The microcephaly protein WDR62 regulates cellular purine metabolism through the HSP70/HSP90 chaperone machinery

  1. Matthew J. Morris
  2. Yvonne Y. Yeap
  3. Chi Chen
  4. S. Sean Millard
  5. Julia K. Pagan
  6. Dominic C. H. Ng

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Abstract

Inherited mutations in WD repeat-containing protein 62 (WDR62) are associated with microcephaly ( MCPH2 ). While WDR62 plays important roles in mitosis and centriole biogenesis, additional WDR62 functions may cause abnormal brain growth. Here, we reveal a novel WDR62 role in the molecular chaperone network regulating purine metabolism. In response to hyperosmotic stress, WDR62 redistributes to purinosomes—phase-separated membraneless assemblies of purine metabolic enzymes and their chaperones. While WDR62 is not needed for purinosome formation, its loss disrupts purine homeostasis, resulting in the accumulation of purine nucleotide intermediates and a reduction in the levels of hypoxanthine-guanine phosphoribosyl transferase (HPRT), a key purine salvage enzyme. We link this to WDR62’s interaction with Bcl2-associated athanogene 2 (BAG2), a co-chaperone that modulates the function of HSP70/90. In cells lacking WDR62, BAG2 levels are elevated and HPRT stability is reduced. Knocking down BAG2 in these cells restores HPRT levels, underscoring the crucial role of WDR62-BAG2 interactions in chaperone-mediated stability and turnover of metabolic pathway enzymes. Notably, common microcephaly-associated mutations in WDR62 alter its interaction with BAG2, suggesting that purine metabolic defects resulting from WDR62 mutations may underlie microcephaly in humans.

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

    In Morris, M.J. et al., the authors strive to better understand the roles for the microcephaly protein WDR62 in brain growth and function. To accomplish this, an in situ biotinylation assay was performed and identified 42 proteins proximal to WDR62 including the Hsp70 co-chaperone BAG2. Through a series of co-immunoprecipitation assays, the BAG2-WDR62 interaction was shown to be mediated through the structured N-terminal region of WDR62, and it was proposed that common WDR62 mutations disrupt this interaction. In AD293 cells, loss of WRD62 expression resulted in an increase in the expression of BAG2 expression while reducing HPRT expression. Subsequent loss of BAG2 expression by siRNA treatment restored the expression of HPRT suggesting that there is an association between the stability of HPRT and BAG2, likely mediated through its proposed association with Hsp70/90 molecular chaperones. Finally, the authors investigate the subcellular localization and ability of WRD62 to phase separate. WRD62 was shown to form discrete condensates induced by sorbitol-mediated hyperosmotic stress. The formation of WRD62 granules are hypothesized to be driven by cell volume exclusion and macromolecular crowding. These granules appear similar, both in physical appearance and characteristics, to other reported biomolecular condensates such as those reported in metabolism (e.g. purinosomes). WRD62-containing condensates were shown to colocalize with enzymes in de novo purine biosynthesis; however, this association is not required for purinosome formation and/or its stability under both purine-depleted and sorbitol-driven growth conditions. Overall, the manuscript provided a previously unrealized and exciting association between WDR62 and purine metabolism.

    EVIDENCE, REPRODUCIBILITY AND CLARITY Summary: The current manuscript reads as multiple manuscripts with findings that are at times weakly connected (in my opinion). For example, I had a hard time understanding how the BioID results relate to the discovery of WRD62 phase-separation and its colocalization with purinosomes. I would strongly encourage the authors to consider dividing the results into separate manuscripts to strengthen their claims and create a more focused and cohesive manuscript (or series of manuscripts). I believe then several of my reservations associated with the current manuscript will be addressed, and in my opinion, the hard work from the authors will be better received across the scientific community.

    Response: We thank Reviewer #1 for acknowledging the novelty of our work and appreciate the constructive feedback regarding the lack of integration among individual findings. In response, we have removed content related to condensate formation and conducted additional experiments to more thoroughly characterize the mechanisms of WDR62 interaction. These new data, along with revisions to the manuscript text, have strengthened the coherence of our findings. We believe the revised manuscript now presents a more unified narrative, highlighting the complex roles of WDR62 in regulating purine metabolism.

    I would like to commend the authors for all the work that went into the current version of the manuscript. Being part of a biochemistry and cell biology research group, I completely understand how much time and effort must have went into generating these data. That being said, I felt that there were several instances where clarification and additional information is warranted to arrive at the conclusions made by the authors. These points are outlined below.

    Major Comments:

    1. There appears to be a discrepancy between the data presented in Figure 1 and what is stated in the main text. Clarification is necessary to better understand the results:

    • The following statement (and derivatives of it) are repeated throughout the manuscript: "...we found that the WDR62 interactome comprised molecular chaperones such as HSP70, HSP90, and their co-regulators, BAG2, STIP1, and DNAJC7" (lines 91-93, 316-318, 422-425). STIP1 and DNAJC7 were not identified in the list of 42 proximal proteins to WDR62 (Figure 1D). DNAJC7 was included because of a previous report curated in the BioGRID database, and there is no mention of HSP90 in the data produced in Figure 1. Please revise the main text to reflect the data that was generated.

    Response: We thank the reviewer for this valid point and highlighting the instances where our description of results did not accurately reflect the data generated. We have reworded the relevant sections (e.g. lines 105-107) in our revised manuscript to better delineate interactors identified in BioID studies (BAG2) as opposed to those previously reported on protein interaction databases such as BioGRID (DNAJC7).

    Based on the data presented in the Venn Diagrams in Figure 1D, the author's numbers do not seem to be consistent with the sentence on lines 126-128. I count 37 proteins unique to their BioID study, 90 unique to the BioGRID database, and 5 proteins that overlap between the two data sets. This sentence needs to be revised.

    Response: We thank the reviewer for pointing out this inconsistency. There were 95 protein interactors of WDR62 from BioGRID while we identified 42 proteins in our BioID study with 5 proteins overlapping. We have revised the main text (lines 144-146) and Fig. 1D to accurately reflect the protein numbers identified.

    What data were used to generate the interaction map in Figure 1I? Enzymes tied to purine metabolism were not identified from the data presented in Figure 1D but have now appeared. A discussion of this in the main text is warranted.

    Response: We generated the interaction map in Fig. 1I using STRING to visualise WDR62 protein-protein interactions derived from both the BioGRID database and our BioID analysis. As the reviewer correctly points out, purine metabolic enzymes were not direct interactors of WDR62. Purine enzymes are linked to the molecular chaperones which, in turn, associated with WDR62 from our BioID analysis. The links between purine enzymes and chaperones were obtained from the BioGRID database. In response to this feedback, we have revised our manuscript to include a more detailed description of how the interaction map in Fig. 1I was generated, both in the main text (lines 148-157) and the legend for Figure 1. The BioGRID interactions between heat shock proteins and purine enzymes were introduced in the manuscript text at lines 264-266.

    1. This reviewer has several reservations on how the various key players in the manuscript are related to substantiate the conclusions made in the manuscript. For instance, how is HPRT, purinosomes, and WDR62 related? How about HSP90, WRD62, and HPRT? Pairwise connections were made throughout the manuscript; however, trying to tie all three together is difficult with the data presented.

    • The authors tried to connect HPRT, purinosomes, and WDR62 with BAG2; however, this study could greatly improve if we understood how a knockdown of BAG2 impacts purinosome formation and/or WDR62 colocalization with purinosome enzymes.

    Response: We have incorporated additional experiments in our revised manuscript to better connect HPRT, WDR62 and BAG2. Using proximity ligation assays (PLA) we demonstrated endogenous interactions between WDR62 and BAG2 (Fig. 4K), as well as between WDR62/HPRT and BAG2/HPRT (Fig. 6I-J). The interaction between BAG2 and HPRT was decreased in WDR62 KO cells (Fig. 6J), and recent experiments revealed that BAG2 depletion similarly disrupted the WDR62/HPRT interaction. These findings suggest that WDR62 expression, and presumably its interaction with BAG2, is necessary for BAG2-mediated regulation of HPRT.

    Further, we found that the loss of HPRT expression in WDR62 KO cells was reversed by siRNA depletion of BAG2 (Fig. 6K), supporting our model in which elevated BAG2 levels in the absence of WDR62 promote aberrant HPRT degradation. Collectively, our results suggest that proper BAG2 regulation of HPRT requires WDR62.

    To address the reviewer’s suggestion, we also examined WDR62 cytoplasmic localisation following BAG2 depletion and found that BAG2 was not required for WDR62 to form granules in response to osmotic stress. We also show that BAG2 is not responsible for purinosome assembly or for the subcellular distribution/localisation of HPRT.

    Is HPRT a client of HSP90? And how are WRD62 and HSP90 related since they do not associated (based on your BioID data)? These connections would again strengthen the arguments made in the manuscript and help to explain the HSP90 inhibition data presented in Figures 7F and 7G.

    Response: Although our BioID data did not explicitly identify an association between WDR62 and HSP90, we initially focused on HSP90 due to the established role of BAG2 in protein misfolding and degradation through its interaction with HSP90 (doi: 10.7150/thno.78492). We hypothesised that while WDR62 may not directly interact with HSP90, its interaction with BAG2 could provide an indirect link. To strengthen our conclusions and address the limitations of our HSP90 inhibition data (NVP-AUY922), we performed additional experiments using a second HSP90 inhibitor (17-AAG) and an HSP70 inhibitor (MKT-077) across both short (1 h) and long (24 h) treatment durations (Fig 6 and Fig S10). Further details are provided in our response below to minor comment #1.

    Caution is warranted when making conclusions about WDR62 (and its granules) and purinosomes.

    Response: We acknowledge the reviewer’s feedback and have revised our manuscript to focus on the functional characterisation of WDR62 interaction and co-localisation with BAG2 and related HSP co-chaperones. As part of this revision, we removed the FRAP studies and sections discussing WDR62 phase separation and purinosome assembly (further details below). Additionally, we have softened out description of cytoplasmic WDR62 granules as purinosomes. Instead, we describe WDR62 as forming dynamic puncta containing purine enzymes and discuss the possibility that these granules may represent or overlap with bona fide purinosomes.

    The authors describe the association between WDR62 and purinosomes differently throughout the text. I would recommend that the authors come to some conclusion about this and be consistent.

    Response: We thank reviewer #1 for pointing out inconsistencies in our conclusions regarding WDR62 and purinosomes between sections of our manuscript. We have revised our manuscript to ensure our description of these findings are consistent throughout. Specifically, our findings show that WDR62 responds to osmotic and metabolic stress by forming dynamic cytoplasmic granules that share many protein components with purinosomes (Fig. 5). This suggests that WDR62 may be a novel component of bona fide purinosomes or that WDR62 granules substantially overlap with purinosomes both spatially and compositionally. However, the formation of granules by purine enzymes was not perturbed by WDR62 KO (Fig. S6). Thus, we conclude that while WDR62 colocalized with purine enzyme containing granules consistent with purinosomes in response to cell stress, WDR62 was not required for granule formation by purine enzymes such as PFAS and PPAT.

    A. (Lines 339-340) "WDR62 granules represent or overlap substantially with the phase-separated metabolons known as purinosomes". Based on the data presented, it appears that these might still be different entities but either overlap or have similar components. Purinosome localization with mitochondria (approx 60-80%) and microtubules (approx 90-95%) were significantly higher than those reported for WDR62 granules (approx 40%). This comparison would suggest that not all WDR62 granules behave similarly to purinosomes. And from the dot plot in Figure 3G, about half of the characterized WDR62 granules do not align with the previously reported characteristics of purinosomes.

    Response: In Fig. 3G, we measured the diameter and distribution of WDR62 granules and found their size and number per cell closely matched those reported for BAG2 condensates (doi: 10.1038/s41467-022-30751-4). This aligns with our findings that WDR62 interacts with BAG2 and is recruited to similar subcellular compartments. The reviewer correctly notes that WDR62 granules only partially align with previously reported characteristics of purinosomes, suggesting that they may be distinct entities. Our revised manuscript acknowledges this possibility while also emphasising that WDR62 granules share features and colocalise with many purinosome components. To enhance the focus and clarity of the manuscript, we have removed Fig. 3G as the diameter and number of WDR62 granules are already reported in Fig. 3F.

    In the abstract and introduction, the authors state that WDR62 is being recruited to the purinosome and leave out the other possibility. I would recommend that the authors soften this claim in these sections because of the above possibility but also the lack of characterization of the sorbitol-induced "purinosomes". There is little discussion or evidence for how sorbitol induces purinosome formation. Is de novo purine biosynthesis activated upon sorbitol treatment? Are multiple de novo purine biosynthetic enzymes present in the sorbitol-induced "purinosomes"? Further, I agree that there is a tendency for WDR62 to associate with condensates that bear an enzyme within de novo purine biosynthesis; however many of these proteins are known to self-aggregate upon cell stress. Therefore, the entities that are being observing and called purinosomes might not be bone fide purinosomes. Additional care is necessary to make these statements. In my opinion, the current data only suggests that this might be a possibility.

    Response: As indicated, we have softened our claim that stress-induced WDR62 granules represented bona fide purinosomes. Fig. 3 of our revision more precisely describes the characteristics of WDR62 granules while Fig. 4 now reports on the co-localisation of WDR62 granules with protein chaperones and de novo purine synthesis enzymes typically associated with purinosomes. We now conclude that WDR62 may be associated with purinosomes but may also represent distinct entities with shared components and characteristics. Notably, proteins such as BAG2 and PFAS may undergo phase separation in response to stress independently of purinosome assembly.

    In additional work conducted for our revised manuscript, we find that WDR62 loss reduced rates of purine synthesis in cells cultured in the presence of purines (Fig. 5) but was not involved in de novo purine biosynthesis under purine-depleted conditions (Fig. S9). This was consistent with the finding that WDR62 loss did not prevent stress-induced formation of PFAS or PPAT granules (Fig. S6) which are likely to represent purinosomes. We concede that additional investigation is required to determine the functional significance of WDR62 granules in response to stress stimuli and purine depletion.

    (Lines 325-329) The authors reference a previous manuscript demonstrating that co-chaperones co-cluster with purinosomes. Based on this fact, they infer that WDR62 granules might represent purinosomes since WDR62 interacts with these same set of co-chaperones. These co-chaperones interact with a large number of different proteins (in fact, most kinases), so it is uncertain how the authors decided to go down this path to link purine metabolism with WDR62. Discussion of how this connection was made would help elevate the story. What additional insights did they have that lead them down these investigations?

    Response: BAG2 functions as a co-chaperone that regulates the activity of HSP70/90. While the reviewer correctly points out that co-chaperones such as BAG2 have a broad number of clients, numerous studies have established the role of HSP70/90 in purine metabolism (e.g. doi: 10.1016/j.isci.2020.101058, 10.1073/pnas.1300173110) and in neurodevelopment (10.3389/fnins.2018.00821). Moreover, purines are critical for normal brain development and dysregulation is well known to lead to congenital defects including microcephaly. As such, when we identified a role for WDR62 in the chaperone network through interaction with BAG2, it was not a leap to hypothesise that neurometabolic defects stemming from dysregulated purine production or salvage might be involved in WDR62-associated microcephaly.

    Indeed, we show that WDR62 are localised with purine enzymes in response to purine-depletion and that WDR62 depletion leads to metabolic dysregulation. WDR62 has several binding partners with multiple cellular functions, and we do not exclude alternative mechanisms involved in cortical development. However, the mechanistic link with heat shock proteins and purine metabolism is a novel one that would be of broad interest in molecular neurodevelopmental biology. On this feedback, we have revised main text (lines 214-218, lines 260-263, lines 292-295, lines 378-383) to better explain the rationale underlying our experiments and overall study focus.

    If WDR62 is not required for purinosome formation, why would it localize with the purinosome? Is there any hypothesis that could be readily tested to better help understand this observation? Providing a better understanding of this would greatly elevate the work.

    Response: Given the role of HSP70/90 in purinosome assembly and the interaction of WDR62 with BAG2, and purine enzymes PFAS and PPAT, we were initially surprised that WDR62 depletion did not affect stress-induced PFAS and PPAT granule formation (Fig. S6). At the time of writing the original manuscript, we interpreted these granules as purinosomes. However, it remains possible that WDR62 might have a function in purine synthesis or in purinosome assembly that remains unidentified. Indeed, we have not yet tested different cell types or additional conditions that induce purinosome formation or determined the localisation or activity of other purine synthesis enzymes. Thus, we concede our conclusions on WDR62 and purinosome formation were premature.

    As our revised manuscript is now focused on the WDR62-BAG2-HPRT interaction and given the reviewer’s prior comment that PFAS and PPAT colocalization in granules may not represent purinosomes in all contexts, we acknowledge that potential WDR62 functions in purinosomes warrants further investigation beyond this study. In the revised discussion (lines 473-497) we address these limitations and propose alternative interpretations of our findings.

    (OPTIONAL) Please validate that the associations between WDR62 and the purine biosynthetic enzymes occur on the endogenous level (void of transient transfection). Many methods such as immunofluorescence and proximity ligation assays have been used by others to demonstrate protein-purinosome interactions. This result would reduce any concern that the association is a result of overexpression (artifact).

    Response: As suggested, we conducted proximity ligation assays (PLA) to validate endogenous interactions between WDR62 and BAG2, HPRT, and PFAS (Fig. 4K, Fig. 6I-K). Notably, sorbitol treatment increased the interaction between WDR62 and HPRT (Fig. 6H, I), supporting the role of WDR62 in regulating HPRT under cellular stress. Additionally, WDR62 deletion appear to reduce the interaction between BAG2 and HPRT (Fig. 6K), while BAG2 depletion similarly reduces the interaction between WDR62 and HPRT (Fig. 6J). These findings support a model in which WDR62 and BAG2 cooperatively regulate HPRT stability.

    Figures 6F and 6G conclude that nucleosides from purine-depleted growth conditions accumulate while the corresponding monophosphates do not change between WRD62 knock-out and wildtype cells. Given that purine-depleted growth conditions activate de novo purine biosynthesis (uncertain if this has been demonstrated in AD293 cells), could this result simply demonstrate that purine salvage is no longer used and the nucleosides have accumulated and are awaiting degradation (or exportation) rather than a loss of HPRT expression as inferred from the stated conclusions? The conclusions could be better substantiated with the use of a stable isotope incorporation assay.

    Is there a difference in the contribution of de novo purine biosynthesis and purine salvage to the generation of the monophosphates (AMP, GMP) between WDR62 knockout and wildtype AD293 cells? Use of a stable isotope (such as 15N-glutamine) could help to come to the appropriate conclusion.

    __Response: __We thank the reviewer for this helpful suggestion to better characterize WDR62-dependent purine defects in more detail. In our revised manuscript we performed targeted metabolomics experiments and tracked the incorporation of 13C2-glycine and 13C5-hypoxanthine into purine nucleosides to assess purine synthesis and purine salvage flux between WT and WDR62 KO cells (n=5). Indeed, purine nucleotides in KO cells showed a significant loss of incorporation of 13C2 from 13C2-glycine, consistent with impaired *de novo *synthesis in cells cultured in presence of purines. In contrast, labelling from 13C5-hypoxanthine showed no overt differences between WT and KO cells, suggesting that incorporation via the salvage pathway is not grossly altered under these conditions. We have subsequently added a section to the discussion (lines 498-521) to discuss these results which suggest that the reduced HPRT levels in KO cells may be sufficient to sustain rates of purine salvage which are not altered with WDR62 loss. Thus, the accumulation of nucleosides is most likely due to increase conversion from monophosphates or reduced degradation to uric acid. Nonetheless, we show that WDR62 is required for purine synthesis under basal conditions and has a complex role in regulating purine metabolism.

    (Lines 483-485) If there is a change in de novo purine biosynthesis, are there any detectable changes in AICAR levels that might influence purine metabolism at the transcriptional level?

    __Response: __This remains a possibility. However, we did not detect the AICAR intermediate in our untargeted LC-MS/MS metabolomics analysis perhaps due to low relative abundance and/or low stability. As a result, we were unable to comment on AICAR levels but this would be an interesting research direction to pursue in subsequent follow up studies.

    Are the data and the methods presented in such a way that they can be reproduced? Are the experiments adequately replicated and statistical analysis adequate?

    1. For purine-depleted studies (metabolite analyses, microscopy), how long were the cells grown in purine-depleted medium before the analysis? And how was the purine-depleted medium generated? Please reference any source that might have been used.

    __Response: __We removed purines from the cell culture environment by incubating cells for 7 days with DMEM supplemented with FBS dialyzed to remove small molecules such as nucleosides and nucleobases. This important methodological detail was omitted in error in our original submission. Our revised manuscript includes description of how we depleted cells of purines in the Materials & Methods at Lines 636-640 with reference to source materials and prior studies.

    Details describing the BioID experiment are minimal. How many replicates were performed, was label-free or TMT quantitation used for the protein identification. Further the data analysis and mining of the proteins from the BioID study are missing - What database was used to identify the proteins from the peptides? Please include this information in the Materials and Methods section as well as a link to a repository where the LC-MS/MS data generated can be found. Additionally, it would be very helpful to have a spreadsheet or table that lists the biotinylated proteins and expectant or p values for each.

    __Response: __We performed three independent biological replicates (*n *= 3) for the BioID experiment. We apologise for the omission and have now included this information in the Fig. 1 legend. Label-free quantitation was used for protein identification, and peptides were identified using the ProteinPilot™ Software (v. 4.5) database. As part of our revision, we have updated the Materials and Methods section to include these details and will also provide a spreadsheet listing all biotinylated proteins across replicates, including their p-values. Furthermore, we have submitted our LC-MS/MS data as supplementary files associated with this manuscript.

    Please include information about the streptavidin pulldown presented in Figure 1C.

    __Response: __Streptavidin pulldown followed by immunoblot for known WDR62 interacting proteins is described in our Materials & Methods section at line 753-759. __ __Proteins bound to Streptavidin agarose beads were eluted with Laemmli buffer following washing. Pulldown fractions and total lysates were then resolved on SDS-PAGE, transferred to PVDF and blotted with primary antibodies to detect WDR62 interacting proteins such as CEP170, JNK and AURKA. We also used this method to confirm biotin-labelling and affinity isolation of BAG2 in Fig. 1C.

    Many of the figure legends could benefit from a statistical description.

    Response: As requested, we have updated the legends for all relevant figures and supplementary figures to include statistical descriptions, specifying analyses used and replicate (n) numbers. These additions complement the detailed description of our statistical methods provided in the Materials & Methods section (line 884).

    There seems to be only two data points for Figure S3A. While there is no significant difference observed, it would be ideal to have additional replicates.

    Response: We have completed an additional replicate and updated Fig. S3A for our revised submission. This study which now includes* n *= 3 independent biological replicates. While we observed a slight increase in the proportion of cells with MAPKBP1 granules in response to sorbitol stress, this change was not statistically significant. In contrast, WDR62 formed granules in a much larger proportion of cells (~90%) in response to stress (Fig. 3E).

    (Figure 5I) Please provide statistical analysis to demonstrate the colocalization between FGAMS and WDR62 is robust in purine-depleted AD293 cells.

    Response: Our revised manuscript now includes three independent replicates assessing WDR62 co-localisation with PFAS in purine-depleted AD293 cells (Fig. 4I in revision). We consistently observed a high degree of co-localisation, as quantified by Pearson’s correlation coefficient (mean = 0.8), which was significantly different from control conditions.

    1. The use of HSP90 inhibitors is a little confusing given the connections being made with BAG2 and other HSP70 co-chaperones in Figure 1.

    • Does the same conclusions hold true with an HSP70 inhibitor or siRNA?

    (OPTIONAL) There are a lot of discrepancies between Hsp90 inhibitors and effective treatment concentrations. For example, NVP-AUY922 caused purinosomes to disassemble whereas STA9090 cause purinosomes to change morphology and adopt a more aggregated state. Do other Hsp90 inhibitors share the same phenotypic response as NVP-AUY922 in this study.

    The treatment time (24 h) with NVP-AUY922 is very long. Given that Hsp90 interacts with hundreds of proteins, it is hard to understand whether the effect of Hsp90 inhibition is direct or indirect. Shorter times (1 h or less) would be more insightful.

    __Response: __To address these specific comments on the specificity of effects from HSP90 inhibitor treatment, we have conducted additional experiments using NVP-AUY922, in addition to another HSP90 inhibitor, 17-AAG, and the HSP70 inhibitor, MKT-077, at both 24-hour and 1-hour timepoints.

    Our results demonstrate that NVP-AUY922 can rescue the aggregated HPRT phenotype in WDR62 KO cells even after 1 hour of treatment (Fig. 6F, G). Similarly, 17-AAG exhibits a comparable effect, reinforcing the role of HSP90 inhibition in modulating the spatial distribution of HPRT in the cytosol (Fig. 6F, G). Additionally, we found that MKT-077, a HSP70 inhibitor, also rescues the aggregated HPRT phenotype, with the effect being most pronounced at 24 hours but still evident at 1 hour (Fig. S10A, B). We also utilized BAG2 siRNA but determined that BAG2 depletion rescued WDR62 KO effects on HPRT expression (Fig. 6L) but did not reverse the effect on HPRT spatial distribution (Fig. S10C).

    (OPTIONAL) Does the 2.6-fold increase in BAG2 increase its association with WDR62?

    Response: We observed a ~2.6-fold increase in BAG2 levels following WDR62 deletion (Fig. 6A). However, as WDR62 is not present in KO cells, it is not possible to verify whether there would be an increase association with WDR62 and we did not conduct an experiment to overexpress BAG2 in WT cells. However, we presume that increased cellular levels of BAG2 would lead to increased pulldown with WDR62 by immunoprecipitation for example.

    Is the degradation of HPRT occurring through BAG2-mediated proteasomal degradation? Showing HPRT recovery by treating the cells with MG132 along with CHX would provide meaningful clues as to how BAG2 and HPRT might be related - Is BAG2 concentration increasing to facilitate the enhanced degradation of HPRT?

    __Response: __We thank the reviewer for this useful suggestion. However, our initial experiments with MG132 and chloroquine to inhibit proteosomal and autophagic pathways respectively gave mixed results. Our preliminary findings suggest neither was sufficient to substantially rescue HPRT levels in WDR62 KO cells. However, this needs extensive follow up with more precise dissection of cell degradation pathways with additional inhibitor or genetic targeting of degradation machinery. Thus, we did not include these studies in the revision and will instead include this in a follow up paper once we have completed a more comprehensive investigation.

    Does HPRT colocalize with WDR62 in cells?

    __Response: __ In response to this comment, we have demonstrated that osmotic stress induces the spatial reorganisation of endogenous HPRT into puncta that juxtapose and co-localize with WDR62 granules in a stress-dependent manner (Fig. 6H). This was further validated by examining the endogenous WDR62-HPRT interaction using PLA, which also revealed a stress-induced increase upon sorbitol treatment (Fig. 6I).

    (OPTIONAL) It would be nice to see validation experiments of some of the hits in Figure 1D or 1E in a co-immunoprecipitation experiment conducted similar to Figure 1C.

    __Response: __Our BioID assay, presented in Fig. 1D and E, identified WDR62 interactors, such as AURKA, JNK, CEP170 and MAPKBP1, that have been previously validated by co-IP by our group and others. Among the chaperones identified, we focused on BAG2 in this particularly study and validated BAG2-WDR62 interactions between by coIP (Fig. 2) and by proximity ligation assays (Fig. 4).

    The authors presented the findings that suggest that BAG2 interacts differently with commonly observed WDR62 mutations in MCPH2? How do these mutations affect WDR62 condensation, colocalization with purinosomes, or alter HPRT activity? Tying back the observations to something clinical would help elevate the overall significance of the findings.

    Response: We investigated the condensation of mutant WDR62. Interestingly, R438H mutant, which binds BAG2 (Fig. 2), forms granules constitutively prior to stress treatment while the 3936dupC mutant, which does not bind BAG2 (Fig. 2), did not form granules in response to sorbitol stress treatment. We also find that PFAS is colocalized with R438H granules in the absence of stress, although this requires repeated analysis and quantification. However, WDR62 deletion does not prevent PFAS or PPAT granule formation (Fig. S6) and, given reviewer advice to focus the topic of our revised manuscript, we have not included the effects of WDR62 mutations on granule formation in our revised manuscript.

    However, in response to these comments, we have conducted rescue experiments with patient-identified MCPH mutant variants of WDR62. Expression of the R438H or 3936dupC mutant in WDR62 KO cells did not rescue HPRT to the same extent as full-length WDR62 with wild-type sequence (Fig. 6B). Additionally, attempts to restore BAG2 levels in WDR62 KO cells by expressing mutant WDR62 showed no discernible difference from full-length WDR62. Thus, mutations to WDR62 associated with MCPH alters binding to BAG2 (Fig. 2, increased with R438H and decreased with 3936dupC), this was associated with dysregulated levels of BAG2 and HPRT. In our revised manuscript, we also examined the effect of HPRT depletion on neurodevelopment in vivo (Fig. 7) and included description of these findings at lines 417-442.

    Are the text and figures clear and accurate?

    1. There are many times throughout the manuscript that the wrong figure is being referenced. These mistakes caused significant confusion at many times while reviewing the manuscript. Please double check all in-text references to figures. For example, I believe that you meant to use Figure S1C instead of Figure 2E with the statement on lines 183-185. Again, I believe that correct figure reference on line 501 is Figure 7G not Figure 7E.

    Response: We apologize for this oversight. We have amended the errors indicated by the reviewer. Line 544 (501 in first submission) now refers to the correct figure (Fig. 6F) and lines 204-206 (183-185 in first submission) correctly refers to Fig. S1C in addition to Fig 2E. Each of the authors have also revised the rest of the manuscript to ensure all figures are correctly referenced in the main text.

    The figure legend on Figure S4 does not match the figure and the main text references. Please verify that the text in the figure legends correspond correctly to the figure.

    Response: We apologize for these inconsistencies in the figure legend relating to Fig S4 in our original submission. In the revised manuscript, we have amended the figure legend and the main text referencing Fig. S4 to correctly correspond to order of data panels in this figure.

    Please provide this data for the sentence on lines 399-400 in the supplemental file.

    __Response: __As requested, we have revised the manuscript to include results on HPRT cytoplasmic localisation following osmotic stress. We show that osmotic stress induces the spatial reorganisation of HPRT into puncta that juxtapose and co-localize with WDR62 granules in a stress-dependent manner (Fig. 6H). This was further validated by examining the endogenous WDR62-HPRT interaction using PLA, which also revealed a stress-induced increase upon sorbitol treatment (Fig. 6I).

    I believe that the authors use the phrase "cell proliferation" to describe cell viability in the main text. In the Materials and Methods section, the authors state "The XTT cell proliferation assay enables quantification of cellular redox potential, providing a colorimetric readout of cell viability." Cell proliferation, viability, and cytotoxicity are different measurements, so please revise to reflect the correct experiment that was performed.

    __Response: __The XTT colorimetric assay can be used to determine cell proliferation or loss of cell viability depending on the specific experiment. The reviewer is correct in pointing out that our study using XTT to measure cell numbers in the context of purine-depletion (Figure 5B) is a measure of cell viability. We apologize for the misleading text in our description of the XTT methods in our original submission. In our revised manuscript, we have amended our description of the XTT assay in our methods and in the figure legend to more accurately reflect the experiment performed.

    Other Minor Comments:

    1. Move the sentence "In contrast, despite reduced mRNA..." (lines 387-388) to the last section when a reduction in PFAS expression was first mentioned.

    __Response: __As requested, we have moved this line referring to PFAS protein levels in WDR62 KO cells to the previous section to when a reduction in PFAS mRNA was first mentioned.

    1. Please reference the following in the manuscript: • BioGRID database in the main text and Materials and Methods section • The reported study showing the DNAJC7-WDR62 interaction (as curated from BioGRID) • Fiji in the Materials and Methods section

    __Response: __We have now included references to these in our revised manuscript. References to BioGRID database are in the main text (line 146) and in the Materials and Methods (line 765). The report of DNAJC7-WDR62 interaction (Ref #37) curated from BioGRID was added at line 157 and reference (Ref #82) to Fiji plug-in was indicated at line 690 in Materials & Methods.

    (Line 461-463) The authors state the following: "the loss of WDR62 leads to an increase in BAG2 and vice-versa (Fig. 7A) (Fig. S9B). I am not sure that the vice-versa (i.e. loss of BAG2 increases WDR62) is true. From the data presented in Figure 7H, I do not see a significant change in WDR62 expression upon BAG2 siRNA treatment.

    __Response: __We apologize for the incorrect use of the term “vice versa” in this context. We had meant that while WDR62 loss led to an increase in BAG2, the converse increased expression in WDR62 resulted in a decrease in BAG2 levels. The reviewer is correct that the siRNA knockdown in BAG2 did not substantially alter WDR62 levels. We have amended the text at lines 465-467 to clarify this statement.

    For your BioID study, do you know how many or the proportion of cells that were mitotically arrested with the low dose of nocodazole (200 ng/mL)? Given the small number of unique proteins that were in the mitotic only population, it is curious to know how enriched the cells were and whether WDR62 localization is important in the context of this study.

    __Response: __The overnight treatment with low dose nocodazole results in an enrichment of cells arrested in late prometaphase which we estimate at 50-60% of AD293 cells compared to

    1. Just to clarify, the WDR62-HA lane (third in each set) in Figure 1C is not WDR62-BirA*-HA and that it is only being used as a control.

    Response: This is correct. To improve clarity, we have amended the labels on the WDR62-HA lanes in Figure 1C to say “WDR62-HA only”.

    1. In the Discussion (lines 439-441) "We also show that WDR62 forms dynamic, phase-separated granules that co-localise with chaperones and purine metabolic enzymes, resembling purinosomes." I believe that the authors meant to say co-chaperones instead of chaperones given no microscopy data was presented showing the colocalization of HSP70/90 with WDR62 granules. Please revise.

    __Response: __This sentence (line 473) has been revised as suggested.

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

    Summary:

    The authors provide evidence to reveal the novel functions of WDR62 protein in maintaining the stability and activity of purine metabolic enzymes and overall purine homeostasis. WDR62 interacted with BAG2, and they are recruited to purinosome. WDR62 loss caused accelerated degradation of purine salvage enzyme HPRT, and led to the accumulation of purine nucleotide intermediates.

    While this study is compelling and significant for the field of neurodevelopmental disorders including microcephaly and purine metabolism, there are several concerns that should be addressed before publication.

    __Response: __We thank reviewer #2 for their constructive criticisms and supportive comments noting the statement reinforcing significance of our study in the field. We have made a meaningful and concerted effort to address the reviewer comments with extensive additional experimental work and substantial revision of our manuscript.

    Major comments:

    Although all experiments are conducted using non-neuronal cultured cells, does this phenomenon also occur in neuronal cells?

    __Response: __To address this comment and reviewer concerns regarding the links between WDR62 and HPRT in a neuronal context, we performed in utero-electroporation to determine the effects of HPRT depletion on formation of neocortex in mouse embryos. We electroporated embryonic day 14 (E14) mouse brains with siRNA targeting Wdr62, and Hprt and assessed neural progenitor proliferation, migration and differentiation using immunofluorescence. We find that the loss of both WDR62 and HPRT leads to a similar precocious delamination of neural progenitors from the apical ventricular surface (Fig. 7). This process is the first step in neural migration and required to generate a diversity of cells, both self-renewed (eg. outer radial glia) and differentiated neurons and glial cells in the developing neocortex (doi.org/10.1146/annurev-cellbio-101011-155801). Interestingly, we also uncovered that HPRT loss promoted the self-renewal of delaminated intermediate progenitors (IPs) which is unlike impaired the self-renewal of neural progrenitors observed following WDR62 depletion (Fig. 7). Thus, brain development is sensitive to HPRT levels and the HPRT depletion phenocopies WDR62 in cell delamination which supports a neural role for WDR62-HPRT. Moreover, our findings suggest WDR62 loss has more severe neurodevelopmental defects with hints at the complex metabolic functions of WDR62 (discussed in lines 563-577).

    What is the interaction between endogenous WDR62 and Bag2? This is because in overexpression systems, multiple chaperones may interact with the target protein during protein folding.

    Is endogenous WDR62 also present in the purinosome in purine depleted or sorbitol condition?

    __Response: __In response to these comments and similar concerns by reviewer #1, we examined interactions between WDR62, BAG2, HPRT, and PFAS at the endogenous level by utilising proximity ligation assays (PLA, Fig. 4+6). We determined a robust interaction between endogenous WDR62 and BAG2 (Fig. 4K), evident by abundant PLA puncta which were nuclear excluded and localised to the cytosol, consistent with our results in overexpression systems (Fig. 4). We also confirmed endogenous WDR62 interactions with purine enzymes PFAS (Fig. 4K) and HPRT (Fig. 6I) in a similar fashion. To determine whether sorbitol stress promotes their interaction, we assessed changes in the per cell numbers of these puncta in response to sorbitol stress. We confirmed that endogenous WDR62 interaction with HPRT was dependent on BAG2 (Fig. 6J). WDR62-HPRT interactions increased with sorbitol stress (Fig. 6I).

    Regarding Fig6 and Fig7, when HPRT decreases and inosine accumulates in WDR62-KO condition, did the levels of hypoxanthine, xanthine, and uric acid change?

    __Response: __ In Fig. 5G we used an untargeted metabolomics approach that relies on identification databases such as MS-DIAL and associated spectral libraries. Unlike targeted approaches, this method does not always allow for the confident identification of all metabolites of interest. As a result, hypoxanthine, xanthine, uric acid, and other purine intermediates (e.g., AICAR) were not positively identified. This is likely due to limitations in database coverage, spectral similarity to other compounds, or constrains related to our extraction method.

    Does HPRT and the three microcephaly-associated WDR62 mutants also recruited in the purinosome in purine depleted or sorbitol condition?

    __Response: __In response to this, and a similar comment by reviewer #1, we examined whether endogenous HPRT co-localised with WDR62 granules induced by sorbitol. We show that hyperosmotic stress induces the spatial reorganisation of HPRT into puncta that juxtapose and co-localize with WDR62 granules in a stress-dependent manner (Fig. 6H). This was further validated by examining the endogenous WDR62-HPRT interaction using PLA, which also revealed a stress-induced increase upon sorbitol treatment (Fig. 6I).

    As to whether mutant WDR62 was recruited to purinosomes, as detailed in our response to reviewer #1 above (minor comment #6), we find that R438H mutant formed condensed granules prior to stress treatment while 3936dupC mutant did not form granules in response to stress. Therefore, MCPH mutations appear to disrupt the stress-induced formation of WDR62 granules in the cytoplasm. Since we also find that WDR62 KO did not prevent stress-induced formation of PFAS and PPAT granules, which may represent or overlap with purinomes, we chose to not include our findings on granule localization of mutant WDR62 localization in our current revised manuscript. We instead focused on rescue of HPRT and BAG2 levels with patient-derived MCPH mutant variants of WDR62. We confirmed that, unlike WT WDR62, mutant WDR62 could not fully return HPRT or BAG2 levels in WDR62 KO cells (Fig. 6B).

    In Fig7C, HPRT/tubulin ratio appears to decrease in WT from 0hr to 24h, but the graph does not show this decrease. Additionally, quantification of PFAS(FGAMS) and BAG2/tubulin should be performed.

    Response: While slight variations in HPRT signals are visible from 0 h to 24 h in the representative blot, quantification across the n = 9 biological replicates do not support a significant decrease, with these variations falling within the SEM shown in the graph. This representative blot was selected for its clarity and since it most clearly depicts the key trend which is the increasing difference in the HPRT/Tubulin ratio between WT and KO cells with increased duration of CHX treatment. Additionally, in response to this comment, we have now quantified PFAS and BAG2/Tubulin and have inserted these data into Fig. 6C.

    Fig7D is problematic. HPRT in WDR62-KO cells seems to localize in the nucleus, possibly due to stronger exposure in KO conditions compared to WT. Also, the line scan is drawn in areas with low signal in WT. The comparison should be performed in areas with high perinuclear signal.

    __Response: __We appreciate the reviewer’s feedback and acknowledge their concern of an apparent differences in fluorescence intensity in WDR62 KO vs WT cells. In the original submission, slight differences in fluorescence intensity between the WT and WDR62 KO panels may have exaggerated differences in HPRT levels in the nucleus. To address this, we have replaced the representative images with those with more consistent fluorescence intensity across conditions and better represent the average population of sampled cells. Regardless, quantified the change in HPRT cytoplasmic redistribution in response to WDR62 loss across multiple independent biological replicates (n=4) and multiple cells (>12 cells per repeat) within each biological replicate to confirm a change in HPRT distribution in KO cells (Fig. 6E+G).

    The localization of HPRT should be compared in WT and WDR62-KO with BAG2 siRNA. It is also possible to confirm whether the phenotypes observed in KO, such as cell proliferation and xanthosine/inosine levels, are rescued.

    __Response: __We conducted a series of immunofluorescence experiments to assess the impact of BAG2 knockdown (siRNA) on the spatial distribution of HPRT in WT and WDR62 KO cells. BAG2 depletion had no effect on HPRT distribution and did not rescue its aggregated-like appearance in WDR62 KO cells (Fig. S10C). Thus, while abnormal HPRT localization in absence of WDR62 was due to excessive of HSP70/90 activity (Fig. 6F), this was not reversed by BAG2 siRNA. However, BAG2 siRNA reduced BAG2 levels to below wild-type cells (Fig. 6I). An imbalance of HSP and co-chaperone levels are known to be involved in aggregation of cytoplasmic proteins. (doi.org/10.1096/fj.202002645R). Therefore, while BAG2 siRNA may have returned HPRT levels, it may not have appropriately corrected the levels of HSP70/90 activity required for normal HPRT localization (lines 407-413 in revision).

    We did not attempt to rescue cell proliferation and xanthosine/inosine levels with BAG2 siRNA in order to prioritize other studies requested by reviewers such as neurodevelopment function of HPRT and flux analysis of purine synthesis/salvage.

    It should be considered that the induction of Bag2 in WDR62-KO might allow purinosome formation to proceed normally due to compensation. The co-localization of WDR62 and purine enzymes during purinosome formation should be compared when BAG2 expression is suppressed. Similarly, any changes in BAG2 localization in WDR62-KO should be examined. Furthermore, the purinosome formation ability should be compared in WDR62KO + Bagl2 siRNA condition.

    __Response: __To address these insightful comments and requests by reviewer #2 response, we have performed additional experiments to assess whether BAG2 facilitates WDR62 granule assembly, purinosome assembly, and the WDR62-HPRT interaction. siRNA-mediated BAG2 depletion did not prevent stress-induced assembly of WDR62 or PFAS granules (Fig. S6D+E). Thus, unlike HSP70/90 activity, purinosome assembly and WDR62 localization to purinosomes did not appear to require BAG2. Rather we demonstrated a role for WDR62-BAG2 in regulating HPRT (Fig. 6, lines 400-411).

    The reduction of HPRT in WDR62-KO should be examined for potential effects of enhanced degradation via the ubiquitin-proteasome system or the autophagy-lysosome system.

    __Response: __See our response to reviewer #1, minor comment #3. Briefly, neither MG132 blockade of proteosomal degradation nor chloroquine inhibition of autophagy was sufficient to return HPRT levels in WDR62 KO cells. However, these studies are not exhaustive and we are currently pursuing alternative and more specific inhibitors of UPS or lysosomal degradation. As this is not essential for the main findings of the current manuscript, we will include delineation of HPRT degradation pathway in a future publication.

    Although it is known that HPRT-KO mice do not exhibit any effects on normal brain development except in some dopaminergic neurons, what are your thoughts on this?

    Response: We thank the author for raising this interesting point. While global HPRT KO mice appear not to exhibit widespread brain development defects (doi: 10.1007/s00018-022-04326-x) this does not preclude a role for impaired HPRT to contribute to specific neurodevelopmental defects in context of WDR62 mutation or loss. In utero electroporation studies, we find that WDR62 or HPRT depletion results in precocious delamination of apical precursors which may trigger premature differentiation. However, while WDR62 depletion led to reduced proliferation of delaminated radial glia ventricular/subventricular zone, we observed increased proliferation with HPRT loss (Fig. 7). Our findings are in good concordance with the study mentioned by reviewer #2, Witteveen et al. 2022 (doi: 10.1007/s00018-022-04326-x), who similarly reported an increase in proliferation and abnormal cell migration patterns which may be attributed to apical delamination of radial glia. The increased proliferation of progenitors in the intermediate zone or outer ventricular/subventricular zone may compensate for premature differentiation of apical progenitors to explain the lack of overall reduction in brain size with HPRT deficiency alone. Thus, our findings indicate that defects in WDR62-HPRT may contribute to the premature apical delamination of radial glia but WDR62 has additional functions that are indispensable for normal brain development. This may include complex functions in regulating purine metabolism independent of HPRT. We have now included the paper by Witteveen et al. 2022 in our revised manuscript and the above was discussed in detail at lines 565-577.

    Minor comments: • Please write the full name before the abbreviation of the gene. • There is no measurement data for Fig7C, and a measurement line is drawn only in one panel of the ROI. • The line 488 "Fig11" looks like a typo.

    __Response: __As requested by the reviewer, we have included the full name of genes before their abbreviation and corrected the typographical error (line 548 in revision). For Fig S7C (Fig. S6B in revision), we have removed the measurement line which was included in error in our original manuscript. This supplementary figure demonstrates that the stress-stimulated granule assembly of ectopically expressed PFAS and PPAT was not altered or appreciably different in WDR62 KO cells. We quantified this for sorbitol treatment (Fig S6A). We performed the purine-depletion experiment twice with identical results. Given this was a negative result we focused our efforts elsewhere.

    The table could not be found.

    __Response: __We apologise for this oversight. The Supplementary Information file containing Tables S1-3 was excluded from the original submission has now been included in our revised submission.

    It is strange that all measurement values for WT or control in Fig2, Fig7, and FigS9 are exactly 1.0 without any variation. Please check the measurement method again.

    __Response: __Our densometric band measurements in western blots within the indicated figures are normalized against WT control cells as a reference condition. This removes variation in arbitrary densitometric values that changes from blot to blot even for identical samples. Thus, values are fold-change in protein levels relative to WT control conditions. Hence values for WT or control cells are 1 (no change relative to itself) as the reference points and there is no variation between replicate experiments. We apologize for not explaining this in our original submission. Our revision now describes this quantification and processing of raw data in methods and materials (lines 668-671).

    Please write the method for purine depleted medium.

    __Response: __Our revised manuscript includes description of how we depleted cells of purines in the Materials & Methods at lines 636-640 with reference to source materials and prior studies.

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

    Summary:

    In the present work, authors describe a novel role of the microcephaly associated protein WDR62 in purine metabolism under cell stress conditions. In the proposed cellular model (AD293 WDR62 overexpression system), the WDR62 proximity binding partners are firstly identified and categorized according to their functional role in the cell (protein folding, purine metabolism, and stress granules). Among them, authors focus on BAG2 - a HSP70/90 co-chaperone involved in cellular stress responses. After the characterization of the WDR62-BAG2 physical interaction sites, suggested to be disrupted by WDR62 pathogenic mutations, their functional interaction in cellular stress responses is investigated. WDR62-associated granules are extensively characterized for their physical and dynamic properties under different conditions (i.e., hyper-osmotic stress). Further, through the evaluation of N-and C-terminally truncated form of WDR62 authors characterize the protein regions responsible for WDR62-containing granule condensation - suggesting a potential mechanism disruption in the event of pathological WDR62 mutations. Lastly, authors provide evidence that WDR62 condensation does not occur in canonical stress granules but in the so called-purinosomes, where it participates in the regulation of purine metabolic pathway stabilizing HPRT (purine salvage enzyme) via WDR62-BAG2-HSP70/90 axis.

    Major comments:

    Overexpression system and the employed cell line are a major limitation of the study. There is no experimental data on human neural cells and on endogenous WDR62, underestimating the potential difference in cell type-specific metabolism. In light of this consideration, the provided introduction and conclusions on neural development and microcephaly have to be re-formulated. I suggest providing a more general introduction/conclusions on WDR62 role (and alterations) in cell division and cell metabolism (neurodevelopment and cancer share common patterns) since purine homeostasis is not exclusive of neural progenitor cells.

    This reviewer thinks that the structure of the work is a bit convoluted (too many results in main figures that are not substantial). I suggest to re-organize and to prioritize the most relevant results. Further, it would be clinically relevant to add WDR62 mutant constructs in the functional evaluation of purine metabolism to better dissect the physiological role of WDR62 and the impact of the mutations on cellular physiology.

    Response: We are appreciative for this constructive evaluation of our manuscript and frank comments on the limitations of our study from reviewer #3. We have now included extensive new studies that provide evidence supporting endogenous mechanisms and insights into in vivo functions in neurodevelopment. We have also removed and combined several figures relating to the stress-induced purinosome assembly of WDR62 to better focus our manuscript on WDR62 interaction mechanisms and their purine metabolic function.

    Fig. 1: Overexpressed WDR62 fluorescence signal might be artifactual and may hide more detailed localization pattern during interphase. Authors should also provide endogenous WDR62 immunofluorescence panel in AD293 cells. Additionally, the "cytosolic" localization of WDR62 during interphase (indicated in the introduction, lines 88-89) has been re-defined in recent works pointing out that the protein is dynamically associated with the interphasic centrosome, the Golgi apparatus, and spindle poles during mitosis.

    __Response: __In response to this point, we have added text in the introduction (line 100-102) to clarify the dynamic association of WDR62 in cytoplasmic compartment during interphase includes the golgi apparatus. We have also added reference to the study by Dell’Amico and co-workers (doi: 10.7554/eLife.81716, Ref #24 in revision) alluded to by reviewer #3.

    We utilized ectopic expression of tagged WDR62 constructs to determine redistribution to stress-responsive cytoplasmic granules and co-localization with purine enzymes. Immunofluorescence staining of endogenous WDR62 also appears to reveal granule assembly but these findings are not as clear as the primary antibodies also detect additional proteins independent of WDR62 (validated using our KO cells). We agree that protein overexpression may result in artificial localization patterns but this can be mitigated with careful controls. We find that stress-induced WDR62 granule localization is highly dynamic and reversible. We observe the same response with full-length protein using different fluorescent protein or small affinity tags at either N- or C-terminus. High expression of mutant WDR62 (e.g. 3936dupC) or a closely related family member (MAPKBP1) do not form the same purinosome-associated granules. Moreover, in response to related comments by reviewer #1 and #2, we have now included proximity ligation assays confirm interactions between WDR62, BAG2 and purine enzymes (Fig. 3 and Fig. 6).

    Fig. 1C lacks quantification of BAG2/CEP170/AURKA signal. Further, how can authors exclude that is not nocodazole effect on microtubules disruption which impairs WDR62 spindle pole localization and therefore protein-protein interactions? A panel showing that "low dose" nocodazole do not impinge endogenous and exogenous WDR62 localization in mitotic cells is needed.

    __Response: __WDR62-BirA specific biotinyation and affinity isolation of BAG2, CEP170 and AURKA, compared to BirA or WDR62-HA only controls, was very clear in Fig. 1C. We did not quantify the extent that mitotic synchronization increased or decreased binding to WDR62 as the mitosis specific context was not a focus in our subsequent figures. Rather we focused on and quantified in detail WDR62-BAG2-HPRT mechanisms in response to cell stress.

    We are also very confident that low dose nocodazole treatment does not prevent spindle pole localization. This treatment impinges on microtubule dynamics to trigger spindle checkpoints, arresting cells in mitosis. The bipolar organization of spindles is lost but spindle microtubules and minus-end microtubule directed localization of WDR62 at spindle asters are retained under these conditions and is specific to mitotic cells. The robust WDR62-BirA biotinylation of AURKA, which is spindle pole-associated, specifically in mitotic arrested cells further confirms WDR62 is retained at the spindle. We demonstrated this in our previous papers (Ref. 5+6). Others have also shown that both endogenous (doi: 10.7554/elife.81716) and exogenous WDR62 (doi: 10.1083/jcb.202007167, doi: 10.1242/jcs.157537) retain spindle pole localisation under similar conditions.

    Fig. 3 H-J: The fluorescence signals are saturated (also evident in the intensity profile plot) and thus not applicable for any analysis. Further, how these linear ROIs are chosen? The signal pattern is not homogenously distributed in those images. Please provide a more consistent fluorescence analysis.

    __Response: __We acknowledge reviewer #3 concerns but while some granules, particularly those expressing G3BP-EGFP, exhibit saturated fluorescence signals, this does not impact or prevent our analysis. Our aim was not to quantify subtle fluorescence intensity changes within individual granules, but rather to compare fluorescence signal between granules across different channels to identify overlap. The linear ROIs were selected at random to illustrate that WDR62 and G3BP signals do not overlap between WDR62 and G3BP-positive granules.

    Minor comments:

    Abstract, line 49: How can these WDR62 mutations can result in a complete loss of the protein ("In cells lacking WDR62") if authors report co-IP experiments (Fig. 2) with clear mutant WDR62 bands? Rephrase accordingly.

    __Response: __The statement in our original abstract referenced by reviewer #3 referred to results presented in Fig 7 (now Fig. 6 in our revision) comparing WDR62 KO with WT cells and not co-IP experiments with mutant WDR62 in Fig 2. We have revised our abstract substantially to incorporate additional experimental work and to ensure clarity in our statements related to KO cells lacking WDR62 and cells expressing WDR62 mutants.

    Result referred to Fig. 2D reports that "BAG2 co-immunoprecipitated with WDR62(N)-EGFP but not WDR62(C)-EGFP". The blot and the relative quantification in figure 2D instead show BAG2 signal in the WDR62(C)-EGFP - even if significantly lower. Please rephrase accordingly.

    __Response: __We have revised line 192 of the main text to more accurately state the reduced interaction between WDR62(C)-EGFP and BAG2.

    Lines 186-187: authors declare that the C-terminal tail comprising the helix-loop-helix domain is required for BAG2 to bind full-length WDR62. There are no such data in support of this. The C-terminal fragment includes both the disordered region and the dimerization domain. How can authors conclude that the dimerization domain alone is sufficient to bind BAG2?

    __Response: __In Fig. 2, we show that the co-IP of BAG2 was significantly impaired in cells expressing WDR62(3936dupC), which lacks the C-terminal helix-loop-helix (HLH) domain. Additionally, we demonstrate that the C-terminal half of WDR62, which includes the HLH domain, does not bind BAG2. Based on these findings, we conclude that while the HLH domain is necessary for BAG2 binding to full-length WDR62, it is alone not sufficient. To ensure clarity, we have revised the main text (lines 207-209) to state “…the C-terminal helix-loop-helix domain—required for WDR62 dimerisation—is necessary but not sufficient for BAG2 to bind full-length WDR62.”

    Lines 189-190: results in AD293 cell line are not directly applicable in demonstrating that poor WDR62-BAG2 interaction can lead to alterations in brain development. Please rephrase.

    __Response: __We established that WDR62 interacts with BAG2 co-chaperone and MCPH mutations in WDR62 disrupt this interaction. Although our results were performed in AD293 cells, it seemed reasonable to speculate that WDR62 interactions with chaperones might contribute to brain development given well established WDR62 functions in this context. However, we acknowledge that this speculation may not be appropriate at this point of the manuscript, so we have removed this text (line 210) in our revised manuscript.

    Line 196: Indicate here, as the first mention, stress granules as "SGs" and use the abbreviation consistently throughout the manuscript.

    __Response: __We have abbreviated stress granules as suggested (first mentioned at line 102) and utilized this abbreviation consistently throughout the manuscript.

    Line 255: are human neural progenitor cells enough sensitive to sorbitol? If not, the proposed experimental design is a bit artifactual and the results/conclusions cannot be related to neural development alterations. I suggest applying more "physiological" stressors and frame the results in meaningful neurodevelopmental/tumorigenic environment. Please add this point to the discussion.

    __Response: __Neural progenitors are likely sensitive to sorbitol, as hyperosmotic stress has been used to induce phase separation of a wide variety of proteins in neural contexts (doi: doi.org/10.1038/s41598-023-39090-w, doi.org/10.1016/j.celrep.2018.06.094). In this study, we leveraged sorbitol-induced hyperosmotic stress as a controlled and reproducible means of triggering WDR62 phase separation, enabling us to examine its downstream interactions with BAG2, HPRT, and other purine enzymes. We further extend these observations to metabolic cell stress with purine-depletion.

    We found that WDR62 phase separation occurs rapidly at low sorbitol concentrations (~50 mM) (Fig. 3B), suggesting that even milder osmotic stress, particularly under prolonged exposure, could similarly drive WDR62 condensation in physiological settings. As requested by the reviewer, we have added a small section to the discussion (lines 472-480) to discuss the physiological implications of sorbitol stress on WDR62 granule assembly.

    Line 240: WDR62 granules association with microtubules and especially mitochondria is not convincing (Fig. S5). This data seems to be a bit qualitative, please provide more detailed quantification of this parameter.

    __Response: __The association of WDR62 granules with microtubules and mitochondria is quantitatively assessed using two methods, as shown in the graphs to the right of the images. One graph presents the proportion of WDR62 granules overlapping with CytC/Tubulin, providing a binary measure of colocalization. We also examined the degree of signal correlation across the entire ROI by calculating Pearson’s correlation coefficient. In response to sorbitol, we showed a higher association of WDR62 with Tubulin and CytC compared to randomised controls. We have updated the Materials and Methods to include a detailed description of this analysis (lines 708-720).

    Fig. 4 is convoluted. I suggest moving some data to supplementary to improve the clarity of the figure.

    __Response: __In addressing this comment and related comments from other reviewers to focus our manuscript, we have removed our data on fluorescence recovery and post-stress disassembly of WDR62 granules from what was Fig. 4 in our original submission and combined remaining components with Fig. 3 to centre on stress-induced assembly of WDR62 granules for our revised manuscript.

    Line 273: "Liquid-like protein condensates also exchange their contents with the bulk cytosol [52]". Reference 52 reviews the existing literature referred to biomolecular condensates that exert nuclear function (e.g., genome organization, gene expression, and DNA repair). No mention on events involving cytoplasm. Please add a more relevant reference.

    __Response: __We thank the reviewer for highlighting this inconsistency. However, this reference is no longer required and has been removed from our revised manuscript as the section of the main text has been deleted in alignment with the above response where figure panels relating to WDR62 phase separation were removed for focus and clarity.

    Lines 290-291: have authors considered the effect of sorbitol on microtubules dynamic that might reflect in granules dynamic changes?

    __Response: __We thank the authors for this insightful comment. Hyperosmotic stressors such as sorbitol are known to reduce microtubule dynamicity (doi.org/10.1016/j.devcel.2022.02.001), likely due to increased cytoplasmic viscosity and crowding effects. While we have not directly assessed microtubule dynamics in our study, it is certainly possible that these changes could influence WDR62 granule dynamics, given their association with microtubules (Fig. S6). While we have reduced emphasis on the dynamic nature of WDR62 granules in our revision, a useful direction for future studies to explore how alterations in microtubule dynamics induced by physiological stressors facilitate changes in WDR62 granule assembly or dynamics (e.g., fission, fusion).

    Line 295: I suggest moving the prediction of the disordered region of WDR62 when first mentioned (e.g., Supplement referred to Fig. 2)

    __Response: __This text is no longer required as we have removed this dataset from our revised manuscript to address reviewer consensus feedback to enhance cohesiveness and clarity.

    Fig. S6C-E, I: Unclear which is the criterion by which a cell is marked as "with" or "without" granules.

    __Response: __This text is no longer required as we have removed this dataset from our revised manuscript to address reviewer consensus feedback to enhance cohesiveness and clarity.

    Fig. S8: Unclear, also from the micrograph showed in the figure, how authors have counted/considered the microtubules/mitochondria associated purinosomes. Seems very qualitative and observer dependent. Please provide a more reliable analysis.

    __Response: __We apologise for omitting a description of the methodology used in the analysis of the images in Fig. S8 (now Fig. S6 in revision). We have now provided a detailed description in the Materials and Methods section (lines 709-721) of how microtubule- and mitochondria-associated purinosomes were identified and quantified.

    Fig. 6A: The same blot of WDR62 KO is shown in Fig. S7. Please remove one.

    __Response: __As requested, we have removed a set of blots demonstrating WDR62 protein deletion in KO cells from Fig. S7 (Fig. S6 in revision).

    Fig. 6C, D: Method for cell proliferation measure is indirect and "rounded cells" as indicator of cell death is sub-optimal. Analysis with specific markers would be preferable in both cases.

    Response: We used an XTT assay to measure cell viability as a function of cell number. In revised text, and also detailed in our response to reviewer #1 (point 4 under Text and Figures), we clarified that this was a measure of cell viability in response to purine-depletion as oppose to a direct measure of cell proliferation. Our amended text attributes the results in Fig 6C (now Fig. 5B in revision) to changes in cell viability rather than proliferation.

    With regards to additional measure of cell death, we had also performed LDH release assays to quantify cell death in addition to our measurement of cell rounding. The LDH assay is widely used and accepted measure of cell death or cytotoxicity and is indicated in Figure 5D in the revision.

    Fig.7B: Why the transfection control vector "EGFP only" significantly increases/decreases the BAG2/HPRT expression with respect to the negative control?

    __Response: __The reviewer comment here on Fig. 7B (now Fig. 6B in revision) refers to the control vector (EGFP only) transfected into WDR62 KO cells, as opposed to WT cells. Therefore, the difference in protein expression in this condition does not match the WT cells in the first lane as BAG2 and HPRT are increased and decreased respectively in KO cells compared to WT. This aligns with results presented in Fig. 6A.

    Paragraph from line 410 to 434: very confusing, the reported results are not well conveyed and therefore not convincing. To be reformulated.

    __Response: __We thank the reviewer for the direct and constructive feedback. The revised section (lines 378–416) addresses whether WDR62-BAG2 regulates HPRT levels. It has been substantially updated to include new experimental data and to reflect our latest findings and conclusions. We believe these revisions have significantly improved the logical flow and clarity of the discussion.

    Lines 524-526: the author's conclusion that: "...the loss of purine metabolic enzymes, including HPRT, disrupts neurogenesis, resulting in microcephaly, cell cycle defects, ciliopathies, and abnormalities in proliferation and neural progenitor fate decisions, mirroring the loss of WDR62." is not supported by the cited literature [29] and by the results presented in this work. Please provide additional references or remove the statement.

    __Response: __ As requested by the reviewer, we have removed the statement and substantially revised this section of the discussion (lines 563-677) to incorporate findings from our additional studies such as in utero electroporation.

    Lines 527-529: if authors state that "...other WD repeat-containing and microcephaly-associated proteins interact with purine enzymes..." have to provide additional references in addition to the NWD1 one. Otherwise, these lines should be rephrased as "another WD repeat containing and microcephaly-associated protein...".

    __Response: __We have amended this statement (line 589-592 in revision) as requested.

    Reference 62 is not well indexed in the Reference section. Please adjust.

    __Response: __We thank the reviewer for pointing out this error. The reference to Rauch et al. (2014) [Ref. 60 in the revised manuscript] has been corrected and now includes the complete bibliographic details.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In the present work, authors describe a novel role of the microcephaly associated protein WDR62 in purine metabolism under cell stress conditions. In the proposed cellular model (AD293 WDR62 overexpression system), the WDR62 proximity binding partners are firstly identified and categorized according to their functional role in the cell (protein folding, purine metabolism, and stress granules). Among them, authors focus on BAG2 - a HSP70/90 co-chaperone involved in cellular stress responses. After the characterization of the WDR62-BAG2 physical interaction sites, suggested to be disrupted by WDR62 pathogenic mutations, their functional interaction in cellular stress responses is investigated. WDR62-associated granules are extensively characterized for their physical and dynamic properties under different conditions (i.e., hyper-osmotic stress). Further, through the evaluation of N-and C-terminally truncated form of WDR62 authors characterize the protein regions responsible for WDR62-containing granule condensation - suggesting a potential mechanism disruption in the event of pathological WDR62 mutations. Lastly, authors provide evidence that WDR62 condensation does not occur in canonical stress granules but in the so called-purinosomes, where it participates in the regulation of purine metabolic pathway stabilizing HPRT (purine salvage enzyme) via WDR62-BAG2-HSP70/90 axis.

    Major comments:

    Overexpression system and the employed cell line are a major limitation of the study. There is no experimental data on human neural cells and on endogenous WDR62, underestimating the potential difference in cell type-specific metabolism. In light of this consideration, the provided introduction and conclusions on neural development and microcephaly have to be re-formulated. I suggest providing a more general introduction/conclusions on WDR62 role (and alterations) in cell division and cell metabolism (neurodevelopment and cancer share common patterns) since purine homeostasis is not exclusive of neural progenitor cells.

    This reviewer thinks that the structure of the work is a bit convoluted (too many results in main figures that are not substantial). I suggest to re-organize and to prioritize the most relevant results. Further, it would be clinically relevant to add WDR62 mutant constructs in the functional evaluation of purine metabolism to better dissect the physiological role of WDR62 and the impact of the mutations on cellular physiology.

    Fig. 1: Overexpressed WDR62 fluorescence signal might be artifactual and may hide more detailed localization pattern during interphase. Authors should also provide endogenous WDR62 immunofluorescence panel in AD293 cells. Additionally, the "cytosolic" localization of WDR62 during interphase (indicated in the introduction, lines 88-89) has been re-defined in recent works pointing out that the protein is dynamically associated with the interphasic centrosome, the Golgi apparatus, and spindle poles during mitosis.

    Fig. 1C lacks quantification of BAG2/CEP170/AURKA signal. Further, how can authors exclude that is not nocodazole effect on microtubules disruption which impairs WDR62 spindle pole localization and therefore protein-protein interactions? A panel showing that "low dose" nocodazole do not impinge endogenous and exogenous WDR62 localization in mitotic cells is needed.

    Fig. 3 H-J: The fluorescence signals are saturated (also evident in the intensity profile plot) and thus not applicable for any analysis. Further, how these linear ROIs are chosen? The signal pattern is not homogenously distributed in those images. Please provide a more consistent fluorescence analysis.

    Minor comments:

    Abstract, line 49: How can these WDR62 mutations can result in a complete loss of the protein ("In cells lacking WDR62") if authors report co-IP experiments (Fig. 2) with clear mutant WDR62 bands? Rephrase accordingly.

    Result referred to Fig. 2D reports that "BAG2 co-immunoprecipitated with WDR62(N)-EGFP but not WDR62(C)-EGFP". The blot and the relative quantification in figure 2D instead show BAG2 signal in the WDR62(C)-EGFP - even if significantly lower. Please rephrase accordingly.

    Lines 186-187: authors declare that the C-terminal tail comprising the helix-loop-helix domain is required for BAG2 to bind full-length WDR62. There are no such data in support of this. The C-terminal fragment includes both the disordered region and the dimerization domain. How can authors conclude that the dimerization domain alone is sufficient to bind BAG2?

    Lines 189-190: results in AD293 cell line are not directly applicable in demonstrating that poor WDR62-BAG2 interaction can lead to alterations in brain development. Please rephrase.

    Line 196: Indicate here, as the first mention, stress granules as "SGs" and use the abbreviation consistently throughout the manuscript.

    Line 255: are human neural progenitor cells enough sensitive to sorbitol? If not, the proposed experimental design is a bit artifactual and the results/conclusions cannot be related to neural development alterations. I suggest applying more "physiological" stressors and frame the results in meaningful neurodevelopmental/tumorigenic environment. Please add this point to the discussion.

    Line 240: WDR62 granules association with microtubules and especially mitochondria is not convincing (Fig. S5). This data seems to be a bit qualitative, please provide more detailed quantification of this parameter.

    Fig. 4 is convoluted. I suggest moving some data to supplementary to improve the clarity of the figure.

    Line 273: "Liquid-like protein condensates also exchange their contents with the bulk cytosol [52]". Reference 52 reviews the existing literature referred to biomolecular condensates that exert nuclear function (e.g., genome organization, gene expression, and DNA repair). No mention on events involving cytoplasm. Please add a more relevant reference.

    Lines 290-291: have authors considered the effect of sorbitol on microtubules dynamic that might reflect in granules dynamic changes?

    Line 295: I suggest moving the prediction of the disordered region of WDR62 when first mentioned (e.g., Supplement referred to Fig. 2)

    Fig. S6C-E, I: Unclear which is the criterion by which a cell is marked as "with" or "without" granules.

    Fig. S8: Unclear, also from the micrograph showed in the figure, how authors have counted/considered the microtubules/mitochondria associated purinosomes. Seems very qualitative and observer dependent. Please provide a more reliable analysis.

    Fig. 6A: The same blot of WDR62 KO is shown in Fig. S7. Please remove one.

    Fig. 6C, D: Method for cell proliferation measure is indirect and "rounded cells" as indicator of cell death is sub-optimal. Analysis with specific markers would be preferable in both cases.

    Fig.7B: Why the transfection control vector "EGFP only" significantly increases/decreases the BAG2/HPRT expression with respect to the negative control?

    Paragraph from line 410 to 434: very confusing, the reported results are not well conveyed and therefore not convincing. To be reformulated.

    Lines 524-526: the author's conclusion that: "...the loss of purine metabolic enzymes, including HPRT, disrupts neurogenesis, resulting in microcephaly, cell cycle defects, ciliopathies, and abnormalities in proliferation and neural progenitor fate decisions, mirroring the loss of WDR62." is not supported by the cited literature [29] and by the results presented in this work. Please provide additional references or remove the statement.

    Lines 527-529: if authors state that "...other WD repeat-containing and microcephaly-associated proteins interact with purine enzymes..." have to provide additional references in addition to the NWD1 one. Otherwise, these lines should be rephrased as "another WD repeat containing and microcephaly-associated protein...".

    Reference 62 is not well indexed in the Reference section. Please adjust.

    Referees cross-commenting

    This reviewer thinks that the points raised by reviewer #1 and #2 are very accurate and significant. Some of them are also shared between our three review reports and in general are referred to: clarity of the manuscript improvement, little consistency between the results displayed in the figures and the text/conclusions in some points, concerns about the reliability of some measurements/result and the employed cellular model, and the lack of endogenous protein data.

    Significance

    General assessment:

    The here described new role of WDR62 in purine metabolism and the proposed pathway are novel and relevant to shed light on pathophysiological cellular and molecular mechanisms that potentially underlie neurodevelopmental defects and carcinogenesis - processes in which WDR62 is implicated. The experimental design is extended and generally well-conceived even though quite dispersive in some points.

    The strength of the work resides in its versatility - making these findings potentially applicable to different cell types and different contexts (e.g., from neural development to malignancies) - and in the protein-protein interactions characterization under several conditions.

    Similarly, the major weakness is the generalist trait of the findings that describes WDR62 cellular behavior mostly in an over-expression system in an immortalized cell line, underestimating the intrinsic metabolic and protein expression-level differences among cell types.

    Advance:

    WDR62 is a scaffold protein with pleiotropic functions and a plethora of molecular interactors. Literature reports many molecular pathways involving WDR62 mainly in cell cycle progression, primary cilia biogenesis and centrosomal functions in a neurodevelopmental context. In the present work, authors describe mechanistic insights of a never reported WDR62-BAG2-HSP70/90 molecular pathway shedding new light on the role of this protein in cellular metabolism thus providing a new perspective on WDR62 pathophysiological functions.

    Audience:

    Basic research audience will be interested in this research work. The described molecular pathway involving WDR62 in purine metabolism might be relevant to other research on how WDR62 cellular and molecular dynamics are impactful on neural development and malignancies.

    Expertise:

    Human neural development and alterations, iPSCs, neural stem cells, CRISPR-Cas9

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    Referee #2

    Evidence, reproducibility and clarity

    Summary: The authors provide evidence to reveal the novel functions of WDR62 protein in maintaining the stability and activity of purine metabolic enzymes and overall purine homeostasis. WDR62 interacted with BAG2, and they are recruited to purinosome. WDR62 loss caused accelerated degradation of purine salvage enzyme HPRT, and led to the accumulation of purine nucleotide intermediates.

    While this study is compelling and significant for the field of neurodevelopmental disorders including microcephaly and purine metabolism, there are several concerns that should be addressed before publication.

    Major comments:

    • Although all experiments are conducted using non-neuronal cultured cells, does this phenomenon also occur in neuronal cells?
    • What is the interaction between endogenous WDR62 and Bag2? This is because in overexpression systems, multiple chaperones may interact with the target protein during protein folding.
    • Is endogenous WDR62 also present in the purinosome in purine depleted or sorbitol condition?
    • Regarding Fig6 and Fig7, when HPRT decreases and inosine accumulates in WDR62-KO condition, did the levels of hypoxanthine, xanthine, and uric acid change?
    • Does HPRT and the three microcephaly-associated WDR62 mutants also recruited in the purinosome in purine depleted or sorbitol condition?
    • In Fig7C, HPRT/tubulin ratio appears to decrease in WT from 0hr to 24h, but the graph does not show this decrease. Additionally, quantification of PFAS(FGAMS) and BAG2/tubulin should be performed.
    • Fig7D is problematic. HPRT in WDR62-KO cells seems to localize in the nucleus, possibly due to stronger exposure in KO conditions compared to WT. Also, the line scan is drawn in areas with low signal in WT. The comparison should be performed in areas with high perinuclear signal.
    • The localization of HPRT should be compared in WT and WDR62-KO with BAG2 siRNA. It is also possible to confirm whether the phenotypes observed in KO, such as cell proliferation and xanthosine/inosine levels, are rescued.
    • It should be considered that the induction of Bag2 in WDR62-KO might allow purinosome formation to proceed normally due to compensation. The co-localization of WDR62 and purine enzymes during purinosome formation should be compared when BAG2 expression is suppressed. Similarly, any changes in BAG2 localization in WDR62-KO should be examined. Furthermore, the purinosome formation ability should be compared in WDR62KO + Bag2 siRNA condition.
    • The reduction of HPRT in WDR62-KO should be examined for potential effects of enhanced degradation via the ubiquitin-proteasome system or the autophagy-lysosome system.
    • Although it is known that HPRT-KO mice do not exhibit any effects on normal brain development except in some dopaminergic neurons, what are your thoughts on this?

    Minor comments:

    • Please write the full name before the abbreviation of the gene.
    • There is no measurement data for Fig7C, and a measurement line is drawn only in one panel of the ROI.
    • The line 488 "Fig11" looks like a typo.
    • The table could not be found.
    • It is strange that all measurement values for WT or control in Fig2, Fig7, and FigS9 are exactly 1.0 without any variation. Please check the measurement method again.
    • Please write the method for purine depleted medium.

    Referees cross-commenting

    I concur with the accurate point observations by the other reviewers. The authors should address the most of the comments provided, as many of the suggested experiments are feasible. If the paper aims to elucidate the one of the causes of microcephaly, specifically, the issues related to cell type and endogenous proteins experiments need to be resolved, and addressing these issues would substantially enhance its quality and impact.

    Significance

    Most of the roles of purinosomes in the central nervous system remain unknown. The discovery that the WDR62/MCPH2 gene, responsible for microcephaly, is related to purinosomes will have a major impact on this field. Additionally, the ability to easily induce purinosomes through sorbitol phase separation is a significant technical advance in terms of cost and simplicity. Furthermore, many genes related to microcephaly, such as MCPH, are factors directly involved in cell division by regulating the mitotic spindle and centrosomes. This study has revealed a new role for WDR62, uncovering part of a novel molecular mechanism for microcephaly.

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    Referee #1

    Evidence, reproducibility and clarity

    In Morris, M.J. et al., the authors strive to better understand the roles for the microcephaly protein WDR62 in brain growth and function. To accomplish this, an in situ biotinylation assay was performed and identified 42 proteins proximal to WDR62 including the Hsp70 co-chaperone BAG2. Through a series of co-immunoprecipitation assays, the BAG2-WDR62 interaction was shown to be mediated through the structured N-terminal region of WDR62, and it was proposed that common WDR62 mutations disrupt this interaction. In AD293 cells, loss of WRD62 expression resulted in an increase in the expression of BAG2 expression while reducing HPRT expression. Subsequent loss of BAG2 expression by siRNA treatment restored the expression of HPRT suggesting that there is an association between the stability of HPRT and BAG2, likely mediated through its proposed association with Hsp70/90 molecular chaperones. Finally, the authors investigate the subcellular localization and ability of WRD62 to phase separate. WRD62 was shown to form discrete condensates induced by sorbitol-mediated hyperosmotic stress. The formation of WRD62 granules are hypothesized to be driven by cell volume exclusion and macromolecular crowding. These granules appear similar, both in physical appearance and characteristics, to other reported biomolecular condensates such as those reported in metabolism (e.g. purinosomes). WRD62-containing condensates were shown to colocalize with enzymes in de novo purine biosynthesis; however, this association is not required for purinosome formation and/or its stability under both purine-depleted and sorbitol-driven growth conditions. Overall, the manuscript provided a previously unrealized and exciting association between WDR62 and purine metabolism.

    EVIDENCE, REPRODUCIBILITY AND CLARITY

    Summary: The current manuscript reads as multiple manuscripts with findings that are at times weakly connected (in my opinion). For example, I had a hard time understanding how the BioID results relate to the discovery of WRD62 phase-separation and its colocalization with purinosomes. I would strongly encourage the authors to consider dividing the results into separate manuscripts to strengthen their claims and create a more focused and cohesive manuscript (or series of manuscripts). I believe then several of my reservations associated with the current manuscript will be addressed, and in my opinion, the hard work from the authors will be better received across the scientific community.

    I would like to commend the authors for all the work that went into the current version of the manuscript. Being part of a biochemistry and cell biology research group, I completely understand how much time and effort must have went into generating these data. That being said, I felt that there were several instances where clarification and additional information is warranted to arrive at the conclusions made by the authors. These points are outlined below.

    Major Comments:

    1. There appears to be a discrepancy between the data presented in Figure 1 and what is stated in the main text. Clarification is necessary to better understand the results:
      • The following statement (and derivatives of it) are repeated throughout the manuscript: "...we found that the WDR62 interactome comprised molecular chaperones such as HSP70, HSP90, and their co-regulators, BAG2, STIP1, and DNAJC7" (lines 91-93, 316-318, 422-425). STIP1 and DNAJC7 were not identified in the list of 42 proximal proteins to WDR62 (Figure 1D). DNAJC7 was included because of a previous report curated in the BioGRID database, and there is no mention of HSP90 in the data produced in Figure 1. Please revise the main text to reflect the data that was generated.
      • Based on the data presented in the Venn Diagrams in Figure 1D, the author's numbers do not seem to be consistent with the sentence on lines 126-128. I count 37 proteins unique to their BioID study, 90 unique to the BioGRID database, and 5 proteins that overlap between the two data sets. This sentence needs to be revised.
      • What data were used to generate the interaction map in Figure 1I? Enzymes tied to purine metabolism were not identified from the data presented in Figure 1D but have now appeared. A discussion of this in the main text is warranted.
    2. This reviewer has several reservations on how the various key players in the manuscript are related to substantiate the conclusions made in the manuscript. For instance, how is HPRT, purinosomes, and WDR62 related? How about HSP90, WRD62, and HPRT? Pairwise connections were made throughout the manuscript; however, trying to tie all three together is difficult with the data presented.
      • The authors tried to connect HPRT, purinosomes, and WDR62 with BAG2; however, this study could greatly improve if we understood how a knockdown of BAG2 impacts purinosome formation and/or WDR62 colocalization with purinosome enzymes.
      • Is HPRT a client of HSP90? And how are WRD62 and HSP90 related since they do not associated (based on your BioID data)? These connections would again strengthen the arguments made in the manuscript and help to explain the HSP90 inhibition data presented in Figures 7F and 7G.
    3. Caution is warranted when making conclusions about WDR62 (and its granules) and purinosomes.
      • The authors describe the association between WDR62 and purinosomes differently throughout the text. I would recommend that the authors come to some conclusion about this and be consistent.

    A. (Lines 339-340) "WDR62 granules represent or overlap substantially with the phase-separated metabolons known as purinosomes". Based on the data presented, it appears that these might still be different entities but either overlap or have similar components. Purinosome localization with mitochondria (approx 60-80%) and microtubules (approx 90-95%) were significantly higher than those reported for WDR62 granules (approx 40%). This comparison would suggest that not all WDR62 granules behave similarly to purinosomes. And from the dot plot in Figure 3G, about half of the characterized WDR62 granules do not align with the previously reported characteristics of purinosomes.

    B. In the abstract and introduction, the authors state that WDR62 is being recruited to the purinosome and leave out the other possibility. I would recommend that the authors soften this claim in these sections because of the above possibility but also the lack of characterization of the sorbitol-induced "purinosomes". There is little discussion or evidence for how sorbitol induces purinosome formation. Is de novo purine biosynthesis activated upon sorbitol treatment? Are multiple de novo purine biosynthetic enzymes present in the sorbitol-induced "purinosomes"? Further, I agree that there is a tendency for WDR62 to associate with condensates that bear an enzyme within de novo purine biosynthesis; however many of these proteins are known to self-aggregate upon cell stress. Therefore, the entities that are being observing and called purinosomes might not be bone fide purinosomes. Additional care is necessary to make these statements. In my opinion, the current data only suggests that this might be a possibility.

    • (Lines 325-329) The authors reference a previous manuscript demonstrating that co-chaperones co-cluster with purinosomes. Based on this fact, they infer that WDR62 granules might represent purinosomes since WDR62 interacts with these same set of co-chaperones. These co-chaperones interact with a large number of different proteins (in fact, most kinases), so it is uncertain how the authors decided to go down this path to link purine metabolism with WDR62. Discussion of how this connection was made would help elevate the story. What additional insights did they have that lead them down these investigations?
    • If WDR62 is not required for purinosome formation, why would it localize with the purinosome? Is there any hypothesis that could be readily tested to better help understand this observation? Providing a better understanding of this would greatly elevate the work.

    A. (OPTIONAL) Please validate that the associations between WDR62 and the purine biosynthetic enzymes occur on the endogenous level (void of transient transfection). Many methods such as immunofluorescence and proximity ligation assays have been used by others to demonstrate protein-purinosome interactions. This result would reduce any concern that the association is a result of overexpression (artifact).

    B. Figures 6F and 6G conclude that nucleosides from purine-depleted growth conditions accumulate while the corresponding monophosphates do not change between WRD62 knock-out and wildtype cells. Given that purine-depleted growth conditions activate de novo purine biosynthesis (uncertain if this has been demonstrated in AD293 cells), could this result simply demonstrate that purine salvage is no longer used and the nucleosides have accumulated and are awaiting degradation (or exportation) rather than a loss of HPRT expression as inferred from the stated conclusions? The conclusions could be better substantiated with the use of a stable isotope incorporation assay.

    Is there a difference in the contribution of de novo purine biosynthesis and purine salvage to the generation of the monophosphates (AMP, GMP) between WDR62 knockout and wildtype AD293 cells? Use of a stable isotope (such as 15N-glutamine) could help to come to the appropriate conclusion.

    (Lines 483-485) If there is a change in de novo purine biosynthesis, are there any detectable changes in AICAR levels that might influence purine metabolism at the transcriptional level?

    Are the data and the methods presented in such a way that they can be reproduced? Are the experiments adequately replicated and statistical analysis adequate?

    1. For purine-depleted studies (metabolite analyses, microscopy), how long were the cells grown in purine-depleted medium before the analysis? And how was the purine-depleted medium generated? Please reference any source that might have been used.
    2. Details describing the BioID experiment are minimal. How many replicates were performed, was label-free or TMT quantitation used for the protein identification. Further the data analysis and mining of the proteins from the BioID study are missing - What database was used to identify the proteins from the peptides? Please include this information in the Materials and Methods section as well as a link to a repository where the LC-MS/MS data generated can be found. Additionally, it would be very helpful to have a spreadsheet or table that lists the biotinylated proteins and expectant or p values for each.
    3. Please include information about the streptavidin pulldown presented in Figure 1C.
    4. Many of the figure legends could benefit from a statistical description.
    5. There seems to be only two data points for Figure S3A. While there is no significant difference observed, it would be ideal to have additional replicates.
    6. (Figure 5I) Please provide statistical analysis to demonstrate the colocalization between FGAMS and WDR62 is robust in purine-depleted AD293 cells.

    Minor Comments:

    Do you have suggestions that would help the authors improve the presentation of their ideas and conclusions?

    1. The use of HSP90 inhibitors is a little confusing given the connections being made with BAG2 and other HSP70 co-chaperones in Figure 1.
      • Does the same conclusions hold true with an HSP70 inhibitor or siRNA?
      • (OPTIONAL) There are a lot of discrepancies between Hsp90 inhibitors and effective treatment concentrations. For example, NVP-AUY922 caused purinosomes to disassemble whereas STA9090 cause purinosomes to change morphology and adopt a more aggregated state. Do other Hsp90 inhibitors share the same phenotypic response as NVP-AUY922 in this study?
      • The treatment time (24 h) with NVP-AUY922 is very long. Given that Hsp90 interacts with hundreds of proteins, it is hard to understand whether the effect of Hsp90 inhibition is direct or indirect. Shorter times (1 h or less) would be more insightful.
    2. (OPTIONAL) Does the 2.6-fold increase in BAG2 increase its association with WDR62?
    3. Is the degradation of HPRT occurring through BAG2-mediated proteasomal degradation? Showing HPRT recovery by treating the cells with MG132 along with CHX would provide meaningful clues as to how BAG2 and HPRT might be related - Is BAG2 concentration increasing to facilitate the enhanced degradation of HPRT?
    4. Does HPRT colocalize with WDR62 in cells?
    5. (OPTIONAL) It would be nice to see validation experiments of some of the hits in Figure 1D or 1E in a co-immunoprecipitation experiment conducted similar to Figure 1C.
    6. The authors presented the findings that suggest that BAG2 interacts differently with commonly observed WDR62 mutations in MCPH2? How do these mutations affect WDR62 condensation, colocalization with purinosomes, or alter HPRT activity? Tying back the observations to something clinical would help elevate the overall significance of the findings.

    Are the text and figures clear and accurate?

    1. There are many times throughout the manuscript that the wrong figure is being referenced. These mistakes caused significant confusion at many times while reviewing the manuscript. Please double check all in-text references to figures. For example, I believe that you meant to use Figure S1C instead of Figure 2E with the statement on lines 183-185. Again, I believe that correct figure reference on line 501 is Figure 7G not Figure 7E.
    2. The figure legend on Figure S4 does not match the figure and the main text references. Please verify that the text in the figure legends correspond correctly to the figure.
    3. Please provide this data for the sentence on lines 399-400 in the supplemental file.
    4. I believe that the authors use the phrase "cell proliferation" to describe cell viability in the main text. In the Materials and Methods section, the authors state "The XTT cell proliferation assay enables quantification of cellular redox potential, providing a colorimetric readout of cell viability." Cell proliferation, viability, and cytotoxicity are different measurements, so please revise to reflect the correct experiment that was performed.

    Other Minor Comments:

    1. Move the sentence "In contrast, despite reduced mRNA..." (lines 387-388) to the last section when a reduction in PFAS expression was first mentioned.
    2. Please reference the following in the manuscript:
      • BioGRID database in the main text and Materials and Methods section
      • The reported study showing the DNAJC7-WDR62 interaction (as curated from BioGRID)
      • Fiji in the Materials and Methods section
    3. (Line 461-463) The authors state the following: "the loss of WDR62 leads to an increase in BAG2 and vice-versa (Fig. 7A) (Fig. S9B). I am not sure that the vice-versa (i.e. loss of BAG2 increases WDR62) is true. From the data presented in Figure 7H, I do not see a significant change in WDR62 expression upon BAG2 siRNA treatment.
    4. For your BioID study, do you know how many or the proportion of cells that were mitotically arrested with the low dose of nocodazole (200 ng/mL)? Given the small number of unique proteins that were in the mitotic only population, it is curious to know how enriched the cells were and whether WDR62 localization is important in the context of this study.
    5. Just to clarify, the WDR62-HA lane (third in each set) in Figure 1C is not WDR62-BirA*-HA and that it is only being used as a control.
    6. In the Discussion (lines 439-441) "We also show that WDR62 forms dynamic, phase-separated granules that co-localise with chaperones and purine metabolic enzymes, resembling purinosomes." I believe that the authors meant to say co-chaperones instead of chaperones given no microscopy data was presented showing the colocalization of HSP70/90 with WDR62 granules. Please revise.

    Referees cross-commenting

    I agree with the comments and recommendations by the other reviewers. Many of our shared comments are those that need to be addressed to substantiate the claims made by the authors throughout the manuscript. The proposed experiments across the reviewer comments appear feasible given that similar experiments have already been presented in this version of the manuscript. I strongly encourage the authors to consider these comments when revising their manuscript to help strengthen their claims and boost its overall significance and impact.

    Significance

    Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field. Place the work in the context of the existing literature (provide references, where appropriate).

    The work presented explains a previously unknown role for WDR62 in the regulation of purine metabolism. Despite all the hard work that was performed to reach their conclusions, the use of the AD293 cell line and the lack of correlating the common WDR62 disease-promoting mutations to the observed findings throughout the entire manuscript slightly reduced my enthusiasm for this work. The presented study leverages a lot of existing literature to establish connections between WR62, co-chaperones, and purine metabolic enzymes, with an emphasis on purinosome metabolon, a condensate comprised of the enzymes in de novo purine biosynthesis.

    State what audience might be interested in and influenced by the reported findings.

    The audience that might be interested in the reported findings would likely be those tied to biomolecular condensates in cellular metabolism and their connection to disease. I also feel that researchers that study microcephaly might be interested in this work. In my opinion, I believe that a broader readership could happen if additional studies were performed to make stronger connections between studies presented.

    Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    My field of expertise is tied to understanding the regulation of cellular metabolism through the use of biochemical and biophysical techniques. I am not as familiar with the in depth details of proteomic analysis such as those required for accurate reporting of data tied to protein proximity labeling (BioID) methods.

  5. Version published to 10.1101/2024.07.01.601630v1 on bioRxiv