PLEKHA5, PLEKHA6, and PLEKHA7 bind to PDZD11 to target the Menkes ATPase ATP7A to the cell periphery and regulate copper homeostasis

  1. Sophie Sluysmans
  2. Isabelle Méan
  3. Tong Xiao
  4. Amina Boukhatemi
  5. Flavio Ferreira
  6. Lionel Jond
  7. Annick Mutero
  8. Christopher J. Chang
  9. Sandra Citi

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Abstract

The WW-PLEKHA proteins PLEKHA5, PLEKHA6, and PLEKHA7 coordinate together with PDZD11 the anterograde traffic of the copper pump ATP7A from the trans-Golgi network to the cell periphery. WW-PLEKHAs promote PDZD11 interaction with the C-terminus of ATP7A and are required for maintaining low intracellular copper levels when cells face elevated copper.

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  1. Version published to 10.1091/mbc.e21-07-0355
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    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity):

    This study reveals the role of WW-PLEKHAs (PLEKHA5, 6 and 7) in the basolateral targeting of copper (Cu) transporter ATP7A. The Authors suggest that the WW-PLEKHAs/PDZD11/ATP7A interaction directs Cu-induced trafficking of ATP7A to the basolateral surface of epithelial cells. Suppression of WW-PLEKHAs impairs basolateral delivery of ATP7A and causes increased intracellular Cu levels. On the contrary, WW-PLEKHAs do not seem to participate in the retrieval of ATP7A back to the Golgi once the Cu levels return to basal values. To support these notions the manuscript provides a substantial set of the data, which were achieved with a wide repertoire of methods. In my view, this manuscript could be of interest to a broad readership, ranging from cells biologists to medical doctors. However, further revision should address the concerns outlined below.

    Major points:

    1. The Authors claim that at basal Cu conditions ATP7A resides in the TGN regardless of PDZD11 or WW-PLEKHAs depletion (Figs. 3, 4 and Fig. S6, S7). However, colocalization with TGN marker and its quantification are not shown. Thus, the colocalization of ATP7A with TGN marker (Golgin 97 should work in all cell types) has to be shown and its quantification (Pearson coefficient) has to be provided for control and all KO cells.

    Response: We thank the Reviewer for this comment. We plan to carry out the IF colocalization of ATP7A with Golgin97 and quantifications for WT and KO clonal lines at basal Cu conditions.

    1. Along the same line, ATP7A colocalization with TGN marker and its quantification also has to be conducted for the Cu washout experiments.

    Response: We plan to carry out the IF colocalization of ATP7A with Golgin97 and quantifications for WT and KO clonal lines for the Cu washout conditions.

    1. The authors say that upon addition of Cu ATP7A labeling was detected along lateral contacts, and near the apical and basal plasma membranes (Fig. 3B, WT). Here again "near apical" localization of ATP7A has to be clarified. This could either represent the ATP7A pool that still remains in the Golgi (which is usually close to apical surface in polarized epithelial cells) or the ATP7A pool delivered to the apical membrane of the cells. However, apical targeting of ATP7A would be odd considering previously published data that shows basolateral localization in polarized epithelial cells. Thus, the authors have to show whether "apical" ATP7A overlaps with TGN marker or with an apical marker (Gp135).

    Response. This Reviewer is correct. Effectively we believe that the localization of ATP7A that we observe in cysts is not apical, but sub-apical, as shown for the localization of PLEKHA5, where colocalization with the apical marker gp135 clearly shows a different localization (Fig. 2I).

    Therefore, we will carry out co-localization of ATP7A with gp135 in MDCK cells (the monoclonal antibody does not work on mCCD cells) and the labeling of the micrographs in Fig. 3B and 4B will be revised (sub-apical instead of apical). The labeling of PLEKHA5 sub-apical pool will also be revised (sub-apical and not apical) in Fig. 2.

    1. PDZD11 or PLEKHA6/7 KOs lead to an ATP7A pattern, which looks like pretty large scattered vesicles that do not overlap with basolateral marker. What are these round ATP7A structures, endosomes? Colocalization assessments with EEA1 (early endosomes), VPS35 (sorting endosome) and LAMP1 (late endosomes) would be needed to clarify this. Alternatively, these vesicles could represent a fragmented Golgi with ATP7A inside. To establish this, labelling with TGN marker at these conditions is required.

    Response: We thank the Reviewer for this comment. To clarify the nature (endosomes, Golgi, etc) of the membrane vesicles where ATP7A is localized in KO lines we will carry out double IF colocalization of ATP7A with either Golgin97 or early/sorting (which are mostly overlapped) endosome or the late endosome markers.

    1. Biotinylation experiments. The Authors say that KO of either PDZD11, or PLEKHA7, or both PLEKHA6 and PLEKHA7, but not PLEKHA6 alone, decreased ATP7A levels at the basolateral surface of mCCD cells (Fig. 3G), while a small decrease in the basolateral levels of ATP7A is observed in PLEKHA5-KO, but not PLEKHA6-KO MDCK cells (Fig. 4G). Honestly, it is tough to see this. In Fig. 4G all ATP7A bands in the biotinylated fraction look similar. In Fig. 3G, the P11 and P6/7 KO bands of biotinylated ATP7A might be a bit less intense than in WT, while the P6 KO signal looks even more intense that WT. More convincing blots with quantification have to be provided for both figures.

    Response: We will carry out additional immunoblots and quantifications of the biotinylation experiments results.

    1. Along the same line. Why was apical biotinylation of ATP7A not included? It absolutely should be done to understand whether any KO induces apical mistargeting of ATP7A.

    Response: The levels of ATP7A at the apical surface upon basal or elevated copper are negligible and not physiologically relevant, as established by previous biotinylation studies (for example Greenough et al AJP 2004, and Nyasae et al AJP-Gastrointest Liver Physiol 2007). We will carry out IF analysis of WT and KO cells with ATP7A and apical markers (ex. gp135) to clarify if the subapical labeling for ATP7A is or not on the apical membrane. Importantly, LESS, and not more subapical labeling is detected in KO lines (Fig. 3B, Fig. 4B), as we pointed out in the results section. Therefore, the KO lines do not show increased apical (mistargeting of) ATP7A.

    1. Copper metabolism. The authors say that KO of either PDZD11 or PLEKHA6/7 results in higher Cu levels. What does this mean in terms of physiology and pathology? In the context of Menkes disease one has to show that this intracellular Cu increase is due to a reduction in Cu release from the cells. So, Cu release from the cells into medium has to be measured by ICP-MS or Cu64. On the other hand, it would be important to understand whether Cu accumulation in KO cells is toxic. To this end viability of KO cells should be tested in Cu dose-response experiments.

    Response: The focus of this paper is the molecular mechanisms of ATP7A targeting to the BL plasma membrane, rather than a quantitative analysis of copper transport by and analysis of physiology/pathology of copper homeostasis ATP7A in our WT and KO cell lines. Our measurement of the intracellular copper using the CF4 probe was designed as a physiological readout to confirm that altered localization at the BL plasma membrane correlates with reduced copper extrusion, as it can be hypothesized. This said, to address this point, we plan to carry out an ICP-MS analysis of intracellular copper in selected WT and KO lines, after loading cells with different amounts of copper, and at different times after return to basal copper levels. CF4 and ICP-MS generally track, but they do measure distinct copper pools: CF4 measures exchangeable Cu pools while ICP-MS measures total Cu pools. We will also carry out a crystal violet analysis (see Gudekar et al, Scient. Reports, 2020) of the viability of WT and KO cells in the absence and presence of low or elevated copper levels, as suggested by the Reviewer.

    1. How critical is WW-PLEKHAs or PDZD11 deficiency in terms of Cu metabolism? Are there genetic disorders or mouse phenotypes associated with their loss of function? If yes, do these phenotypes include any impairment of Cu metabolism?

    Response: To our knowledge, no genetic study has addressed the role of WW-PLEKHAs and PDZD11 in Cu metabolism in vivo. PLEKHA7-KO mice are viable and were not reported to display any phenotype consistent with grossly altered Cu metabolism (Popov et al 2015). Mice KO for either PLEKHA5 or PLEKHA6 or PDZD11 have not been described. However, if WW- PLEKHAs have redundant functions in the trafficking of ATP7A, one would expect that mutation/KO of only one of them may not yield a significant phenotype. Furthermore, we cannot exclude that additional PDZ-containing proteins may participate in the trafficking of ATP7A, compensating a pathological or experimental loss of PDZD11. So, answering this question will require to generate single, double and triple KO mice for WW-PLEKHAs, and carry out a detailed analysis of in vivo Cu metabolism. This is beyond the scope of this paper. The text of the Discussion will be revised to address this comment.

    1. Discussion. Could PH domains of WW-PLEKHAS be involved in their basolateral localization, thereby generating a targeting patch for ATP7A? Some publications suggest that the basolateral membrane might be enriched in specific PIPs, which in turn generates a favorable environment for some PH domains. Is this the case for PH domains of WW-PLEKHAS?

    Response: This is an interesting hypothesis that should be investigated in future studies (lipidomic analysis of KO lines, overexpression studies, etc), but is outside of the scope of the present manuscript.

    Minor points:

    1. Fig. 6C. CFP-HA is a negative control but still gives a band (although of lower intensity). So how can one be sure that other interactions are specific? This is particularly worrying because the quantification shows a very minor (less than 1.5) increase in the intensity of bands corresponding to specific interactors.

    Response: CFP-HA is used as a “negative control” 3__rd_ _protein, added to bait (GST-PDZD11) and prey (GFP-ATP7A-Cter) (Fig. 6C). The IB shows that in the presence of CFP-HA the bait binds the prey, which is in agreement with the previously reported interaction between PDZD11 and the C-terminal region of ATP7A (Stephenson et al JBC, 2005). The point of the Figure is to show that the interaction between bait and prey is enhanced in the presence of HA-tagged WW- PLEKHAs (again, CFP-HA is the negative control). We agree that the increase is not huge, but it is nevertheless statistically significant, based on several experiments (Fig. 6E).

    1. Page 11. The result section title "WW-PLEKHAs promote PDZD11 binding to ATP7A through PDZD11 (Figure 6)" does not sound right and has to be corrected.

    Response: The text was revised ("WW-PLEKHAs promote PDZD11 binding to ATP7A”).

    Reviewer #1 (Significance):

    Delivery of copper transporter ATP7A to the basolateral surface of epithelial cells is of great importance for maintenance of copper metabolism and, hence for human health in general. Impairment of this process in enterocytes causes fatal Menkes disease. However, the mechanisms driving basolateral targeting of ATP7A remained poorly characterized. This study provides a significant advance in our understanding of these mechanisms and opens new avenues for investigation of how WW-PLEKHAs/PDZD11-mediated targeting of ATP7A might be affected in the context of inherited disorders of copper metabolism.

    Reviewer #2 (Evidence, reproducibility and clarity):

    This manuscript uncovers new PDZD11 interactors that participate in trafficking of the copper transporter ATP7A from the Golgi/TGN to the cell periphery in response to high copper concentrations. These interactors named PLEKHA5, PLEKHA6, and PLEKHA7 interact with the N-terminal Pro-rich domain of PDZD11 through their WW domains. As PDZD11 interacts with the C-terminal region of ATP7A, the authors investigated the hypothesis that WW-PLEKHAs are required for copper-induced relocalization of ATP7A from the TGN to the plasma membrane where it functions in copper efflux. In vitro pull down experiments verified the formation of ATP7A-, PLEKHAs-, and PDZD11-containing complexes. Using using CRISPR/Cas9 technology, the authors have generated PDZD11-, PLEKHA5-, PLEKHA6-, and PLEKHA6/7-KOs cell lines.

    Cells lacking one (or more) of these proteins were examined by microscopy with respect to their ability of targeting ATP7A to the cell periphery in response to copper. Abnormal trafficking of ATP7A in these mutant cell lines (PDZD11-, PLEKHA5-, PLEKHA6-, PLEKHA7-, and PLEKHA6/7-KOs) presumably prevented copper efflux since elevated intracellular copper was detected using the fluorescent copper probe CF4.

    Although it is difficult to read across the article's figures and supporting figure files (going back- and-forth repeatedly), the manuscript is generally clear and well written, and the results seem well documented accompanied by a tremendous amount of work.

    Comments.

    1. Two-hybrid screen occurs in the nucleus. How the authors could explain the fact that the use of PDZD11 as a bait exhibited an interaction with PLEKHA5 and PLEKHA6 (as well as PLEKHA7) in this system? Microscopic analysis of PLEKHA5 showed a cytoplasmic submembrane localization with E-cadherin, whereas PLEKHA6 exhibited a localization along the plasma membrane at apical junctions. In the case of PLEKHA7, it is an adherens junction protein. Furthermore, these three proteins are quite big (1116, 1297, and 1121 AAs, respectively) with their WW regions at their N termini, which involved the expression of very long cDNAs fused to the TA domain. As truly membrane-associated proteins, isn't surprising that a two hybrid approach worked?

    Response: We carried out several Y2H screens with a number of different baits, and we have always validated the physiological significance of the high score interactions (Pulimeno et al JBC 2011, Guerrera et al, 2016 JBC, and other unpublished data). So, it is an approach that reliably works very well. The Hybrigenics human placenta library that we used contains fragments of proteins, not the full-length proteins. Fig. 1A shows the preys identified with the Y2H using PDZD11 as a bait. The preys that were found comprise only the N-terminal regions of WW- PLEKHAs, not the FL proteins.

    1. Fig. 1C, what are the 4 bands seen for the second blot (anti-HA) in lines 1, 2, 9 and 10? The blot was cut in a way that not enough of the membrane can be evaluated. Why using Ponceau for GST-baits and not using anti-GST antibodies? It would be much better having an uniform method (Western blot assays) to show the data.

    This latter comment is true for Fig 1D, E, and Fig 6.

    Response: The 4 bands seen for the second blot (anti-HA) in lanes 1, 2, 9 and 10 are non- specific cross-reaction of the antibodies with the baits, that are present in high concentration (and present also where there is no CFP-HA, in lanes 1 and 9). The preys can be identified on the basis of their molecular size. For example, no CFP-HA prey is detected, since its size is intermediate between the baits, thus the negative control is validated. We use Ponceau for 2 reasons: 1) Ponceau can detect very well baits on nitrocellulose membranes; 2) to use GST antibodies we would need to cut the membranes. But cutting membranes is not possible when the size of some preys (in this case, the negative control) is in the same range of sizes as the baits. Thus, if we used anti-GST antibodies we would have to strip and re-probe the membranes, which is not optimal in our experience to elicit good signals.

    1. Fig 1B, why Caco-2 cells? All the other experiments were conducted with other cell lines such as mCCD and MDCK. Using different cell lines could give different results.

    Response: We used Caco2 cells because the Y2H was carried out with a human bait on a human placental library, and Caco2 are human cells. We also tried to use MDCK cells, but the efficiency of the IP was lower.

    1. Fig 1D, it is unclear whether the GST-PDZD11 fusion protein (bait) was present or not when used in pull down assays with GFP alone. This is a clear disadvantage of Ponceau, immunoblot would be much better to use.

    Response: The labeling by Ponceau is not optimal in one image (Fig. 1D), probably due to a problem of transfer. But a clearer image for the same pulldown with the same bait is shown in the bottom panel of Fig. 1E (where we show 3 PDZD11 baits, FL, N-term and delta-24), and it clearly shows good normalization of baits. We stain baits with Ponceau for normalization.

    1. In Fig 3A, under basal copper conditions, microscopic image of the PLEKHA6/7-KO seems indicate a distinct pattern of localization for ATP7A in comparison to that of WT. However, this difference does not seem to be highlighted in Fig 3E.

    Response: We will re-examine all the micrographs used for the quantification and integrate the data with the results of the colocalization between ATP7A and TGN marker. This should allow us to establish whether there is a dissociation of ATP7A labeling from TGN marker labeling in KO cells, or else a fragmentation of the TGN in the double-KO mCCD cells.

    In Figs 3F and 4F, what was the method for quantification?

    Response: The methods for quantifications are described in the “Image quantification” section of the Methods. We will add new data about the quantification of co-localization of ATP7A with TGN and endosomal markers.

    Along these lines, what is the copper concentration under basal conditions? How much copper was used for elevated copper conditions and what was the time of treatment?

    Response: Basal conditions refers to normal cell culture medium (“Cell culture” section of the Methods), and elevated copper is 315 µM of CuCl2 dissolved in culture medium. Cells were treated for 4hr (MDCK) or 5hr (mCCD) when cells were cultured on Transwells, overnight in the case of cysts.

    1. Is there any evidence for Atp7A-PDZD11-PLEKHAs association in vivo? Do the authors have assessed these protein-protein interactions using methods such as bimolecular fluorescence in cells?

    Response: We have attempted co-IP experiments with endogenous proteins, but they were inconclusive, probably due to the different extraction conditions required to solubilize membrane (ATP7A) and cytoplasmic (WW-PLEKHAs, PDZD11) proteins, and a disassembly of the complex under the conditions required to solubilize ATP7A. We have not tried bimolecular fluorescence, but for the revision we plan to carry out Proximity Ligation Assay (PLA) experiments, which in our hands are very effective in assessing physiological proximity of proteins in cells. Our pulldown experiments however provide evidence that the three proteins form a complex, and that WW- PLEKHAs enhance the interaction between PDZD11 and ATP7A (Fig. 6C-E). This is a mechanism that we have shown occurs also for the complex between PLEKHA7, PDZD11 and Tspan33 (Shah et al, 2018 Cell Rep, Rouaud et al, 2020 JBC).

    1. In Fig 5, do the authors have verified the mRNA (or/and protein) steady-state levels of metallothioneins? Probing whether metallothioneins are induced would strongly reinforced their conclusion as to whether an increase intracellular copper levels occurred in PDZD11-, PLEKHA5-, PLEKHA6-, PLEKHA7-, and PLEKHA6/7-Kos cell lines.

    Response: We thank the Reviewer for this comment. In the revision we will carry out RT-PCR analysis of the levels of expression of mRNAs for Metallothioneins I and II.

    1. In Fig 6E, what was the method for quantitative immunoblot assays? Have you used an Odyssey infrared imaging system (Li-Cor). What was the loading (internal) control under the same analytical method?

    Response: The Li-Cor imaging system was used to capture the signals, and intensities were measured in Image Studio Lite program (Li-cor). Signals from the prey (C-terminus of ATP7A) were normalized to signals from the bait (GST-PDZD11) which were used as loading control.

    1. In the case of the manuscript section entitled " PLEKHA5, PLEKHA6 and PLEKHA7 show distinct localizations in cells and tissues and define cytoplasmic...", (pages 5 to 7) the reader would benefit having a Table that would summarize all the data. It would be more understandable.

    Response: We thank the Reviewer for this suggestion. We will include a Table in the revision.

    1. Do PDZD11, PLEKHA5, PLEKHA6, and PLEKHA7 proteins exist as multiple isoforms? If that is the case, for each of them, are they exhibiting the same tissue-specific expression profiles as shown in Fig S3? For each protein, if different isoforms exist, perhaps some of them participate in a different way for the targeting of ATP7A?

    Response: No PDZD11 isoforms are known, but 15, 5, and 9 different protein-coding transcripts are reported (ensemble.org) for PLEKHA5, PLEKHA6 and PLEKHA7, respectively, the largest ones being the WW-containing transcripts. We focused exclusively on the WW-containing isoforms of PLEKHAs because PDZD11 binds to the WW domains, and the Y2H identified only the WW-containing isoforms of PLEKHA5, PLEKHA6 and PLEKHA7. The observation that the phenotype of PDZD11-KO cells is similar to that of either PLEKHA6-KO, PLEKHA7-KO or double-KO mCCD cells suggests that PLEKHA5, PLEKHA6 and PLEKHA7 WW-containing isoforms act in a complex with PDZD11. This is consistent with the previous observations that highlight a role of the C-terminal region of ATP7A in regulating its traffic, and the binding of the same region to PDZD11. However, we cannot exclude that PLEKHA5/6/7 isoforms that lack the WW domains could participate in the regulation of the targeting of ATP7A, through other, PDZD11-independent mechanisms. The text of the Discussion will be revised to clarify this point.

    1. Is it known whether PDZD11, PLEKHA5, PLEKHA6, and PLEKHA7 proteins participate in the copper-regulated trafficking of the ATP7B (Wilson) protein? In Fig S3, it is shown that they are expressed in liver, with PLEKHA7 exhibiting a slower migration (protein modification?). Alternatively, are they strictly involved in the regulation of ATP7A (Menkes)? Could the authors discuss about it?

    Response: ATP7B lacks the PDZ-binding motif that is responsible for PDZD11 binding, and the C-terminus of ATP7B does not interact with PDZD11 (AIPP1) by beta-galactosidase assays in yeast, unlike ATP7A (Stephenson et al, JBC 2005). For this reason, ATP7B is not expected to be regulated by PDZD11 and WW-PLEKHAs. However, analysis of the localization of ATP7B in our cell lines could be done in future studies. The text of the Discussion will be revised to make this point.

    1. The proposed model in Fig 7 is unclear illustrating a nucleus that consumes a lot of space while it is not involved in the proposed mechanism. Cellular proteins that are involved in the proposed mechanism should be bigger and their interactions that lead to formation of protein complexes must be better illustrated as a function of copper availability.

    Response: The model of Fig. 7 will be re-drawn to take into account these suggestions.

    1. Typo. Line 320: remove "or" and replace it by "and" : ...both PLEKHA6 and PLEKHA7 (Fig. 5A-D).
    1. Typo. Line 329: remove (Figure 6) in the title.

    Response: The typos were corrected.

    Reviewer #2 (Significance ):

    This study represents a significant advanced in the copper field.

    Reviewer #3 (Evidence, reproducibility and clarity):

    Summary

    The authors identified some major interesting findings including the key role of WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) in the recruitment of PDZD11 targeting ATP7A to the cell periphery in response to elevated copper. Generating the antibodies against PLEKHAs and PDZD11 and various knock out cell lines and validating their expression in these cell lines and tissues is innovative. Further, the authors showed that copper dependent WW-PLEKHAs and PDZD11 regulate the localization and function of ATP7A to modulate cellular copper homeostasis.

    Major comments:

    We are in agreement with the manuscript conclusions. Based on the presented studies, the authors propose the in-vivo role of WW-PLEKHAs and PDZD11 in ATP7A trafficking, and how microtubule dynamics and trafficking machinery regulate ATP7A localization. Additionally, investigating the effects of the cell membrane trimolecular complexes ATP7A-PDZD11-WW- PLEKHA on elevated copper would be impactful.

    Additional notes:

    1. Figure 4, the authors provide excellent data and images showing localization of PLEKHA, PDZD11 and ATP7A within different cell lines. Nevertheless, showing PLEKHA, PDZD11 and ATP7A localization on membrane of the cell surfaces at elevated copper condition with cell fractionation technique and their interaction through co-immunoprecipitation (co-IP) could validate author's hypothesis. At least the authors should comment on this.

    Response: As stated in the response to comment n.6 from Reviewer #2, we attempted co-IP experiments with endogenous proteins, which were inconclusive. Our pulldown experiments provide evidence that the three proteins form a complex, and that WW-PLEKHAs enhance the interaction between PDZD11 and ATP7A (Fig. 6C-E). This is a mechanism that we have shown occurs also for the complex between PLEKHA7, PDZD11 and Tspan33 (Shah et al, 2018 Cell Rep, Rouaud et al, 2020 JBC). We plan for the revision to carry out Proximity Ligation Assay (PLA) experiments, which in our hands are very effective in assessing proximity of proteins in cells, when co-IPs are technically difficult or impossible.

    1. Figure 5, Alternatively, intracellular copper levels by ICPMS in the cell lines would strengthen the results. As author's treated the cell lines with very high copper concentration, copper concentration dependent studies would be appreciated to verify how PLEKHA's and PDZD11 response depends on copper concentration

    Also, the authors should clearly mention the number of replicates for each experiment and indicate in the figure legends.

    Response. We will carry out ICP-MS to evaluate intracellular copper levels as a function of genotype. Depending on the results, we will carry out studies about the dose-dependence of the effects of copper. The number of replicates of the experiment will be mentioned in the Figure legend in the revised text.

    Minor comments:

    1. Figure1B, co-IP efficiency is lower in Caco-2 cells, therefore endogenous levels of PLEKHA5, PLEKHA6 and PDZD11 in Caco-2 should be checked and shown. Mention the number of replicates for the experiments.

    Response. Endogenous levels of proteins are shown in the Input lanes. The low levels of PLEKHA5 in Caco2 cells are consistent with the IB analysis of tissue lysates, showing relatively low levels in intestine (Fig. S3D). The number of replicates of the experiment will be mentioned in the Figure legend in the revised text.

    1. Figure 2A, as per result of 2A, E-cadherin labeling is missing. Figure 2M and 2N, author analyzed the co-localization of PLEKHA-5 in presence of nocodazole but not PDZD11. It would be interesting to see the PDZD11 as well after nocodazole treatment.

    Response. E-cadherin-labelled panel will be added to Fig. 2A in the revision. We will also show the effect of nocodazole on the localization of PDZD11.

    1. The result section title for figure 6 (line 329) is misleading. Also, trimolecular complex PLEKHA's, PDZD11 and ATP7A membrane localization at elevated copper concentration could be shown by immunofluorescence, if possible.

    Response. The title of the section was revised, to reflect more accurately the results of Figure 6. It is now “WW-PLEKHAs promote the binding of the C-terminal region of ATP7A to PDZD11”.

    Triple IF colocalization of endogenous PLEKHAs, PDZD11 and ATP7A is not possible for 2 reasons: 1) PDZD11 antibodies can only reveal endogenous junctional (clustered) labeling (Guerrera et al JBC2016); the lateral and cytoplasmic labeling is too weak, and can only be appreciated upon overexpression of PDZD11, as shown in Fig. 2B-E (co-expression with selected WW-PLEKHAs highlights how each PLEKHA directs PDZD11 to a different pool). 2) Both antibodies against PDZD11 and ATP7A were raised in rabbits, which makes it technically impossible to do triple labeling. We will address the question of the existence of the ATP7A- containing trimolecular complex by PLA analysis (ATP7A+PDZD11 and ATP7A+WW-PLEKHAs)._

    1. General comment: it would be interesting to see the hypothesis and finding in mice model with copper accumulation (for example Atp7b KO mice) as PLEKHA's and PDZD11 are sensitive to copper concentration. Or at least the authors can comment on this future possibility.

    Response. We agree with the Reviewer that mouse models could be useful to test the relevance of WW-PLEKHAs and PDZD11 as targets or effectors of copper-sensing mechanisms in vivo.

    The text of the Discussion will be modified to envisage these possible future studies

    Reviewer #3 (Significance):

    In conditions including Menkes disease, occipital horn syndrome (OHS), and ATP7A-related distal motor neuropathy (DMN), characterized by altered intestinal copper metabolism, the new knowledge ATP7A associates with WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) and PDZD11 is an important finding for the study of copper homeostasis.

    As ATP7A is structurally similar to ATP7B (60% homology), the current study opens the area of the research where WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) and PDZD11 could also play role in ATP7B trafficking to address not only Menkes disease but also Wilson disease and other diseases related to altered copper levels.

    This is a well written and presented manuscript with excellent mechanistic work utilizing molecular imaging techniques and several confirmatory experiments. I recommend the manuscript to be accepted for publication with minor modifications.

  3. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    The authors identified some major interesting findings including the key role of WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) in the recruitment of PDZD11 targeting ATP7A to the cell periphery in response to elevated copper. Generating the antibodies against PLEKHAs and PDZD11 and various knock out cell lines and validating their expression in these cell lines and tissues is innovative. Further, the authors showed that copper dependent WW-PLEKHAs and PDZD11 regulate the localization and function of ATP7A to modulate cellular copper homeostasis.

    Major comments:

    We are in agreement with the manuscript conclusions. Based on the presented studies, the authors propose the in-vivo role of WW-PLEKHAs and PDZD11 in ATP7A trafficking, and how microtubule dynamics and trafficking machinery regulate ATP7A localization. Additionally, investigating the effects of the cell membrane trimolecular complexes ATP7A-PDZD11-WW-PLEKHA on elevated copper would be impactful.

    Additional notes:

    1. Figure 4, the authors provide excellent data and images showing localization of PLEKHA, PDZD11 and ATP7A within different cell lines. Nevertheless, showing PLEKHA, PDZD11 and ATP7A localization on membrane of the cell surfaces at elevated copper condition with cell fractionation technique and their interaction through co-immunoprecipitation (co-IP) could validate author's hypothesis. At least the authors should comment on this.
    2. Figure 5, Alternatively, intracellular copper levels by ICPMS in the cell lines would strengthen the results. As author's treated the cell lines with very high copper concentration, copper concentration dependent studies would be appreciated to verify how PLEKHA's and PDZD11 response depends on copper concentration Also, the authors should clearly mention the number of replicates for each experiment and indicate in the figure legends.

    Minor comments:

    1. Figure1B, co-IP efficiency is lower in Caco-2 cells, therefore endogenous levels of PLEKHA5, PLEKHA6 and PDZD11 in Caco-2 should be checked and shown. Mention the number of replicates for the experiments.
    2. Figure 2A, as per result of 2A, E-cadherin labeling is missing. Figure 2M and 2N, author analyzed the co-localization of PLEKHA-5 in presence of nocodazole but not PDZD11. It would be interesting to see the PDZD11 as well after nocodazole treatment.
    3. The result section title for figure 6 (line 329) is misleading. Also, trimolecular complex PLEKHA's, PDZD11 and ATP7A membrane localization at elevated copper concentration could be shown by immunofluorescence, if possible.
    4. General comment: it would be interesting to see the hypothesis and finding in mice model with copper accumulation (for example Atp7b KO mice) as PLEKHA's and PDZD11 are sensitive to copper concentration. Or at least the authors can comment on this future possibility.

    Significance

    In conditions including Menkes disease, occipital horn syndrome (OHS), and ATP7A-related distal motor neuropathy (DMN), characterized by altered intestinal copper metabolism, the new knowledge ATP7A associates with WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) and PDZD11 is an important finding for the study of copper homeostasis.

    As ATP7A is structurally similar to ATP7B (60% homology), the current study opens the area of the research where WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) and PDZD11 could also play role in ATP7B trafficking to address not only Menkes disease but also Wilson disease and other diseases related to altered copper levels.

    This is a well written and presented manuscript with excellent mechanistic work utilizing molecular imaging techniques and several confirmatory experiments. I recommend the manuscript to be accepted for publication with minor modifications.

  4. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    This manuscript uncovers new PDZD11 interactors that participate in trafficking of the copper transporter ATP7A from the Golgi/TGN to the cell periphery in response to high copper concentrations. These interactors named PLEKHA5, PLEKHA6, and PLEKHA7 interact with the N-terminal Pro-rich domain of PDZD11 through their WW domains. As PDZD11 interacts with the C-terminal region of ATP7A, the authors investigated the hypothesis that WW-PLEKHAs are required for copper-induced relocalization of ATP7A from the TGN to the plasma membrane where it functions in copper efflux. In vitro pull down experiments verified the formation of ATP7A-, PLEKHAs-, and PDZD11-containing complexes. Using using CRISPR/Cas9 technology, the authors have generated PDZD11-, PLEKHA5-, PLEKHA6-, and PLEKHA6/7-KOs cell lines. Cells lacking one (or more) of these proteins were examined by microscopy with respect to their ability of targeting ATP7A to the cell periphery in response to copper. Abnormal trafficking of ATP7A in these mutant cell lines (PDZD11-, PLEKHA5-, PLEKHA6-, PLEKHA7-, and PLEKHA6/7-KOs) presumably prevented copper efflux since elevated intracellular copper was detected using the fluorescent copper probe CF4.

    Although it is difficult to read across the article's figures and supporting figure files (going back-and-forth repeatedly), the manuscript is generally clear and well written, and the results seem well documented accompanied by a tremendous amount of work.

    Comments.

    1. Two-hybrid screen occurs in the nucleus. How the authors could explain the fact that the use of PDZD11 as a bait exhibited an interaction with PLEKHA5 and PLEKHA6 (as well as PLEKHA7) in this system? Microscopic analysis of PLEKHA5 showed a cytoplasmic submembrane localization with E-cadherin, whereas PLEKHA6 exhibited a localization along the plasma membrane at apical junctions. In the case of PLEKHA7, it is an adherens junction protein. Furthermore, these three proteins are quite big (1116, 1297, and 1121 AAs, respectively) with their WW regions at their N termini, which involved the expression of very long cDNAs fused to the TA domain. As truly membrane-associated proteins, isn't surprising that a two hybrid approach worked?
    2. Fig. 1C, what are the 4 bands seen for the second blot (anti-HA) in lines 1, 2, 9 and 10? The blot was cut in a way that not enough of the membrane can be evaluated. Why using Ponceau for GST-baits and not using anti-GST antibodies? It would be much better having an uniform method (Western blot assays) to show the data. This latter comment is true for Fig 1D, E, and Fig 6.
    3. Fig 1B, why Caco-2 cells? All the other experiments were conducted with other cell lines such as mCCD and MDCK. Using different cell lines could give different results.
    4. Fig 1D, it is unclear whether the GST-PDZD11 fusion protein (bait) was present or not when used in pull down assays with GFP alone. This is a clear disadvantage of Ponceau, immunoblot would be much better to use.
    5. In Fig 3A, under basal copper conditions, microscopic image of the PLEKHA6/7-KO seems indicate a distinct pattern of localization for ATP7A in comparison to that of WT. However, this difference does not seem to be highlighted in Fig 3E.

    In Figs 3F and 4F, what was the method for quantification?

    Along these lines, what is the copper concentration under basal conditions? How much copper was used for elevated copper conditions and what was the time of treatment?

    1. Is there any evidence for Atp7A-PDZD11-PLEKHAs association in vivo? Do the authors have assessed these protein-protein interactions using methods such as bimolecular fluorescence in cells?
    2. In Fig 5, do the authors have verified the mRNA (or/and protein) steady-state levels of metallothioneins? Probing whether metallothioneins are induced would strongly reinforced their conclusion as to whether an increase intracellular copper levels occurred in PDZD11-, PLEKHA5-, PLEKHA6-, PLEKHA7-, and PLEKHA6/7-KOs cell lines.
    3. In Fig 6E, what was the method for quantitative immunoblot assays? Have you used an Odyssey infrared imaging system (Li-Cor). What was the loading (internal) control under the same analytical method?
    4. In the case of the manuscript section entitled " PLEKHA5, PLEKHA6 and PLEKHA7 show distinct localizations in cells and tissues and define cytoplasmic...", (pages 5 to 7) the reader would benefit having a Table that would summarize all the data. It would be more understandable.
    5. Do PDZD11, PLEKHA5, PLEKHA6, and PLEKHA7 proteins exist as multiple isoforms? If that is the case, for each of them, are they exhibiting the same tissue-specific expression profiles as shown in Fig S3? For each protein, if different isoforms exist, perhaps some of them participate in a different way for the targeting of ATP7A?
    6. Is it known whether PDZD11, PLEKHA5, PLEKHA6, and PLEKHA7 proteins participate in the copper-regulated trafficking of the ATP7B (Wilson) protein? In Fig S3, it is shown that they are expressed in liver, with PLEKHA7 exhibiting a slower migration (protein modification?). Alternatively, are they strictly involved in the regulation of ATP7A (Menkes)? Could the authors discuss about it?
    7. The proposed model in Fig 7 is unclear illustrating a nucleus that consumes a lot of space while it is not involved in the proposed mechanism. Cellular proteins that are involved in the proposed mechanism should be bigger and their interactions that lead to formation of protein complexes must be better illustrated as a function of copper availability.
    8. Typo. Line 320: remove "or" and replace it by "and" : ...both PLEKHA6 and PLEKHA7 (Fig. 5A-D).
    9. Typo. Line 329: remove (Figure 6) in the title.

    Significance

    This study represents a significant advanced in the copper field.

  5. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    This study reveals the role of WW-PLEKHAs (PLEKHA5, 6 and 7) in the basolateral targeting of copper (Cu) transporter ATP7A. The Authors suggest that the WW-PLEKHAs/PDZD11/ATP7A interaction directs Cu-induced trafficking of ATP7A to the basolateral surface of epithelial cells. Suppression of WW-PLEKHAs impairs basolateral delivery of ATP7A and causes increased intracellular Cu levels. On the contrary, WW-PLEKHAs do not seem to participate in the retrieval of ATP7A back to the Golgi once the Cu levels return to basal values. To support these notions the manuscript provides a substantial set of the data, which were achieved with a wide repertoire of methods. In my view, this manuscript could be of interest to a broad readership, ranging from cells biologists to medical doctors. However, further revision should address the concerns outlined below.

    Major points:

    1. The Authors claim that at basal Cu conditions ATP7A resides in the TGN regardless of PDZD11 or WW-PLEKHAs depletion (Figs. 3, 4 and Fig. S6, S7). However, colocalization with TGN marker and its quantification are not shown. Thus, the colocalization of ATP7A with TGN marker (Golgin 97 should work in all cell types) has to be shown and its quantification (Pearson coefficient) has to be provided for control and all KO cells.
    2. Along the same line, ATP7A colocalization with TGN marker and its quantification also has to be conducted for the Cu washout experiments.
    3. The authors say that upon addition of Cu ATP7A labeling was detected along lateral contacts, and near the apical and basal plasma membranes (Fig. 3B, WT). Here again "near apical" localization of ATP7A has to be clarified. This could either represent the ATP7A pool that still remains in the Golgi (which is usually close to apical surface in polarized epithelial cells) or the ATP7A pool delivered to the apical membrane of the cells. However, apical targeting of ATP7A would be odd considering previously published data that shows basolateral localization in polarized epithelial cells. Thus, the authors have to show whether "apical" ATP7A overlaps with TGN marker or with an apical marker (Gp135).
    4. PDZD11 or PLEKHA6/7 KOs lead to an ATP7A pattern, which looks like pretty large scattered vesicles that do not overlap with basolateral marker. What are these round ATP7A structures, endosomes? Colocalization assessments with EEA1 (early endosomes), VPS35 (sorting endosome) and LAMP1 (late endosomes) would be needed to clarify this. Alternatively, these vesicles could represent a fragmented Golgi with ATP7A inside. To establish this, labelling with TGN marker at these conditions is required.
    5. Biotinylation experiments. The Authors say that KO of either PDZD11, or PLEKHA7, or both PLEKHA6 and PLEKHA7, but not PLEKHA6 alone, decreased ATP7A levels at the basolateral surface of mCCD cells (Fig. 3G), while a small decrease in the basolateral levels of ATP7A is observed in PLEKHA5-KO, but not PLEKHA6-KO MDCK cells (Fig. 4G). Honestly, it is tough to see this. In Fig. 4G all ATP7A bands in the biotinylated fraction look similar. In Fig. 3G, the P11 and P6/7 KO bands of biotinylated ATP7A might be a bit less intense than in WT, while the P6 KO signal looks even more intense that WT. More convincing blots with quantification have to be provided for both figures.
    6. Along the same line. Why was apical biotinylation of ATP7A not included? It absolutely should be done to understand whether any KO induces apical mistargeting of ATP7A.
    7. Copper metabolism. The authors say that KO of either PDZD11 or PLEKHA6/7 results in higher Cu levels. What does this mean in terms of physiology and pathology? In the context of Menkes disease one has to show that this intracellular Cu increase is due to a reduction in Cu release from the cells. So, Cu release from the cells into medium has to be measured by ICP-MS or Cu64. On the other hand, it would be important to understand whether Cu accumulation in KO cells is toxic. To this end viability of KO cells should be tested in Cu dose-response experiments.
    8. How critical is WW-PLEKHAs or PDZD11 deficiency in terms of Cu metabolism? Are there genetic disorders or mouse phenotypes associated with their loss of function? If yes, do these phenotypes include any impairment of Cu metabolism?
    9. Discussion. Could PH domains of WW-PLEKHAS be involved in their basolateral localization, thereby generating a targeting patch for ATP7A? Some publications suggest that the basolateral membrane might be enriched in specific PIPs, which in turn generates a favorable environment for some PH domains. Is this the case for PH domains of WW-PLEKHAS?

    Minor points:

    1. Fig. 6C. CFP-HA is a negative control but still gives a band (although of lower intensity). So how can one be sure that other interactions are specific? This is particularly worrying because the quantification shows a very minor (less than 1.5) increase in the intensity of bands corresponding to specific interactors.
    2. Page 11. The result section title "WW-PLEKHAs promote PDZD11 binding to ATP7A through PDZD11 (Figure 6)" does not sound right and has to be corrected.

    Significance

    Delivery of copper transporter ATP7A to the basolateral surface of epithelial cells is of great importance for maintenance of copper metabolism and, hence for human health in general. Impairment of this process in enterocytes causes fatal Menkes disease. However, the mechanisms driving basolateral targeting of ATP7A remained poorly characterized. This study provides a significant advance in our understanding of these mechanisms and opens new avenues for investigation of how WW-PLEKHAs/PDZD11-mediated targeting of ATP7A might be affected in the context of inherited disorders of copper metabolism.

  6. Version published to 10.1101/2021.06.17.448791 on bioRxiv