication. Instead of "% internalized" it should be mentioned as "% Internalized Robo1".
    3. I suggest authors to include the simple schematic of the mechanism they are proposing in the manuscript.
    4. The authors should mention the rationale or the function of using the acid wash method for their experimental conditions for a better understanding of the reader.
    5. siRNA-mediated knockdown of specific genes should be correctly denoted in the figure. For example, instead of "CLTC", it should be "siCLTC" for easy understanding. The same correction has to be done in all the figures with siRNA data.

    Referees cross-commenting

    Reviewer 1 comments and suggestions are valid and carry significant weight in improving the manuscript.

    Significance

    Strengths: The manuscript writing is good and the authors have generated a lot of constructs for a thorough understanding of Robo1 internalization events under different conditions. Studying the differential protein interactions with and without Slit2 with the Robo1-Bir*Flag method is convincing.

    Limitations: The representation of figures and their labels, Figure resolution, poor quality, missing important controls and experiments.

    Advance: Not very conceptual

    Audience: Broad and basic research

    My field of expertise: Endocytosis, receptor surface labeling studies, ligand mediated receptor signaling and its effect on axon guidance during embryonic development.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The authors investigate the endocytosis of the axon guidance receptor ROBO1 that is triggered by binding to its ligand SLIT2 and required for its ligand-induced inhibition of cell spreading. For this, they perform internalization experiments in a cell line expressing extra- and intracellularly tagged ROBO1. Using BioID they identify the protein APC as novel regulator of ROBO1 endocytosis. Their main finding is that SLIT2 induces the dissociation of APC thereby promoting endocytosis.

    Major comments:

    The data and key conclusions of the paper are convincing. However, the reliability of the findings in terms of the new interaction could be improved by not relying solely on proximity ligation approaches (BioID, PLA), but employing a complementary biochemical strategy.

    The authors state that an immunoprecipitation (IP) was not possible due to a lack of antibodies for IP. This does not seem convincing since in the paper Saito-Diaz et al which they cite commercial antibodies were used to immunoprecipitate APC. Alternatively, the cell line expressing tagged ROBO1 could be used together with endogenous or tagged APC for an biochemical interaction experiment.

    Regarding the PLA experiment, I was very surprised by the very strong labeling for Clathrin+ROBO1 shown in the representative image. It is hard to believe that this image is representative when the average number of dots in the quantification is about 100. From the image it is also hard to see how it would be possible to quantify individual dots. For this, a zoom would be helpful.

    Clathrin and ROBO1 are likely not even direct interactors but come together by their common interaction with AP2. Therefore, to back this surprisingly strong result up, I would recommend to include one more control such as another rabbit antibody recognizing a protein that does not associate with clathrin or use e.g. the ROBO1 wildtype vs the ROBO1 mutant, that does not bind AP2 and therefore should also not associate with clathrin, for the experiment. Even better, the authors could confirm the PLA results by the mentioned complementary biochemical experiments to bolster the findings by an independent approach.

    Minor comments:

    In general, data and methods are presented in a manner that should make them reproducible by others. Some small things to improve are:

    • In the paragraph on antibodies the used concentrations for the different applications should be provided.
    • It should be described how the poly-D-lysine coating was exactly performed.

    The statistical analysis looks adequate. There are just some minor things that should be specified:

    • Just to make sure: Is it really always SD which is provided and not SEM? Sometimes the error bars look so small that I was wondering about this.
    • It should be specified for each experiment which post-hoc test is used or stated that one is always used for the One-Way ANOVA and the other for the Two-Way ANOVA resp. a rationale should be provided why two different post-hoc tests are used.
    • When using the t-test, it should be stated whether it is paired or unpaired and one- or two-tailed.
    • It should be stated whether it was tested that the data fulfill the requirements for parametric tests (normal distribution).

    Text and figures are mostly clear, apart from some small things:

    • I was wondering about figure 1B. If I understand the methods description right, all cells were permeabilized prior to secondary antibody application. Why then is so little fluorescence for Flag visible in the first PBS row at 30 min? That would only make sense for me if the cell was not permeabilized and the protein internalized. So where did the majority of the protein end up after 30 min since you should see the entire population in a permeabilized cell? Could you please comment on this?
    • Fig. 2A the upper left image (0 min PBS) should be very similar to the upper left image in Fig. 1B, shouldn´t it? But it looks quite different to me in terms of surface amount of ROBO1-Flag. Could you please comment on this?
    • Please explain what the molecular difference between bio-active NSLIT2 and bio-inactive CSLIT2 is. Please provide a rationale why you sometimes use CSLIT2 as negative control and sometimes D2SLIT2. In Fig. 3G you are using D2SLIT2. Even though there is no significance reached with the analyzed n, it is very striking that the bars are consistently higher upon D2SLIT2 application. Can you comment on this effect? Or am I misunderstanding the labeling of the figure?
    • On page 3 it states "...endocytosis of ROBO1...requires...APC": I found this confusing since it is the dissociation of APC that is required for promoting endocytosis. Therefore, it would be good to rephrase this sentence.
    • On page 8 is written "...cells surface ROBO1 [is] removed". Please be more accurate since the acid wash does not remove ROBO1, but only the antibody bound to the extracellular epitope.
    • On page 8 provide an explanation for the abbreviation HAC.
    • On page 15 you speak of "mutant AP2". Please be more accurate since there is no mutant AP2 involved, but you are refering to ROBO1 with mutations in its AP2 binding motifs.
    • On page 14 you speak of "cells expressing the mutant alleles of AP2". As above, please be more accurate and replace with "cells expressing ROBO1 harboring mutations in both AP2 binding sites".
    • On page 19 you write: "Using proximity ligation assays, we observed that ROBO1, APC and clathrin interact with one another". I am maybe a bit picky here, but in my eyes with these assays you only show that they are very close together and might be in a complex, but you do not show (direct) interaction in a strict sense. Therefore, I would downtone this a bit.
    • In Fig. 5B I would find it easier for the reader if siRNA and control were shown side by side for the different conditions.
    • Between the internalization assays and the spreading assays, you switch from HEK293 cells to COS7 cells. Please provide a rationale for this for the reader.
    • You provide a table with putative interactors within the paper and as supplementary table. Could you please explain better to the reader what your criteria were for including hits into the "short-list" presented in Table1.

    Typos

    • p. 6: CO2 instead of CO2
    • p21 last line: Immunoblotting should not be capitzalized.
    • Figure s1 legend: full-lenth is missing a g

    Referees cross-commenting

    I find the comments of Reviewer 2 productive and reasonable, and they overlap in part with mine (e.g. regarding biochemical approaches such as co-IP).

    Significance

    It was already known from Drosophila and for mammalian cells that SLIT2 induces the endocytosis of ROBO1 and that this is necessary for its repulsive function in axon guidance as the authors point out. The key advance of the study is the identification of APC as an interactor of ROBO1 which decreases its endocytosis until it dissociates upon SLIT2 binding to ROBO1. This is an interesting aspect which opens up parallels to the regulation of Wnt signaling by APC as the authors discuss. The significance of this finding would be even greater if it would have been shown that this mechanism actually operates in axon guidance. That not being the case, the authours might want to discuss in more detail if APC has previously been implicated to affect axon guidance. Researchers working on endocytosis, adhesion, cellular signaling and the development of the nervous system will be interested in these findings.

  5. Version published to 10.1101/2024.01.12.574717v2 on bioRxiv
  6. Version published to 10.1101/2024.01.12.574717v1 on bioRxiv