Paladin is a phosphoinositide phosphatase regulating endosomal VEGFR2 signalling and angiogenesis

Abstract Cell signalling governs cellular behaviour and is therefore subject to tight spatiotemporal regulation. Signalling output is modulated by specialized cell membranes and vesicles which contain unique combinations of lipids and proteins. The phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2), an important component of the plasma membrane as well as other subcellular membranes, is involved in multiple processes, including signalling. However, which enzymes control the turnover of non‐plasma membrane PI(4,5)P2, and their impact on cell signalling and function at the organismal level are unknown. Here, we identify Paladin as a vascular PI(4,5)P2 phosphatase regulating VEGFR2 endosomal signalling and angiogenesis. Paladin is localized to endosomal and Golgi compartments and interacts with vascular endothelial growth factor receptor 2 (VEGFR2) in vitro and in vivo. Loss of Paladin results in increased internalization of VEGFR2, over‐activation of extracellular regulated kinase 1/2, and hypersprouting of endothelial cells in the developing retina of mice. These findings suggest that inhibition of Paladin, or other endosomal PI(4,5)P2 phosphatases, could be exploited to modulate VEGFR2 signalling and angiogenesis, when direct and full inhibition of the receptor is undesirable.


5th Mar 2020 1st Editorial Decision
Dear Mats, Thank you for the submission of your research manuscript to our journal, which was now seen by two referees, whose reports are copied below.
As you can see, the referees express interest in the proposed role of Paladin in regulation of angiogenesis as a phosphoinositide phosphatase.However, they also raise a number of concerns that need to be addressed to consider publication here.In particular, the referees point out -That deeper analysis on the role of Paladin in regulation of VEGFR2 recycling/trafficking is required (ref #1 paragraphs 6, 7 and ref #2 point 1) -That better characterization of the role of Paladin in VEGFA signalling is necessary (ref #1 paragraph 8, ref #2 point 2).
-To some discrepancies in the data, missing controls and quantifications.I find the reports informed and constructive, and believe that addressing the concerns raised will significantly strengthen the manuscript.
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Deniz
Deniz Senyilmaz Tiebe, PhD Editor EMBO Reports Referee #1: In this paper the authors provide evidences that paladin dephosphorylates PIPs and this activity contributes both in vitro and in vivo to regulate VEGFR2 activity through affecting its trafficking.The data presented are interesting but some of them require more controls and experiments to better define their meaning.
Fig 1 .In this figure the authors show that paladin dephosphorylates both PIP 2 and PIP3 .Because PIP3 is mainly active at plasmamembrane and paladin is present in endosomal compartment, I think that it is quite surprising that paladin work s on PIP3.This point has to be explained.I suggest to define the Km of these substrates .I think this data is vey important to identify the relevant physiologic substrate of paladin.Furthermore it is necessary to add PI 5 phosphatase as a second positive control specific for PIP2.
The absence of colocalization between paladin and VE-cadherin is well documented in S1.However PIP2 may accumulate in caveolae (e.g.10.1073/pnas.0900216106;10.1074/jbc.M110.196022)and it is well established that VEGFR2 is present in this kind of plasma membrane domain type (e.g.Mol Biol Cell.14,334,2003) .So I suggest to verify this possible localization of paladin in plasmamebrane .The experiments shown in Figure 3 are fine but they need a biological counterpart, which is also useful to interpret the in vivo data.Do silencing and over-expression of paladin modify the mitogen and motogen activity of VEGFA?The manuscript demonstrates that Paladin can bind to phosphoinositides (PIs) and acts as a phosphatase for PI(4,5)P2 and PI(3.4,5)P3.Authors also revealed that Paladin fine-tunes VEGFR2 intracellular trafficking in endothelial cells.In addition, Paladin was found to negatively regulate activation of VEGFR2 and its downstream target ERK1/2 in endothelial cells in vitro and in vivo.In accordance, Paladin germ-line KO animals showed defects in both developmental and pathological angiogenesis in the mouse retina.These observations are novel and describe new roles for Paladin.Moreover, authors highlighted the relevance of Paladin for VEGFR2 signaling.However, there are several major and minor issues that need to be addressed prior to publication: Major issues: 1.The authors show that Paladin is a phosphatase for PIs and that Paladin affects VEGFR2 trafficking.However, importantly, the authors fail to demonstrate that the effect of Paladin on VEGFR2 trafficking is through its phosphatase activity on PIs.To test this, the authors should perform experiments confirming that Paladin regulates VEGFR2 signalling and trafficking via its activity on PIs. 2 A, B, C and lines 161-163 of the main text: "We observed the formation of a Paladin-VEGFR2 complex in response to VEGF-A treatment both in vitro (in primary endothelial cells) and in vivo (in a mouse model)."This claim is not supported by data.Both in vivo and in vitro data show that VEGF alone is not sufficient to induce complex formation.This only occurs in case of peroxivanadate treatment.Moreover, peroxivanadate alone was sufficient to induce complex formation in vivo.This condition, is a important missing control in Figure 2A and 2B.Input samples should also be included.Along the same line of thought, based on Figure 2D, authors claimed that "Accordingly, super-resolution microscopy analysis confirmed VEGF-A induced co-localization of Paladin and VEGFR2.This indicated that Paladin could be involved in VEGF-A/VEGFR2 signaling."(lines 165-167).Yet, authors do not show conditions with or without VEGF stimulation, and no quantification is provided to support the claim on the levels of co-localization.2H shows that Paladin KD cells have higher levels of internalised VEGFR2 upon 5-15min of VEGFA stimulation than control cells.However, quantification in Figure 2J and Sup.Figure2F shows that both the number of VEGFR2 vesicles and the number of VEGFR2 vesicles colocalising with EEA1 at 10min is equivalent to those in Paladin KD cells, whilst one would expect to have higher levels.Can authors comment on these contradictory results?Can authors provide additional confirmatory experiments to clarify this important claim in the manuscript?Moreover, the authors should complement their analysis for VEGFR2 trafficking with additional vesicular markers (Rab5+, Rab7+, Rab4+ or Rab11+ vesicles) in a time course manner to assess the fate of VEGFR2-positive vesicles in control and Paladin KD cells.4. In Figure 3, authors show that Paladin KD cells have higher signal transduction in response to VEGFA stimulation in vitro and in vivo.However, data presentation is very puzzling.For instance, Figure3i,j has different merged time points than Figure3f-h.Authors should provide a consistent way to display time in their analyses.The reviewer suggests that every time point should be displayed individually.In addition, quantification for pVEGFR2 levels in vivo is missing.4: To understand the extent of the observed rescue upon use of U0126 in Paladin KO animals, it would be important to provide representative images of those that were used for the quantifications represented in these graphs.Also, in figure 4I it is not clear in which condition CyclinD1 stainings were performed (WT or KO?).Representative images for both WT and KO animals should be shown.Moreover, authors could use ERG staining to label endothelial nuclei and thus increase the precision of the quantification (CyclinD1/ERG Double-positive cells), thus excluding confounding contribution of pericytes and microglia.

Figure
6.In general, it would be more informative to show quantification of vascular parameters in Paladin WT and KO retinas in absolute numbers and not as relative to control.Moreover, authors should show all data points (such as in Figure 1B/C), avoiding bars, and privileging scatter dot plots or box and whiskers.
7. Discussion (line 297-299): "Our data suggest that Paladin is a part of a VEGF-driven negative feedback loop in retinal angiogenesis where VEGF-A upregulates Paladin which acts to dampen VEGFR2 driven signaling and endothelial sprouting" The authors show that VEGF increases Paladin expression in HUVECS and in the mouse retina upon tail vein injection of VEGF.They do not show the existence of a loop that is be interrupted if Paladin is knocked down/ knocked out.Therefore, we believe that in the discussion, the authors should rephrase their sentence to avoid overstating the significance of their findings.
Minor issues: 1. Figure S1E: It was shown that recombinant Paladin binds specifically to PI(3)P, PI(4)P, and PI(5)P, and PI(3,5)P2.It is somehow surprising not to find Paladin targets [PI(4,5)P2 and PI(3,4,5)P3] as binders.Could the authors explain these results? 2. Line 172-173: "However, the receptor was degraded at the same rate as control-treated cells after VEGF-A stimulation (Figure 2e, Suppl Figure 2b)."It is unclear how the authors concluded about degradation rates.Could the authors clarify? 3. It would be interesting to provide images of P5 retinas images with higher magnification of tip cells from lacZ-stained Paladin het animals, similar to Figure 5a/c.4: To strengthen their findings, it would be relevant to show the expression pattern of VEGFR2 and pVEGFR2 in the wt and Pald1 KO retinas.
6. Line 147-148: "Super-resolution microscopy revealed that one-quarter of the Rab4-or Rab11positive structures were also positive for Paladin".The quantification for the number of vesicles that are double positive for Paladin/Rab4 and for Paladin/Rab11 should be showed in a graph.
7. Line 170-171: "siRNA-mediated knockdown of PALD1 in HDMEC resulted in a 35-51% increase of the total basal VEGFR2 pool".It is unclear what technique was used to obtain this result.It is not stated in the main text nor in the figure legend.If the technique used was Western Blot, it would be important to show the image with the bands that allowed to make this quantification.
8. Line 237-238: For clarity, the authors should clarify in the main text which animal model was used.From the main text it seems that an inducible endothelial specific knock out animal was used.But from reading the materials and methods, that does not seem to have been the case.9. Line 342: this reference "Lanahan, 2010" has not been included in the bibliography section.10.Line 346: "We also observed increased pTyr1173 phosphorylation in HDMEC".For clarity, the authors should rephase this sentence to be more accurate, namely increased in which condition as compared with what.
11. Figure 1D, right panel: the expression pattern of paladin and PI4P seem to be heterogeneous across the cell population and perhaps inversely correlated.Is that the case?Could the authors show separate panels for each colour?Could the authors comment on this? 12. Figure 4K and L: Could the authors clarify why two regimes of U0126 administration were used?13.Line 271: "VEGF-A induced production of the Paladin protein in endothelial cells in vitro and in the retinal vasculature in vivo, as indicated by LacZ reporter expression (Figure 5b,c and Suppl Figure 5a)."Authors should clarify this text as it suggests that quantifications were performed on LacZ reporter, yet, Figure 5B seems to be WB from bands showed in Sup Figure5a.Thank you for your email of March 5 2020, with the reviewers' report on our manuscript by Nitzsche et al.We sincerely appreciate the constructive comments from yourself and the reviewers.We have performed an extensive revision resulting in a very substantial consolidation of the finding that Paladin is a phosphoinositide phosphatase which targets PI(4,5)P 2 , with important consequences for endothelial cell biology which we investigate using in vitro and in vivo models.In the revision, we have focused on the impact of Paladin on the early events of VEGFR2 trafficking after VEGF-A stimulation.The major findings supporting an important role for Paladin in the early steps of VEGFR2 internalization are the following: • Rapid VEGF-A induced Paladin and VEGFR2 colocalization in the cell periphery but not at junctions/membrane.• VEGF-A-induced co-localization between Paladin and the early endosome marker EEA1.• Augmented VEGFR2 internalization in response to VEGF-A in Paladin-deficient conditions, accompanied by increased levels of pVEGFR2 and pErk1/2.• Marked accumulation of PI(4,5)P 2 already at 2 min of VEGF-A stimulation in Paladinknockdown cells, supporting an important role for Paladin in PI(4,5)P 2 dephosphorylation.
In the initial submission of our work, there were some concerns relating to different effect of Paladin-deficiency when comparing in vitro and in vivo models.Overall, the in vitro and in vivo data from Paladin loss of function models are consistent with a few exceptions: 1) elevated baseline VEGFR2 in vitro under Paladin-deficiency but unchanged in vivo, 2) delayed VEGFR2 degradation in vivo when compared to in vitro and 3) delayed pErk1/2 increase in vivo compared to in vitro.Given the different contexts and signaling kinetics of the models, it is in our view still compelling that lack of Paladin consistently promoted VEGF-A production, Erk1/2 activation and angiogenesis across models in vitro and in vivo and of both physiological and pathological angiogenesis.This is also discussed in the manuscript on page 15.

Summary of changes to Main Figures:
Figure 1: previous 1b is now 1a and 1b-h are new.Figure 2: previous 2e-h are now 2a-d and 1e-h are new.Figure 3: 3a-e remain, previous 3f and g have been merged to 3f and one new graph added to 3f. Figure 4: 4a-h remain, previous 4j is now 4i.Previous 4k,l are now 4l,m, Previous 4i has been modified and moved to EV 4. Figure 4j,k are new.Figure 5: No changes.
Please find a point-by-point response to the reviewer's questions.
Referee #1: 15th Oct 2020 1st Authors' Response to Reviewers 2 In this paper the authors provide evidences that paladin dephosphorylates PIPs and this activity contributes both in vitro and in vivo to regulate VEGFR2 activity through affecting its trafficking.The data presented are interesting but some of them require more controls and experiments to better define their meaning.Because PIP3 is mainly active at plasmamembrane and paladin is present in endosomal compartment, I think that it is quite surprising that paladin works on PIP3.This point has to be explained.I suggest to define the Km of these substrates .I think this data is very important to identify the relevant physiologic substrate of paladin.Furthermore it is necessary to add PI 5 phosphatase as a second positive control specific for PIP2.
The in vitro substrate specificity of phosphoinositide phosphatase is often not absolutely strict due to structural constraints.For example, myotubularins dephosphorylate PI3P and PI(3,5)P2 (Berger et al., Hum Mol Genet.2002 PMID: 12045210).The in vivo specificity also depends on the local context availability of the substrate.We saw efficient dephosphorylation (using Paladin wt compared to C/S) mainly for PI(4,5)P 2 .Moreover, we also noted colocalization of Paladin with early endosomes.From this we conclude that PI(4,5)P 2 , which is transported from the plasma membrane to early endosomes, is probably the main in vivo substrate of paladin.In line with this, the PI(4,5)P 2 signal increased in intact PALD1 knock-down cells treated with VEGF-A, compared to control cells (new Figure 2g,h).Although interesting and important for further understanding of the biochemical properties of Paladin's phosphatase activity, we consider it beside the scope of our study and also not within our expertise, to do in vitro measurements of Paladin's catalytic activity.
As the reviewer rightly points out, PI(4,5)P2 (and PIP3) is considered to be mainly active at the plasma membrane.However, it is also important to note that those phosphoinositides are also present at other location in the endosome compartment, albeit at lower levels as also discussed in the manuscript.
The suggestion to use additional positive controls for PIP2 is good, however, our positive control PTEN shows that the assay per se works and also generates a value we can bench mark to.Instead our efforts in the revision have been on defining the role of Paladin in VEGFR2 turnover and signaling in endothelial cells.
The absence of colocalization between paladin and VE-cadherin is well documented in S1.However PIP2 may accumulate in caveolae (e.g.10.1073/pnas.0900216106;10.1074/jbc.M110.196022)and it is well established that VEGFR2 is present in this kind of plasma membrane domain type (e.g.Mol Biol Cell.14,334,2003) .So I suggest to verify this possible localization of paladin in plasmamebrane .This is a very good suggestion and we stained HDMEC for Paladin and Caveolin 1 +/-VEGF-A but failed to detect co-localization.Moreover, in-depth analyses shown in Figures 1b,c,g convince us that Paladin is localized in intracellular vesicles, not at the plasma membrane.Fig 2A .May the authors show the Co-IP experiment without overexpressing paladin?I'm aware that sometimes is very difficult to show a protein-protein interaction in native conditions.However, it's the most convincing experiment to support a relevant biological interaction.Perhaps another technique may help to confirm the interaction between paladin and VEGFR2 shown panel B (Fret, in situ PLA) We thank the reviewer for this suggestion.We have now performed in situ proximity ligation assays (PLA) to confirm a VEGF-A-induced interaction between VEGFR2 and Paladin, see Figure 1e,f, and Figure EV1i.VEGF-A-induced complex formation is significantly induced at 2 min, but less so at 10 min of treatment.Blotting for Paladin on VEGFR2 IPs in vivo and in vitro confirmed complex formation but only when cells were treated with VEGF-A and peroxyvanadate (see Figure EV1).The PLA experiments (Figure 1 e,f) revealed an interaction between VEGFR2 and Paladin that occurs quickly (at 2 min) upon VEGF-A stimulation.Based on this we performed new and improved staining and image analysis for Paladin and EEA1 and can now show VEGF-Ainduced increase in Paladin/EEA1 co-localization (Figure 1g,h).Further studies then focused on the early interaction with VEGFR2, and the localization and function of Paladin (Figure 2).The localization of Paladin in other endosomal compartments is still relevant, but not the focus of the current study and thus we have removed these panels.Yes, pVEGFR2 follows the same trend as the total VEGFR2 with more pVEGFR2 internalized after stimulation, please see included Figure (Y-axis is relative internalization and X-axis time in min, n=2).As pVEGFR2 and total VEGFR2 followed the same pattern and the internalized pVEGFR2 levels are low and therefore difficult to detect and quantify, we decided to focus on the total VEGFR2 levels.Does paladin silencing affect VEGFR2 recycling to the plasma-membrane?In my opinion this issue has to be faced to offer to the readership a more compelling vision of the effect of paladin on VEGFR2 trafficking We chose to focus on the effect of loss of function of Paladin at early time points of VEGFR2 trafficking.We observe an interaction of Paladin and VEGFR2 at 2 min after VEGF-A stimulation.In the absence of PALD1, we detect a rapid increased internalization of the receptor, increased levels of pVEGFR2 and increased co-localization of EEA1/VEGFR2 as well as an increase in PIP2 levels.In addition, the surface levels of VEGFR2 did not differ between control and PALD1 knock-down cells even at late time points, as we followed VEGFR2 levels to 180 min after VEGF-A stimulation.Our conclusion is that Paladin regulates early internalization and trafficking of VEGFR2.We have therefore focused on the early internalization events regulated by Paladin, in this study.However, it does not rule out that Paladin have other effects on subsequence trafficking of VEGFR2 including recycling, which we also discuss, see page [14][15] The experiments shown in Figure 3 are fine but they need a biological counterpart, which is also useful to interpret the in vivo data.Do silencing and over-expression of paladin modify the mitogen and motogen activity of VEGFA?
We have now included data from in vitro studies supporting a role for Paladin in endothelial sprouting and proliferation.VEGF-A induced endothelial spouting was enhanced in the absence of PALD1 as compared to control siRNA, see Figure EV 4b.Endothelial proliferation was increased in PALD1 knock-down cells compared to controls cells when tracked for 72h using Incucyte, Figure EV 4c.The in vivo data from heart shows a transient delay in degradation of VEGFR2 in the Pald1 -/- mice.The kinetics of signaling and receptor degradation are different between cell-and animal experiments, with faster kinetics in vivo compared to cells which is as expected for example due to the higher and more consistent in vivo temperature.On the other hand, handling of the animals which we strive to conduct in a consistent manner, may induce stress hormones that may influence the analyses.Based on our experience from VEGFR2 and many other signaling systems, we do not expect a 100% concordance between cell and animal experiments.However, the in vitro analyses are important as they allow to address mechanistic aspects.In the paper we describe a consistent signaling alteration in PALD1 silenced cells in vitro, and in the Pald1 knock-out in vivo, in the developing retina, in the heart and in the pathological angiogenesis of the eye, allowing the conclusion that Paladin regulates early steps of internalization of VEGFR2 with consequence for signaling preferentially in the Erk1/2 pathway.The faster internalization rate seen in Paladindeficiency agrees with the notion that VEGFR2 needs to escape from cell surface localized protein tyrosine phosphatases in order to preserve phosphorylation on key tyrosine residues such as Y1173, which is a prerequisite for downstream signaling in the Erk1/2 pathway.The discrepancy between kinetics of degradation in vivo and in vitro are likely to be due to differences in internalization kinetics between HDMEC vs heart EC or in vitro vs. in vivo).We believe that further addressing such kinetics is beyond the scope of this manuscript and does not change the overall conclusion drawn from the study.We would like to point out that we already had applied pharmacological inhibition of the Erk1/2 pathway using a MEK inhibitor.We chose this MEK inhibitor for two reasons: 1) there was an in vivo study published (Roth et al., Invest Ophthalmol Vis Sci.2003.PMID: 14638742) in rat showing that it is possible to achieve efficient pErk inhibition in retina with this inhibitor, and 2) we wished to monitor the inhibition of Erk1/2 phosphorylation, since this was the key observation.Erk inhibitors could also be relevant to study, but would not allow us to measure Erk1/2 activation by immunostaining for pT202/pY204 as a way to verify that the inhibitor indeed worked.We regard the data provided as convincing proof of concept.

Referee #2:
The manuscript demonstrates that Paladin can bind to phosphoinositides (PIs) and acts as a phosphatase for PI(4,5)P2 and PI(3.4,5)P3.Authors also revealed that Paladin fine-tunes VEGFR2 intracellular trafficking in endothelial cells.In addition, Paladin was found to negatively regulate activation of VEGFR2 and its downstream target ERK1/2 in endothelial cells in vitro and in vivo.In accordance, Paladin germ-line KO animals showed defects in both developmental and pathological angiogenesis in the mouse retina.These observations are novel and describe new roles for Paladin.Moreover, authors highlighted the relevance of Paladin for VEGFR2 signaling.However, there are several major and minor issues that need to be addressed prior to publication: Major issues: 1.The authors show that Paladin is a phosphatase for PIs and that Paladin affects VEGFR2 trafficking.However, importantly, the authors fail to demonstrate that the effect of Paladin on VEGFR2 trafficking is through its phosphatase activity on PIs.To test this, the authors should perform experiments confirming that Paladin regulates VEGFR2 signaling and trafficking via its activity on PIs.
To more strongly link the novel phosphatase activity of Paladin to its effect on VEGFR2 trafficking we have performed a number of new analysis that follows the effect of Paladin loss of function over time after VEGF-A stimulation.In particular, we have analyzed the effect on PI(4,5)P 2 in intact cells after VEGF-A stimulation +/-PALD1 siRNA, new Figure 2g,h.We observe a sharp accumulation PI(4,5)P 2 in intact cells after PALD1 knock-down as compared to control cells after 2 min VEGF-A stimulation.Overall, we observe a consistent pattern indicating that Paladin plays an essential role early after VEGF-A stimulation.VEGFR2-Paladin interaction is established at 2 min after VEGF stimulation (Figure 1c,d,e,f).The important role for this co-localization is indicated by faster VEGFR2 internalization (Figure 2c,d), elevated EEA1/VEGFR2 co-localization (Figure 2e,f) and the dramatic increases in PI(4,5)P 2 levels (Figure 2g,h) in response to VEGF-A treatment of Paladin-deficient cells.Taken together, biochemical, cellular and in vivo data collectively point to a role for Paladin as a PI(4,5)P 2 phosphate regulating early VEGFR2 trafficking.
In spite of these results, as pointed out be the reviewer, we have not proven that the PI(4,5)P2-levels per se drives this process.Our ambition has been to present the data and conclusions in a balanced manner and we have been careful to not make the claim that the PI(4,5)P2-levels directly drives VEGFR2 trafficking.Not even with a new mouse model e.g.expressing kinase inactivated Paladin, could we make that claim.However, we do believe it is fair to state that in the context of previous literature, our data suggests that Paladin regulates VEGFR2 trafficking via its effect on PI(4,5)P 2 . 2 A, B, C and lines 161-163 of the main text: "We observed the formation of a Paladin-VEGFR2 complex in response to VEGF-A treatment both in vitro (in primary endothelial cells) and in vivo (in a mouse model)."This claim is not supported by data.Both in vivo and in vitro data show that VEGF alone is not sufficient to induce complex formation.This only occurs in case of peroxivanadate treatment.Moreover, peroxivanadate alone was sufficient to induce complex formation in vivo.This condition, is a important missing control in Figure 2A and 2B.Input samples should also be included.Along the same line of thought, based on Figure 2D, authors claimed that "Accordingly, super-resolution microscopy analysis confirmed VEGF-A induced colocalization of Paladin and VEGFR2.This indicated that Paladin could be involved in VEGF-A/VEGFR2 signaling."(lines 165-167).Yet, authors do not show conditions with or without VEGF stimulation, and no quantification is provided to support the claim on the levels of colocalization.

Figure
We apologize and have now addressed the reviewer's concern.We have replaced the super resolution microscopy with immunostaining analyses (Figure 1b) and added proximity ligation assay (PLA) analysis of VEGFR2 and Paladin interactions showing that VEGF-A induced PLA-complexes occur between VEGFR2 and Paladin in intact cells (Figure 1e,f for controls see Figure EV1).Blots have been complemented and moved to the Figure EV 1. Clarification of the role of peroxyvandate has been included in the text.2H shows that Paladin KD cells have higher levels of internalised VEGFR2 upon 5-15min of VEGFA stimulation than control cells.However, quantification in Figure 2J and Sup.Figure2F shows that both the number of VEGFR2 vesicles and the number of VEGFR2 vesicles colocalizing with EEA1 at 10min is equivalent to those in Paladin KD cells, whilst one would expect to have higher levels.Can authors comment on these contradictory results?Can authors provide additional confirmatory experiments to clarify this important claim in the manuscript?Moreover, the authors should complement their analysis for VEGFR2 trafficking with additional vesicular markers (Rab5+, Rab7+, Rab4+ or Rab11+ vesicles) in a time course manner to assess the fate of VEGFR2-positive vesicles in control and Paladin KD cells.

Figure
We agree that the data as presented in the initial submission was unclear.With improved staining protocol for Paladin, higher resolution imaging and shorter time points for VEGF-A stimulation, it is now clear that EEA1+/VEGFR2+ vesicles increase, especially 2 min after VEGF-stimulation in the absence of Paladin (new Figure 2e,f).We have not tracked the fate of VEGFR2 further in the endosomal compartment, as the data is now concordant with biotinylation and signaling experiments, but rather focused on corroborating the early events with PLA-studies and PI(4,5)P 2 stainings as presented in Figure 1 and 2. 4. In Figure 3, authors show that Paladin KD cells have higher signal transduction in response to VEGFA stimulation in vitro and in vivo.However, data presentation is very puzzling.For instance, Figure3i,j has different merged time points than Figure3f-h.Authors should provide a consistent way to display time in their analyses.The reviewer suggests that every time point should be displayed individually.In addition, quantification for pVEGFR2 levels in vivo is missing.
We thank the reviewer for this important comment.We have now changed the time points analyzed to be more consistent.However, depending on which signaling molecule you study, they exhibit different peaks of phosphorylation.Moreover, signaling studies in vivo are very challenging as the signaling kinetics is fast requiring consistent retrieval and freezing of tissues within minutes after VEGF-A injection in the tail vein, and even for experienced experimentalists, this inevitably introduces variability.Depending on the kinetics of the signaling, we chose to bin some of the time points in the experiments.For different signaling molecules, the peak and duration of signaling is different, and therefore it is reasonable to bin them differently.Please also note that none of the old Figure h-j showed any significant changes, so the time courses are merely there to illustrate the dynamics of the signaling.Consequently, we have moved 3h-j to Figure EV 3. We have updated Figure 3 to also include pVEGFR2 data in vivo, Figure 3f.4: To understand the extent of the observed rescue upon use of U0126 in Paladin KO animals, it would be important to provide representative images of those that were used for the quantifications represented in these graphs.Also, in figure 4I it is not clear in which condition CyclinD1 stainings were performed (WT or KO?).Representative images for both WT and KO animals should be shown.Moreover, authors could use ERG staining to label endothelial nuclei and thus increase the precision of the quantification (CyclinD1/ERG Double-positive cells), thus excluding confounding contribution of pericytes and microglia.

Figure
We have now included representative images for the graphs in the previous Figure 4k,l 4j there are now representative images of CyclinD1 from wildtype and KO retinas.We could not perform double stain for Erg and Cyclin D1 as both antibodies are from the same species (rabbit).However, we show a high-power image where the reader can see that the CyclinD1 stain is clearly vascular and appears to be endothelial and not in pericytes (Figure EV4d).6.In general, it would be more informative to show quantification of vascular parameters in Paladin WT and KO retinas in absolute numbers and not as relative to control.Moreover, authors should show all data points (such as in Figure 1B/C), avoiding bars, and privileging scatter dot plots or box and whiskers.
Since developmental angiogenesis in the retina is a very dynamic process and small differences in time point of harvesting the tissue can result in, for example, different vascular outgrowth, we found a normalization to the wild type littermates necessary to pool the data from several litters.However, absolute numbers for vascular parameters were already provided for some of the data in previous Figure EV 4, such as filopodia number, that are less effected by the exact time point of tissue harvest and the data are therefore easier to pool from several litters.We now moved filopodia data to the new Figure 4d.All data are now presented as dot plots.
7. Discussion (line 297-299): "Our data suggest that Paladin is a part of a VEGF-driven negative feedback loop in retinal angiogenesis where VEGF-A upregulates Paladin which acts to dampen VEGFR2 driven signaling and endothelial sprouting" The authors show that VEGF increases Paladin expression in HUVECS and in the mouse retina upon tail vein injection of VEGF.They do not show the existence of a loop that is be interrupted if Paladin is knocked down/ knocked out.Therefore, we believe that in the discussion, the authors should rephrase their sentence to avoid overstating the significance of their findings.
Point well taken; the sentence has been removed.
A reason for this finding could be that Paladin binds to phosphoinositides other than the substrates.For example, it is known that PTEN binds to PI(3)P, which mediates its localization to endosomes, but dephosphorylate PI(3,4,5)P3 (Naguib, Bencze et al.Mol Cell.2015).Importantly, Naguib et al. show that the PIP array does not indicate binding of PTEN to PI(3,4,5)P3, even though it is the substrate.A reason for this could perhaps be that the catalytic site mediates a more transient, low affinity interaction.However, as we don't follow up the potential interaction of Paladin and PIPs with further experiments, it is reasonable to omit the data to avoid confusion.
2. Line 172-173: "However, the receptor was degraded at the same rate as control-treated cells after VEGF-A stimulation (Figure 2e, Suppl Figure 2b)."It is unclear how the authors concluded about degradation rates.Could the authors clarify?
The slope of the curve for the VEGFR2 protein 0-60 min after VEGF stimulation in the previous Suppl Figure 2b (now Figure EV2b) is similar for Paladin knock-down and ctrl treated cells, suggesting that VEGFR2 was degraded at the same rate in the presence and absence of Paladin.However, as we have not formally determined the degradation rate, we have changed the wording to: " However, the receptor was degraded similarly over time after VEGF-A stimulation when comparing PALD1 siRNA and control treated cells (Figure 2a, Figure EV 2b)." 3. It would be interesting to provide images of P5 retinas images with higher magnification of tip cells from lacZ-stained Paladin het animals, similar to Figure 5a/c.Indeed, the Paldin LacZ reporter localizes nicely to tip cells in the sprouting retina.We have published this in Wallgard et al Dev Dyn 2012 Figure 6i and 8c 4. Figure 4: To strengthen their findings, it would be relevant to show the expression pattern of VEGFR2 and pVEGFR2 in the wt and Pald1 KO retinas.
We believe that the analysis suggested would not bring us further in the understanding of Paladin's role on VEGFR2 signaling and trafficking.Moreover, pVEGFR2 detection is technically very challenging, if not impossible to perform in vivo in most tissues, most likely since the phosphorylated receptor pool is small and very unstable.Our in vitro and in vivo signaling studies suggest that we should expect a shift in the rate of internalization of pVEGFR2 rather than any broad changes in the overall pVEGFR2 levels.
The Rab data has now been removed as discussed above, Q3.
6. Line 147-148: "Super-resolution microscopy revealed that one-quarter of the Rab4-or Rab11-positive structures were also positive for Paladin".The quantification for the number of vesicles that are double positive for Paladin/Rab4 and for Paladin/Rab11 should be showed in a graph.
We have now provided extensive quantification of Paladin in relation to VEGFR2 and EEA1 and focused on these interactions and removed the Rab stainings as this is no longer the focus of the paper, as also discussed above 7. Line 170-171: "siRNA-mediated knockdown of PALD1 in HDMEC resulted in a 35-51% increase of the total basal VEGFR2 pool".It is unclear what technique was used to obtain this result.It is not stated in the main text nor in the figure legend.If the technique used was Western Blot, it would be important to show the image with the bands that allowed to make this quantification.
We used western blot to quantify the Paladin levels and we have modified the text to state that clearly, see Figure 2a,c  8. Line 237-238: For clarity, the authors should clarify in the main text which animal model was used.From the main text it seems that an inducible endothelial specific knock out animal was used.But from reading the materials and methods, that does not seem to have been the case.
The text has been updated to state the use of a constitutive Pald1 knock-out mouse.9. Line 342: this reference "Lanahan, 2010" has not been included in the bibliography section.
This has now been corrected.10.Line 346: "We also observed increased pTyr1173 phosphorylation in HDMEC".For clarity, the authors should rephase this sentence to be more accurate, namely increased in which condition as compared with what.
Text has been corrected.11. Figure 1D, right panel: the expression pattern of paladin and PI4P seem to be heterogeneous across the cell population and perhaps inversely correlated.Is that the case?Could the authors show separate panels for each colour?Could the authors comment on this?
The reviewer is correct that there was some variation in the staining intensity, but the images presented exaggerated that difference.Staining homogeneity has been improved.However, the data has been removed as the focus of Figure 1 has shifted (see above).
12. Figure 4K and L: Could the authors clarify why two regimes of U0126 administration were used?
The maximum inhibition of pERK was observed 2-4.5 h after dosing.We used two different regimens as the filopodia that characterize the tip cells are highly dynamic structures.We reasoned that we should observe a potential phenotype with 4h treatment which was also practically feasible.Migration of endothelial cells in the retinal plexus is a slower process and we had to wait 24 h before we could expect to see an effect and therefore, we spaced the injection interval accordingly.A text describing this rationale has been included in M&M. 13.Line 271: "VEGF-A induced production of the Paladin protein in endothelial cells in vitro and in the retinal vasculature in vivo, as indicated by LacZ reporter expression (Figure 5b,c and Suppl Figure 5a)."Authors should clarify this text as it suggests that quantifications were performed on LacZ reporter, yet, Figure 5B seems to be WB from bands showed in Sup Figure5a.
The reviewer is correct, we have now changed it to: "Indeed, VEGF-A, but not other endothelial cell growth factors such as fibroblast growth factor-2 (FGF2) or stromal derived factor 1a (SDF1a) significantly induced expression of Paladin in endothelial cells in vitro (Figure 5b, Figure EV 5a).Moreover, Paladin was induced in the retinal vasculature in vivo, as indicated by LacZ reporter expression (Figure 5c)." --At the end of this email I include important information about how to proceed.Please ensure that you take the time to read the information and complete and return the necessary forms to allow us to publish your manuscript as quickly as possible.
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We replaced Supplementary Information with Expanded View (EV) Figures and Tables that are collapsible/expandable online.A maximum of 5 EV Figures can be typeset.EV Figures should be cited as 'Figure EV1, Figure EV2" etc... in the text and their respective legends should be included in the main text after the legends of regular figures.

Fig 2A .
Fig 2A.May the authors show the Co-IP experiment without overexpressing paladin?I'm aware that sometimes is very difficult to show a protein-protein interaction in native conditions.However it's the most convincing experiment to support a relevant biological interaction.Perhaps another technique may help to confirm the interaction between paladin and VEGFR2 shown panel B (Fret, in situ PLA)

Fig 3e .
Fig 3e.The in vivo analysis clearly demonstrates that paladin deletion in heart EC accelerates VEGFR2 degradation after VEGF stimulation.This effect does not occur in vitro (3A).Why? May it depend on different time courses analyzed?Which is the mechanism sustain the faster degradation observed in vivo in Pdl null mice?

14 .
Figure S1D: it is not very clear what portion of the cell this image is reporting.The authors should provide an additional image with the zoom out of this cell with the location of the zoom in marked. 1 Point-by-point letter MS EMBOR-2020-50218V1 Dear Dr Senyilmaz,

Fig 1 .
Fig 1.In this figure the authors show that paladin dephosphorylates both PIP 2 and PIP3 .Because PIP3 is mainly active at plasmamembrane and paladin is present in endosomal compartment, I think that it is quite surprising that paladin works on PIP3.This point has to be explained.I suggest to define the Km of these substrates .I think this data is very important to identify the relevant physiologic substrate of paladin.Furthermore it is necessary to add PI 5 phosphatase as a second positive control specific for PIP2.

Fig 2D .
Fig 2D.These data have to be quantified.Furthermore the authors have to determine where this interaction occurs ( Rab4, -7, -11 positive vesicles, Golgi membrane)

Fig
Fig 2g,h.When cells are stimulated by VEGF, VEGFR2 undergoes phosphorylation.Does paladin silencing modify the internalization of phosphorylated form of VEGFR2?

Fig 3e .
Fig 3e.The in vivo analysis clearly demonstrates that paladin deletion in heart EC accelerates VEGFR2 degradation after VEGF stimulation.This effect does not occur in vitro (3A).Why? May it depend on different time courses analyzed?Which is the mechanism sustain the faster degradation observed in vivo in Pdl null mice?

Fig 4 & 5 .
Fig 4 & 5. Does Erk inhibition rescue the effect in retina vascularization observed in Pdl null mice?There a lot of compound in vivo tested that could easily exploited (e.g.refametinib, GDC-0994) and placed them in Figure EV 4 f,g.We have updated the Results section and Figure 4 legend to clearly state that the previous Figure 4i (now Figure EV 4d) represents the wildtype condition, to illustrate the CyclinD1 protein localization in the retinal vasculature.In the new Figure and Figure EV 2a.

14 .
Figure S1D: it is not very clear what portion of the cell this image is reporting.The authors should provide an additional image with the zoom out of this cell with the location of the zoom in marked.Previous FigureS1Dhas been replaced with the new Figure1bto better show the overview and zoomed in parts.submitting your revised manuscript.I have now looked at everything and all is fine.Therefore I am very pleased to accept your manuscript for publication in EMBO Reports.

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Computational models that are central and integral to a study should be shared without restrictions and provided in a machine-readable form.The relevant accession numbers or links should be provided.When possible, standardized format (SBML, CellML) should be used instead of scripts (e.g.MATLAB).Authors are strongly encouraged to follow the MIRIAM guidelines (see link list at top right) and deposit their model in a public database such as Biomodels (see link list at top right) or JWS Online (see link list at top right).If computer source code is provided with the paper, it should be deposited in a public repository or included in supplementary information.23.Could your study fall under dual use research restrictions?Please check biosecurity documents (see link list at top right) and list of select agents and toxins (APHIS/CDC) (see link list at top right).According to our biosecurity guidelines, provide a statement only if it could.micewith constitutive deletion of Pald1 (Exon 1-18 replaced by a LacZ reporter cassette) have been generated and backcrossed for at least 10 generations.Pald1+/-intercrosses were performed to generate homozygous and heterozygous littermates (Wallgard et al., Dev Dyn 2012).Mice were used at postnatal days 3-5, or 7-17 (OIR model) or as adult week 6-10.Mice were kept in groups of max 5/cage.In exceptional cases mice were placed alone for brief periods, e.g.aggressive males and in consultation with the University Veterinarian.The cages were made of plastic and floors covered with a layer of wood shavings.The cages contained enrichment (cardboard houses, paper, small nests).Cages, food and water bottles were changed once/week and supervision was carried out daily by trained staff.All animal experiments were performed in compliance with the relevant laws and institutional guidelines and were approved by the Uppsala University board of animal experimentation.