Hydroxyurea induces ER stress and cytoplasmic protein aggregation

  1. Ana Sánchez-Molina
  2. Manuel Bernal
  3. Joel D. Posligua-García
  4. Antonio J. Pérez-Pulido
  5. Laura de Cubas
  6. Elena Hidalgo
  7. Silvia Salas-Pino
  8. Rafael R. Daga

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Abstract

The endoplasmic reticulum (ER) lumen provides the proper redox environment for disulfide bond formation, which is required for the appropriate folding of proteins that enter the secretory pathway and constitute membranes. Defective protein folding in the ER activates proteostatic mechanisms that are now beginning to be elucidated. Here, we show that hydroxyurea (HU) causes ER stress and triggers a transient perinuclear ER expansion, which leads to the clustering of nuclear pore complexes. This striking phenotype is mimicked by diamide (DIA), a specific thiol stress inductor, and prevented or rapidly reverted by dithiothreitol, a dithiol-reducing agent, suggesting that ER expansion is caused by disulfide stress. ER expansion induced by HU or DIA depends on glutathione (GSH), is Ire1-independent, and is associated with a unique transcriptome program that differs from the canonical unfolding protein response (UPR). The ER luminal expansion accumulates Hsp70 Bip1 chaperone, and it evolves parallel with the appearance of cytoplasmic protein aggregates containing heat stress proteins (HSPs), indicating that both HU and DIA are impinging on protein folding. Thus, our data reveal that HU induces disulfide stress that impinges on protein folding in the cytoplasm and ER.

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    Reviewer 1

    Major issue #1. Regarding the conclusions on IRE1 signaling, both yeast species have different IRE1 activities (https://elifesciences.org/articles/00048), the total deletion of IRE1 in S pombe appears to indicate that expansion of perinuclear ER is independent of IRE1, however since IRE1 signaling has exclusively a negative impact on mRNA expression, it might be relevant to identify mRNA whose expression is stabilized under those circumstances and evaluate whether those could confer a mechanism which would also yield perinuclear ER expansion (eg differential deregulation of ER stress controlled lipid biosynthesis required for lipid membrane synthesis). In S. cerevisiae, do the authors observe HAC1 mRNA splicing?

    We have not tested whether HAC1 mRNA is processed in S. cerevisiae.

    In addition, as requested by the reviewers, we reassessed our RNA-seq data and compared it with data from (Kimmig et al., 2012) (UPR activation in S. pombe), which added a new layer of data that reinforces the differences between the transcriptomic responses induced by HU and DIA and the canonical UPR. The following information is now included in the paper (page 26, highlighted in blue):

    “We further compared our transcriptomic data with that obtained by Kimmig et al. from DTT- treated *S. pombe *cells. When we compared the genes that were downregulated in our conditions with the ones described by Kimmig *et al. *(FC≤-1), we found no similarities between HU treatment (75 mM HU for 150 minutes) and UPR-induced downregulation, and only three genes ( ist2, *efn1 *and *xpa1) *all of them encode for transmembrane proteins, were common with DIA treatment (3 mM DIA for 60 minutes). Additionally, ist2 and xpa1, but not efn1, are considered Ire1-dependent downregulated genes and are located in the ER. These results show that HU- or DIA- induced transcriptomic programs are different from UPR, as they do not heavily rely on mRNA decay and favor gene overexpression. Interestingly, we found similarities between genes showed to be upregulated more that twofold by DTT in Kimmig et al., and HU and DIA conditions. When the two N-Cap-inducing conditions were compared with DTT, we found eight common upregulated genes (frp1, plr1, SPCC663.08c, srx1, gst2, str3, *caf5 *and hsp16) mostly involved in reduction processes and the chaperone Hsp16 which suggests folding stress”.

    Major issue #2. The authors indicate that HU and DIA lead to thiol stress, it might be relevant to evaluate the thiol-redox status of major secretory proteins in S. pombe (or even cargo reporters if necessary) to fully document the stress impact on global protein redox status.

    We agree with the reviewer that it is important to determine the redox and the functional state of the secretory pathway in our conditions to fully understand the cellular consequences of these treatments, especially in the case of HU, as it is routinely used in clinics. In this context, we have already included new data showing that HU or DIA treatment leads to alterations in the Golgi apparatus and in the distribution of secretory proteins (Figures 3A-B). In addition, we are currently performing mass spectrometry experiment to detect protein glutathionylation in our conditions, as it has been previously shown that DIA treatment leads to glutathionylation of key ER proteins such as Bip1, Pdi or Ero1 (Lind et al., 2002; Wang & Sevier, 2016), which might by reproduced upon HU treatment. Finally, we plan to test the folding and processing of specific secretory cargoes by western blot in our experimental conditions (See below, Reviewer 2, Major issue #1).

    What happens if HU-treated yeast cells are grown in the presence of n-acetyl cysteine?

    We have tested whether the addition of this antioxidant could prevent and/or revert the N-Cap phenotype. We found that NAC in combination with HU increased N-Cap incidence (Figure 5H). As NAC is a GSH precursor and we find that GSH is required to develop the phenotype of N-Cap (Figure 5A-B, D, G), this result further supports that the HU-induced cellular damage might involve ectopic glutathionylation of proteins.

    Unfortunately, we have not tested NAC in combination with DIA, as NAC seems to reduce DIA as soon as they get in contact, as judged by the change in the characteristic orange color of DIA, the same that happens when we combine GSH and DIA (Supplementary Figure 5A-B).

    In this regard, the following information has been added to the manuscript (page 30, highlighted in blue):

    “We also tested GSH addition to the medium in combination with either HU or DIA. When mixed with DIA, we noticed that the color of the culture changed after GSH addition (Figure S5A), which suggests that GSH and DIA can interact extracellularly, thus preventing us from being able to draw conclusions from those experiments. On the other hand, combining GSH with HU increased N-Cap incidence (Figure 5G), as expected based on our previous observations. Additionally, we checked whether the addition of the antioxidant N-acetyl cysteine (NAC), a GSH precursor, impacted upon the N-Cap phenotype. The results were the same as with GSH addition: when combined with HU, NAC increased N-Cap incidence (Figure 5H), whereas in combination, the two compounds interacted extracellularly (Figure S5B). These data align with NAC being a precursor of GSH, as incrementing GSH levels augments the penetrance of the HU-induced phenotype”.

    Major issue #3. The appearance of cytosolic aggregates is intriguing, do the authors have any idea on the nature of the protein aggregates?

    DIA is a strong oxidant, and HU treatment results in the production of reactive oxygen species (ROS). Therefore, one hypothesis would be that cytoplasmic chaperone foci represent oxidized and/or misfolded soluble proteins. Indeed, in this revised version of the manuscript we have included data showing that guk1-9-GFP and Rho1.C17R-GFP soluble reporters of misfolding accumulate in cytoplasmic foci upon HU or DIA treatment that colocalize with Hsp104 (Figure 4I-J, pages 23-24 and 29), which demonstrate that cytoplasmic chaperone foci contain misfolded proteins. We have also tested if they contain Vgl1, which is one of the main components of heat shock induced stress granules in *S. pombe *(Wen et al., 2010). However, we found that HU or DIA-induced foci lacked this stress granule marker, and indeed Vgl1 did not form any foci in response to these treatments. Therefore, our aggregates differ from the canonical stress-induced granules.

    Are those resulting from proficient retrotranslocation or reflux of misfolded proteins from the ER?

    To test whether these cytosolic aggregates result from retrotranslocation from the ER, we plan to use the vacuolar Carboxipeptidase Y mutant reporter CPY*, which is misfolded. This misfolded protein is imported into the ER lumen but does not reach the vacuole. Instead, it is retrotranslocated to the cytoplasm, where it is ubiquitinated and degraded by the proteasome (Mukaiyama et al., 2012). We will analyze by fluorescence microscopy the localization of CPY*´-GFP and Hsp104-containing aggregates upon HU or DIA treatment and with or without proteasome inhibitors. We can also test the levels, processing and ubiquitination of CPY*-GFP by western blot, as ubiquitination of retrotranslocated proteins occurs once they are in the cytoplasm.

    Are those aggregates membrane bound or do they correspond to aggresomes as initially defined? The Walter lab has demonstrated a tight balance between ER phagy and ER membrane expansion (https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.0040423), which could also impact on the presence of protein aggregates in the cytosol.

    Our results suggest that these aggregates are not bound to ER membranes, as they do not appear in close proximity to the ER area marked by mCherry-AHDL in fluorescence microscopy images.

    To fully rule out this possibility, we have tested whether these Hsp104-aggregates colocalized with ER transmembrane proteins Rtn1 and Yop1, and with Gma12-GFP that marks the Golgi apparatus. In none of the cases the Hsp104-containing aggregates colocalized or were surrounded by membranes. This information will be added to the final version of the manuscript.

    With respect to autophagy, we have tested whether deletion of key genes involved in autophagy affected the N-Cap phenotype. To this end, we used deletions of vac8 and atg8 in strains expressing Cut11-GFP and/or mCherry-AHDL and found that none of them affected N-Cap formation. These data suggest that the core machinery of autophagy is not critical for HU/DIA-induced ER expansion. We plan to include this data in the final version of the manuscript along with the rest of experiments proposed.

    To get deeper insights and to fully rule out a possible contribution of macro-autophagy to the HU- and DIA-induced phenotypes, we plan to analyze by western blot whether GFP-Atg8 is induced and cleaved upon HU or DIA treatments which would be indicative of macroautophagy activation.

    To test whether the cytoplasmic aggregates are the result of an imbalance between ER-expansion and ER-phagy we plan to analyze the localization of GFP-Atg8 and Hsp104-RFP in the atg7Δ mutant, impaired in the core macro-autophagy machinery. In these conditions, the number or size of the cytoplasmic aggregates might be impacted.

    On the other hand, it has been recently shown that an ER-selective microautophagy occurs in yeasts upon ER stress (Schäfer et al., 2020; Schuck et al., 2014). This micro-ER-phagy involves the direct uptake of ER membranes into lysosomes, is independent of the core autophagy machinery and depends on the ESCRT system and is influenced by the Nem1-Spo7 phosphatase. ESCRT directly functions in scission of the lysosomal membrane to complete the uptake of the ER membrane. Interestingly, N-Caps are fragmented in the absence of cmp7 and specially in the absence of vps4 or lem2, the nuclear adaptor of the ESCRT (Figure 3E), We had initially interpreted these results as the need to maintain nuclear membrane identity during the process of ER expansion (Kume et al., 2019); however, the appearance of fragmented ER upon HU treatment in the absence of ESCRT might also be due to an inability to complete microautophagic uptake of ER membranes. To test this hypothesis, we plan to analyze whether the fragmented ER in these conditions co-localize with lysosome/vacuole markers.

    Major issue #4. Nucleotide depletion was previously shown to lead to HSP16 expression through activation of the spc1 MAPK pathway (https://academic.oup.com/nar/article/29/14/3030/2383924), one might think that HU (or diamide) could lead to this through a nucleotide dependent mechanism and not necessary through a thiol-redox protein misfolding stress. This issue has to be sorted out to ensure that the HSP effect is independent of nucleotide depletion.

    As stated in (Taricani et al., 2001), hsp16 expression is strongly induced in a cdc22-M45 mutant background. We performed experiments in this mutant that were included in the original version of the manuscript and remain in the current version (Sup. Fig. 2C) and, under restrictive conditions, we do not see spontaneous N-Cap formation. If Hsp16 overexpression and nucleotide depletion were key to the mechanism triggering N-Cap appearance, we would expect this mutant to eventually form N-Caps when placed at restrictive temperature. Furthermore, Taricani et al. show that Hsp16 expression was abolished in a Δatf1 mutant background in the presence of HU, and we found that this mutant is still able to produce N-Caps in HU; therefore, our results strongly suggest that the phenotype of N-cap is independent on the MAPK pathway and on the expression of hsp16.

    Minor issues

    1. __P1 - UPR = Unfolded Protein Response: __Corrected in the manuscript
    2. 2__. P22 - HSP upregulation "might" be indicative of a folding stress:__ Corrected in the manuscript
    3. __ The abstract does not reflect the findings presented in the manuscript. In addition, I would recommend the authors revise the storytelling in their manuscript to push forward the message on either the specific phenotype associated with perinuclear ER or on the characterization of protein misfolding stress.__ We have modified the abstract to better reflect our findings and will further revise our arguments in the final version of the manuscript once we have the results of the experiments proposed

    Reviewer 2

    Major issue #1. The authors state the cytoplasmic and ER folding are both disrupted. The impact on ER protein biogenesis would be bolstered with some biochemical data focused on the folding of one or more nascent secretory proteins. Is disulfide bond formation and/or protein folding indeed disrupted?

    We have addressed the status of secretion in cells treated with HU or DIA by assessing the morphology of the Golgi apparatus and the localization of several secretory proteins by fluorescence microscopy and found that both HU and DIA treatments impact the secretion system. In addition, we plan on addressing the redox status of ER proteins (Bip1, Pdi or Ero1) by biochemical approaches. Please see the answer to major issue #2 from reviewer 1.

    We will also analyze by western blot the biogenesis and processing of the wildtype vacuolar Carboxypeptidase Y (Cpy1-GFP) and/or alkaline phosphase (Pho8-GFP), two widely used markers to test the functionality of the ER/endomembrane system.

    Major issue #2. Increased signal of Bip1 in the expanded perinuclear ER is shown and is suggested as consistent with immobilization of BiP upon binding of misfolded proteins. The authors suggest that this increased signal must reflect Bip1 redistribution because "Bip1 levels are constant". Yet, the western image (Figure 4B) looks to show increased level of Bip1 protein up HU treatment. Given the abundance of Bip1 in cells, it seems possible that a two-fold increase in newly synthesized proteins in the perinuclear region may account for the increased signal. These original data cited by the authors uses photobleaching (not just fluorescence intensity) to show a change in crowding / mobility, which the authors should consider to support their conclusion. Alternatively, a detected increased engagement of Bip1 with substrates (e.g. pulldown experiment) would be similarly strengthening.

    This same issue arose with reviewer 3, so we decided to change the image of the western blot showing another one with less exposure and added a quantification showing that Bip1-GFP levels remain mostly constant between control conditions and treatments with HU and DIA.

    We have also performed the suggested photobleaching experiment to analyze potential changes in crowding and mobility in Bip1-GFP upon HU treatment. We found that Bip1-GFP signal recovers after photobleaching the perinuclear ER in HU-treated cells that had not yet expanded the ER, showing that Bip1-GFP is dynamic in these conditions. However, Bip1-GFP signal did not recover after photobleaching the whole N-Cap in cells that had fully developed the expanded perinuclear ER phenotype, whereas it did recover when only half of the N-Cap region was bleached. This suggests that Bip1-GFP is mobile within the expanded perinuclear ER but cannot freely diffuse between the cortical and the perinuclear ER once the N-Cap is formed.

    These data have been included in the revised version of the manuscript, in figure 4B, sup. figures 4A-B, and in page 22.

    Major issue #3. It is curious that cycloheximide (CHX) has a distinct impact on HU versus DIA treatment. Blocking protein synthesis with CHX exacerbates the phenotype with DIA, but not HU. The authors use the data with CHX to argue that their drug treatments are interfering with folding during synthesis and translation into the ER. If so, what is the rationale as to why CHX treatment decreases expansion upon HU treatment? Relatedly, is protein synthesis and/or ER import impacted upon treatment with HU and/or DIA?

    As all three reviewers had comments about the CHX and Pm-related data, we revised those experiments and noticed a phenotype occurring upon HU+CHX treatment that had gone unnoticed previously and that changed our understanding about the effect of these drugs on the ER. Briefly, we noticed that, although CHX treatment decreases the HU-induced expansion of the perinuclear ER, it indeed induced expansion but in this case in the cortical area of the ER. This means that the phenotype of ER expansion in HU is not being suppressed by addition of CHX, but rather taking place in another area of the ER (cortical ER). We do not understand why this happens; however, these results show that ER expansion is exacerbated both in DIA and HU when combined with CHX. We have included this data in Figures 3C-D and in page 21.

    We also examined the trafficking of secretory proteins that go from the ER to the cell tips and noticed that this transit was affected under both drugs (Figures 3A-B). This suggests that, although there is still protein synthesis when cells are exposed to the drugs (as can be seen by the higher levels of chaperones induced by both stresses (Figure 4C-E)), their protein synthesis capacity is possibly impinged on to certain degree. All this information is now included in the manuscript (page 18).

    Major issue #4. While the authors suggest that there is disulfide stress in the ER / nucleus, the redox environment in these compartments is not tested directly (only cytoplasmic probes).

    Although we have only included experiments using one redox sensor in the manuscript, we had tested the oxidation of several biosensors during HU and DIA exposure monitoring cytoplasmic, mitochondrial and glutathione-specific probes. We have tried to use ER directed probes however, we have not been successful due to oversaturation of the probe in the highly oxidative environment of the ER lumen.

    Although so far we have not been able to directly test the redox status of the ER with optical probes, we plan to test the folding and redox status of several ER proteins and secretory markers by biochemical approaches, so hopefully these experiments will give us more information on this question (See answer to Reviewer 1, Main Issue #2 and Reviewer 2, Main issue #1).

    Major Issue #5. What do the authors envision is the role of the cytoplasmic chaperone foci? Do CHX / Pm treatment with HU/DIA reverse the chaperone foci?

    Pm causes premature termination of translation, leading to the release of truncated, misfolded, or incomplete polypeptides into the cytosol and the re-engagement of ribosomes in a new cycle of unproductive translation, as puromycin does not block ribosomes (Aviner, 2020; Azzam & Algranati, 1973). This likely decreases the number of peptides entering the ER that can be targeted by either HU or DIA, decreasing in turn ER expansion. Indeed, we have found that Pm treatment alone results in the formation of multiple cytoplasmic protein aggregates marked by Hsp104-GFP (Figure 4K), consistent with a continuous release of incomplete and misfolded nascent peptides to the cytoplasm. This would explain why Pm treatment suppresses N-Cap formation when cells are treated with either HU or DIA.

    To further test this idea, we analyzed the number and size of Hsp104-containing cytoplasmic aggregates in cells treated with HU or DIA and Pm, where N-Caps are suppressed. As expected, we found an increase in the accumulation of proteotoxicity in the cytoplasm in these conditions. This information has now been added to the paper (Figure 4K, pages 23-24 and 29).

    On the other hand, CHX inhibits translation elongation by stalling ribosomes on mRNAs, preventing further peptide elongation but leaving incomplete polypeptides tethered to the blocked ribosomes. This reduces overall protein load entering the ER by blocking new protein synthesis and stabilizes misfolded proteins bound to ribosomes. Accordingly, it has been shown previously that blocking translation with CHX abolishes cytoplasmic protein aggregation (Cabrera et al., 2020; Zhou et al., 2014). Similarly, we have found that Hsp104 foci are not observed when we add CHX alone or in combination with HU or DIA (Figures 4K-L). These results suggest that cytoplasmic foci that we observe upon HU or DIA treatment likely contain misfolded proteins derived from ongoing translation.

    As this question had also been raised by reviewer 1, we further explored the nature of these cytoplasmic foci (please see answer to Reviewer1, Issue 3). Briefly:

    • We tested whether they colocalize with the foci of Guk1-9-GFP and Rho1.C17R-GFP reporters of misfolding that appear upon HU or DIA treatments and, indeed, Hsp104-containing aggregates colocalize with Guk1-9-GFP and Rho1.C17R-GFP. This information has now been added to the paper (Figure 4I-J, pages 23-24 and 29).
    • We tested whether these foci were membrane bound with several ER transmembrane proteins (Tts1, Yop1, Rtn1) and integral membrane protein Ish1, and in none of the cases we detected membranes surrounding the aggregates. This information will be included in the final version of the paper.
    • We plan to test whether the cytoplasmic foci represent proteins retro-translocated from the ER.
    • We will also test whether autophagy or an imbalance between ER expansion and ER-phagy might contribute to the accumulation of cytoplasmic protein foci. The new data regarding the suppression of cytoplasmic foci by CHX treatment has already been included in the current version of the manuscript in Figure 4K and in the text (page 29).

    The authors argue that cytoplasmic foci are "independent" from ER expansion and are "not a direct consequence of thiol stress" based on the observation that DTT does not reverse these foci. This seems like a strong statement based on the limited analysis of these foci.

    We agree with the reviewer. We have toned down our statements about the relationship between thiol stress, the cytoplasmic chaperone foci and their relationship with ER expansion. We have removed from the text the statement that cytoplasmic foci are independent from ER expansion and thiol stress and have further revised our claims about CHX and Pm in the main text and the discussion to address these and the other reviewers’ concerns.

    Major Issue #6. Based on the transcriptional data, the authors speculate a potential role on role on iron-sulfur cluster protein biogenesis. This would seem to be rather straightforward to test.

    To address this issue, we plan to analyze the localization of proteins involved in iron-sulfur cluster assembly and/or containing iron-sulfur clusters by in vivo fluorescence microscopy, such as DNA polymerase Dna2 or Grx5, during HU or DIA treatments.

    Related to this, we have found that a subunit of the ribonucleotide reductase (RNR) aggregated in the cytoplasm upon HU exposure (Figure S2B). It is worth noting that RNR is an iron-containing protein whose maturation needs cytosolic Grxs (Cotruvo & Stubbe, 2011; Mühlenhoff et al., 2020). The catalytic site, the activity site (which governs overall RNR activity through interactions with ATP) and the specificity site (which determines substrate choice) are located in the R1 (Cdc22) subunits, which are the ones that aggregate, while the R2 subunits (Suc22) contain the di-nuclear iron center and a tyrosyl radical that can be transferred to the catalytic site during RNR activity (Aye et al., 2015). The fact that a subunit of RNR aggregates could be related to an impingement on its synthesis and/or maturation due to defects in iron-sulfur cluster formation, as it has been recently published that RNR cofactor biosynthesis shares components with cytosolic iron-sulfur protein biogenesis and that the iron-sulfur cluster assembly machinery is essential for iron loading and cofactor assembly in RNR in yeast (Li et al., 2017). This information has been added to the discussion.

    Major Issue #7. The authors suggest that "pre-treatment" with DTT before HU addition suppresses formation of the N-Caps. However, these samples (Figure 2J) contain DTT coincident with the treatment as well. To say it is the effect of pre-treatment, the DTT should be added and then washed out prior to HU or DIA addition. Alternatively, the language used to describe these experiments and their outcomes could be revised.

    We modified the language used to describe the experiment in the manuscript, as suggested by the reviewer, to clarify that while DTT is kept in the medium, N-Caps never form. In addition, we have also performed a pre-treatment with DTT; adding 1 mM DTT one hour before, washing the reducing agent out and adding HU to the medium then. The result indicates that pre-treating cells with DTT significantly reduces N-Cap formation after a 4-hour incubation with HU, which suggests that triggering reducing stress “protects” cells from the oxidative damage induced by HU and DIA. This information has been also added to the manuscript (Figure 2J).

    Major Issue #8. For a manuscript with 128 references there is rather limited discussion of the data in the context of the wider literature. The discussion primarily focuses on a recap of the results. The authors do cite several prior works focused on redox-dependent nuclear expansion. However, while cited, there is no real discussion of the relationship between this work in the context of that previously published (including several known disulfide bonded proteins that are involved in nuclear/ER architecture).

    We have revised and expanded our discussion. In addition, in the final revision of our work we will increase the discussion in the context of the new results obtained.

    Minor points

    1. __ Figure numbering goes from figure 4 to S6 to 5.__ We have updated the numbering of the figures after merging several supplementary figures, so now this issue is fixed.

    __ It would be helpful to the reader to explain what some of the reporters are in brief. For example, Guk1-9-GFP and Rho1.C17R-GFP reporters__.

    Both the Guk1-9-GFP and Rho1.C17R-GFP are two thermosensitive mutants in guanylate kinase and Rho1 GTPase respectively, that have been previously used in S. pombe as soluble reporters of misfolding in conditions of heat stress. During mild heat shock, both mutants aggregate into reversible protein aggregate centers (Cabrera et al., 2020). This information has now been added to the manuscript.

    __ Supplementary Figure 3. The main text suggests panel 3A is focused on diamide treatment. The figure legend discusses this in terms of HU treatment. Which is correct?__

    We thank the reviewer for pointing out this mistake. The experiment was performed in 75 mM HU, the legend was correct. It has now been corrected in the manuscript.

    __ The authors use ref 110 and 111 to suggest the importance of UPR-independent signaling. However, they do not point out that this UPR-independent signaling referred to in these papers is dependent on the UPR transmembrane kinase IRE1.__

    We have included pertinent clarification in the new discussion.

    Reviewer 3

    Major issue #1. It is hard to see how the claim of ER stress can be supported if BiP levels do not change (Fig. 4B). Also, this figure is overexposed. The RNA-seq data should be able to establish ER stress as well, but no rigorous analysis of ER stress markers is presented.

    Regarding the levels of Bip1, we now show in Figure 4 a less exposed image of the western blot, and a quantification of Bip1-GFP intensity from three independent experiments. We find that, in our experimental conditions, neither HU nor DIA treatments significantly altered Bip1 levels.

    With respect to the RNA-Seq, as we mentioned in the major issue 1 from reviewer 1, we reassessed our data to further clarify and add information about ER stress markers induced or repressed by HU and DIA.

    Major issue #2. The interpretation of the CHX and puromycin experiments of Figure 3A-B is hard to follow. My best guess is that the authors argue that CHX decreases misfolded protein load and that puromycin increases misfolded protein load, and that since DIA is a stronger oxidative stress than HU hence CHX is only protective under HU and not DIA. However, while CHX decreases misfolded protein load, puromycin hasn't been show directly to increase it and I don't see how this explains puromycin being protective at all.

    We have found that puromycin treatment alone results in the formation of cytoplasmic foci containing Hsp104, suggesting that puromycin indeed increases folding stress in the cytoplasm. We have now included this data in Figure 4K (please see Main Issue #5 from Reviewer 2). Pm suppresses the formation of N-caps induced by HU or DIA; however, we have not addressed cell survival or fitness in these conditions and therefore we cannot conclude about being protective.

    In addition, upon the reevaluation of our data, we have realized that CHX treatment suppresses HU-induced perinuclear expansion, although it does not suppress but instead enhances ER expansion in the cortical region. This data has been added to the present version of the manuscript in Figure 3C-D (pages 20-21).

    Furthermore, puromycin causes Ca leakage from the ER (which can be recapitulated with thapsigargin and blocked with anisomycin; easy experiments), which could be responsible for the differences from CHX, and the model does not address the effects on downstream stress signaling. The authors should be much more clear regarding their argument, since this data is used to support the argument of disrupted ER proteostasis.

    Thapsigargin has been described to be ineffective in yeasts as they lack a (SERCA)‐type Ca2+ pump which is the target of this drug (Strayle et al., 1999). However, deletion of the P5A-type ATPase Cta4, which is required for calcium transport into ER membranes (Lustoza et al., 2011), reduced but did not abolish ER expansion. We also tested the effect of anisomycin. We found that anisomycin in combination with HU or DIA mimicked CHX behavior (ER expansion occurrs in both conditions, exacerbating perinuclear ER expansion in combination with DIA and cortical ER expansion when combined with HU). It is difficult to correlate this result with a role of Ca leakage in ER expansion, as there is no recent information regarding CHX and Ca leakage, although it has been indicated that CHX treatment does not increase cytoplasmic Ca levels (Moses & Kline, 1995). As anisomycin, like CHX, blocks protein synthesis and stabilizes polysomes, what we can conclude from this information is that nascent peptides attached to ribosomes during protein synthesis do promote ER expansion when combined with HU or DIA. This information will be added to the final version of the paper.

    Regarding the downstream effects of HU or DIA treatment on ER proteostasis, we plan to further explore the effect of these drugs on the secretory system (please see major issue #2 from Reviewer 1) and to evaluate the redox state and processing of several key ER and secretory proteins. We have also further explored the nature of the aggregates that appear in the cytoplasm in our experimental conditions, which also shed light into the downstream effects of these drugs in cytoplasmic proteostasis (please see answer to issue #5 from Reviewer 2).

    Major issue #3. The claim that a canonical UPR is not induced is weak. First, the transcriptional program of S. cerevisiae from Travers et al is used as the canonical UPR, and compared to HU/DIA induced stress in S. pombe. These organisms may not be similar enough to assume that they have transcriptionally identical UPRs. Second, no consideration is given to the mechanism by which the different transcripts are modulated between "canonical" and HU/DIA induced UPR. Is it solely through RIDD, or does it point to differences in sensing or signaling transduction?

    We readdressed this topic by analyzing the genes that have been described to be differentially expressed during UPR activation in S. pombe and comparing them with our data by reevaluating our transcriptomic data.. The re-analysis of our RNA-Seq data have allowed us to infer the mechanisms that modulate the ER response to HU or DIA treatment and further separate them from UPR. This information has been added to the paper (page 26). As an alternative approach, we will also analyse the levels of UPR targets by western blot upon HU or DIA treatment

    Finally, the p-values used are unadjusted (e.g. by Bonferroni's method or by ANOVA or at least controlled by an FDR approach) and unmodulated (extremely important when n = 3 and variance is poorly sampled), which makes them not dependable. It looks like HSF1 targets are induced, which should be addressed.

    We thank the reviewer for pointing this out. We forgot to include this information which now appears in the M&M section as follows:

    “A gene was considered as differentially expressed when it showed an absolute value of log2FC(LFC)≥1 and an adjusted p-valueIn this regard, we are currently performing proteome-wide mass spectrometry experiments to detect protein glutathionylation in our conditions, as it has been previously shown that DIA treatment leads to glutathionylation of key ER proteins such as Bip1, Pdi or Ero1 (Lind et al., 2002; Wang & Sevier, 2016), which might by reproduced upon HU treatment. We also plan to test the folding and processing of specific secretory cargoes by western blot in our experimental conditions (see below, and Reviewer 2, Major issue #1).

    We have already tested whether mutant strains with deletions of key enzymes in both cytoplasmic and ER redox systems are able to expand the ER upon HU or DIA treatment. We have found that only *pgr1Δ *(glutathione reductase), *gsa1Δ *(glutathione synthetase) and gcs1Δ (glutamate-cysteine ligase) mutants fully suppressed N-Cap formation, which suggests that glutathione has an important role in the phenotype of ER expansion. We have now added the *pgr1Δ *mutant strain to the main text of the manuscript (Figure 5C, page 30).

    Major issue #5. Figure S5 presents weak ER expansion in fibrosarcoma cells in response to HU (at very low concentrations and DIA is not included). The lack of any other phenotypes being presented could suggest that such experiments were done but didn't show any effect. The authors should straightforwardly discuss whether they performed experiments looking for perinuclear ER expansion or NPC clustering, and if not, what challenges precluded such experiments. Given how important this line of experimentation is for establishing generality, much more discussion is needed here.

    We not only investigated the effects of HU on the ER in mammalian cells, but also of DIA. The results from this experiment mimicked the effect of HU (an increase in ER-ID fluorescence intensity in DIA). We merely excluded this information from the manuscript because we were focusing on HU at that point due to its importance as it is used currently in clinics. In this new version of the manuscript, we have included an extra panel in supplementary figure 5 to show the results from DIA in mammalian cells.

    Minor concerns

    1) Figure 1A should show individual data points (i.e. 3 averages of independent experiments) in the bar graph.

    Although we initially changed the graph, we believe the bar plot disposition facilitates its comprehension and went back to the initial one. Also, as the rest of the graphs similar to 1A are all expressed as bar plots. Therefore, we preferred keeping the figure as it was in the original version. However, we include here the graph with each of the averages of the independent experiments.

    2) It is argued that Figure 1B demonstrates that the SPB is clustered with the NPC cluster. However, a single image is not enough to support this claim, as the association could be coincidental.

    We have changed the image to show a whole population of cells, with several of them having NPC clusters, and we have indicated the position of SPB in each of them (all colocalizing with the N-Cap).

    3) Figures 1B through 1D do not indicate the HU concentration.

    We thank the reviewer for pointing out this mistake. Figures 1B and 1C represent cells exposed to 15 mM HU for 4 hours, while the graph in 1D shows the results from cells exposed to 75 mM HU over a 4-hour period. This information has been now added to the corresponding figure legend.

    4) I was confused by the photobleaching experiments of Figure S1. How do the authors know that there is complete photobleaching of the cytoplasm or nucleus in the absence of a positive control? If photobleaching is incomplete, they could be measuring motility without compartments rather than transport between compartments, and hence the conclusion that trafficking is unaffected could be wrong.

    Our control is the background of each microscopy image; we make sure that after the laser bleaches a cell, the bleached area coincides with the background noise. That way, we make sure that fluorescence from any remaining GFP is completely removed from the bleached area.

    5) On page 8, they say "exposure to DIA" when they intend HU.

    This has been corrected in the manuscript.

    6) In Figure S3A, the colocalization of INM proteins with the ER are presented. It is not clearly explained what conclusions are meant to be drawn from this figure, but it seems it would have been more useful to compare INM and Cut11, to see whether the NPCs are localizing at the INM or ONM.

    We have added an explanation in the main text to clarify the main conclusions derived from this figure. We think that NPCs localize in a section of the nucleus where the two membranes (INM and ONM) are still bound together.

    7) I had to read Figure 2C's description and caption several times to understand the experiment. A schematic would be helpful. 20 mM HU is low compared to most conditions used. Does repositioning eventually take place for 75 mM HU or 3 mM DIA treatment, or do the cells just die before they get a chance?

    20 mM HU was used in this experiment to provide a time frame suitable for analysis after HU addition, as a higher HU concentration increases the repositioning time. We found that both HU (75mM 4h) and DIA (3mM 4h)-induced ER expansions are reversible upon drug washout. If HU is kept in the media, ER expansions are eventually resolved. However, DIA is a strong oxidant and if it is kept in the media ER expansions are not resolved and cells do not survive.

    8) Figure 2D shows little oxidative consequence from 75 mM HU treatment until 40 min., the same time that phenotypes are observed (Figure 1D). Is this relationship consistent with the kinetics of other concentrations of HU, or of DIA? Seems like a pretty important mechanistic consideration that can rationalize the effects of the two oxidants.

    Thanks to this comment we realized that the numbering underneath Figure 1D (1E in the new version of the manuscript) was wrongly annotated. The original timings shown in the figure were “random”, meaning that the time stablished as 40 minutes was not measuring the passing of 40 minutes since the beginning of the experiment. We have now corrected this panel: the timings are now normalized to the moment when NPCs cluster. The fact that, before, that moment coincided with “40 minutes” does not mean N-Caps appear at that time point in HU (they indeed appear after a >2 hour incubation).

    9) Figure S4 is missing the asterisk on the lower left cell.

    Fixed in the corresponding figure.

    10) How is roundness determined in Figure S4B?

    Roundness in Figure S4B (now S2E) is determined the same way as in Figure 1D, and as is described in the Method section (copied below). A clarification has been added to the legend to address that.

    The ‘roundness’ parameter in the ‘Shape Descriptors’ plugin of Fiji/ImageJ was used after applying a threshold to the image in order to select only the more intense regions and subtract background noise (Schindelin et al., 2012). Roundness descriptor follows the function:

    where [Area] constitutes the area of an ellipse fitted to the selected region in the image and [Major axis] is the diameter of the round shape that in this case would fit the perimeter of the nucleus.

    11) What threshold is used to determine whether cells analyzed in Figures S4C have "small ER" or "large ER"?

    Large ER are considered when their area along the projection of a 3-Z section is over 4 μm2 (more than twice the mean area of the ER in cells with N-Caps in milder conditions). This has now been clarified in the legend of the corresponding figure.

    __12) The authors interpret Figure 4K as indicating that ER expansion is not involved in the generation of punctal misfolded protein aggregates. However, the washout occurs only after the proteins have already aggregated. The proper interpretation is that the aggregates are not reversible by resolution of the stress, and hence are not physically reliant on disulfide bonds. __

    We agree with the reviewer and have modified the interpretation of the indicated figure accordingly (page 29).

    The speculation that these proteins are iron dependent is a stretch; there is no reason to believe that losses of iron metabolism are the most important stress in these cells. It seems at least as likely that oxidizing cysteine-containing proteins in the cytosol or messing with the GSH/GSSG ratio in the cytosol would make plenty of proteins misfold; oxidative stress in budding yeast does activate hsf1. However, this point could be addresses by centrifugation and mass spectrometry to identify the aggregated proteome. It is also surprising that the authors did not investigate ER protein aggregation, perhaps by looking at puncta formation of chaperones beyond BiP. By contrast, the fact that gcs1 deletion prevents ER expansion but does not prevent Hsp104 puncta does support the idea that cytoplasmic aggregation is not dependent on ER expansion.

    To address this suggestion, we plan to analyze the localization of other chaperones and components of the protein quality control such as the ER Hsp40 Scj1 or the ribosome-associated Hsp70 Sks2.

    13) Figure 4L is cited on page 28 when Figure 4K is intended.

    This has been corrected in the text, although new panels have been added and now it is 4N.

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    Referee #3

    Evidence, reproducibility and clarity

    This article makes the following claims, using S. pombe as their model system. Hydroxyurea (HU) and diamide (DIA) induce ER stress, an atypical UPR, and cytoplasmic protein aggregation. HU and DIA induce IRE1-independent and GSH-dependent reversible ER perinuclear expansion which causes nuclear pore clustering with no effect on protein trafficking, and can be reversed by DTT.

    Major concerns:

    1. It is hard to see how the claim of ER stress can be supported if BiP levels do not change (Fig. 4B). Also, this figure is overexposed. The RNA-seq data should be able to establish ER stress as well, but no rigorous analysis of ER stress markers is presented.
    2. The interpretation of the CHX and puromycin experiments of Figure 3A-B is hard to follow. My best guess is that the authors argue that CHX decreases misfolded protein load and that puromycin increases misfolded protein load, and that since DIA is a stronger oxidative stress than HU hence CHX is only protective under HU and not DIA. However, while CHX decreases misfolded protein load, puromycin hasn't been show directly to increase it and I don't see how this explains puromycin being protective at all. Furthermore, puromycin causes Ca leakage from the ER (which can be recapitulated with thapsigargin and blocked with anisomycin; easy experiments), which could be responsible for the differences from CHX, and the model does not address the effects on downstream stress signaling. The authors should be much more clear regarding their argument, since this data is used to support the argument of disrupted ER proteostasis.
    3. The claim that a canonical UPR is not induced is weak. First, the transcriptional program of S. cerevisiae from Travers et al is used as the canonical UPR, and compared to HU/DIA induced stress in S. pombe. These organisms may not be similar enough to assume that they have transcriptionally identical UPRs. Second, no consideration is given to the mechanism by which the different transcripts are modulated between "canonical" and HU/DIA induced UPR. Is it solely through RIDD, or does it point to differences in sensing or signaling transduction? Finally, the p-values used are unadjusted (e.g. by Bonferroni's method or by ANOVA or at least controlled by an FDR approach) and unmodulated (extremely important when n = 3 and variance is poorly sampled), which makes them not dependable. It looks like HSF1 targets are induced, which should be addressed.
    4. Mechanistically, one would expect effects to be mediated by PDIs and oxidoreductases. No effort is made to characterize the redox state of these molecules, nor how that relates to the kinetics of ER expansion and resolution under HU/DIA treatment. No discussion is made of the existing literature on oxidants and ER stress. A few papers: PMID: 29504610, PMID: 31595201.
    5. Figure S5 presents weak ER expansion in fribrosarcoma cells in response to HU (at very low concentrations and DIA is not included). The lack of any other phenotypes being presented could suggest that such experiments were done but didn't show any effect. The authors should straightforwardly discuss whether they performed experiments looking for perinuclear ER expansion or NPC clustering, and if not, what challenges precluded such experiments. Given how important this line of experimentation is for establishing generality, much more discussion is needed here.

    Minor concerns:

    1. Figure 1A should show individual data points (i.e. 3 averages of independent experiments) in the bar graph.
    2. It is argued that Figure 1B demonstrates that the SPB is clustered with the NPC cluster. However, a single image is not enough to support this claim, as the association could be coincidental.
    3. Figures 1B through 1D do not indicate the HU concentration.
    4. I was confused by the photobleaching experiments of Figure S1. How do the authors know that there is complete photobleaching of the cytoplasm or nucleus in the absence of a positive control? If photobleaching is incomplete, they could be measuring motility without compartments rather than transport between compartments, and hence the conclusion that trafficking is unaffected could be wrong.
    5. On page 8, they say "exposure to DIA" when they intend HU.
    6. In Figure S3A, the colocalization of INM proteins with the ER are presented. It is not clearly explained what conclusions are meant to be drawn from this figure, but it seems it would have been more useful to compare INM and Cut11, to see whether the NPCs are localizing at the INM or ONM.
    7. I had to read Figure 2C's description and caption several times to understand the experiment. A schematic would be helpful. 20 mM HU is low compared to most conditions used. Does repositioning eventually take place for 75 mM HU or 3 mM DIA treatment, or do the cells just die before they get a chance?
    8. Figure 2D shows little oxidative consequence from 75 mM HU treatment until 40 min., the same time that phenotypes are observed (Figure 1D). Is this relationship consistent with the kinetics of other concentrations of HU, or of DIA? Seems like a pretty important mechanistic consideration that can rationalize the effects of the two oxidants.
    9. Figure S4 is missing the asterisk on the lower left cell.
    10. How is roundness determine in Figure S4B?
    11. What threshold is used to determine whether cells analyzed in Figures S4C have "small ER" or "large ER"?
    12. The authors interpret Figure 4K as indicating that ER expansion is not involved in the generation of punctal misfolded protein aggregates. However, the washout occurs only after the proteins have already aggregated. The proper interpretation is that the aggregates are not reversible by resolution of the stress, and hence are not physically reliant on disulfide bonds. The speculation that these proteins are iron dependent is a stretch; there is no reason to believe that losses of iron metabolism are the most important stress in these cells. It seems at least as likely that oxidizing cysteine-containing proteins in the cytosol or messing with the GSH/GSSG ratio in the cytosol would make plenty of proteins misfold; oxidative stress in budding yeast does activate hsf1. However, this point could be addresses by centrifugation and mass spectrometry to identify the aggregated proteome. It is also surprising that the authors did not investigate ER protein aggregation, perhaps by looking at puncta formation of chaperones beyond BiP. By contrast, the fact that gcs1 deletion prevents ER expansion but does not prevent Hsp104 puncta does support the idea that cytoplasmic aggregation is not dependent on ER expansion.
    13. Figure 4L is cited on page 28 when Figure 4K is intended.

    Significance

    This paper is for the most part well-written, presenting a logical chain of experiments that fully support the most important claims that have been made. Specifically, they show that HU and DIA induce reversible perinuclear expansion and nuclear pore clustering in an IRE1-independent and GSH-dependent manner, and that DTT can prevent and accelerate recovery of this phenotype. Both oxidants clearly induce protein aggregation in the cytosol. The evidence that perinuclear expansion is responsible for nuclear pore clustering is compelling, with strong support from the kinetics and the nup120 deletion experiments. Some conclusions are not supported, including the claim of an atypical UPR and of ER stress, but the validity of these claims does not substantively affect the overall importance of the paper and could be handled by withdrawal or tempering of the claims. The lack of a molecular mechanism connecting oxidation with ER expansion moderately detracts from the potential impact. Adequate experimental detail is provided unless otherwise noted

    This paper is likely to be important for cell biologists interested in interorganelle communication and how the cell responds to oxidative stress. Modulating ER oxidoreductase activity has been shown to be a powerful way to regulate ER stress and proteostasis, and this paper shows how specific oxidative stresses that have not widely been investigated in this context, as opposed to the more commonly studied reductive and electrophilic stresses, can remodel the ER with cell-wide consequences. More specifically, the nuclear pore and nuclear morphology phenotypes, while not yet functionally significant in yeast, could be significant in other unexplored ways identified in the future. Towards that end, it would be valuable to see if these gross phenotypes reproduce in any metazoan cell or tissue, rather than just looking at ER expansion as in the current manuscript. My expertise is centered around ER proteostasis and chaperones, and as such I consider this paper important to my field.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript by Sánchez-Molina et al describes a striking time and dose-dependent clustering of nuclear pores and perinuclear ER expansion in response to hydroxyurea (HU) or diamide (DIA) treatment in S. pombe. Using microscopy, the authors establish clustering is reversible upon drug washout or extended drug treatment. Pretreatment or post-treatment with the reductant DTT prevents or reverses the clustering and expansion effects, as does the release of translating polypeptides from ribosomes (with puromycin). The phenotypes were established to occur independent of the established impact of HU on RNR activity and the cell cycle. The authors suggest instead that the phenotypes (referred to as nuclear-cap (N-Cap) formation) are associated with disulfide-based folding stress. Overlapping transcriptional responses for HU and DIA treatment suggest that cells are experiencing folding stress (based on chaperone induction) and/or a disruption in iron homeostasis (induction of genes involved in iron homeostasis). The observed clustering, ER expansion, and transcriptional profiles are independent of the well-established ER stress response pathway: the UPR.

    The manuscript outlines several interesting phenotypic observations, and they establish the potential for conserved of this ER expansion and nuclear pore clustering from yeast (S. cerevisiae) and mammals (HT1080 fibrosarcoma cells). Data clearly establish the time and dose-dependent formation of these interesting structures. Additional experiments with combined drug treatments points towards a role for changes in the redox environment in cells, an impact on cytoplasmic protein aggregation, and a potential impact on the ER folding environment / ER redox environment.

    Data obtained with thiol oxidants and reductants, alongside translation inhibitors, suggest a potential connection between the N-Cap phenotype and oxidative folding within the ER. Yet, this latter observation remains a suggestive link with less clear mechanistic connections. Some experiments that would more directly assess the suggested changes within the nuclear/ER region are outlined below.

    1. The authors state the cytoplasmic and ER folding are both disrupted. The impact on ER protein biogenesis would be bolstered with some biochemical data focused on the folding of one or more nascent secretory proteins. Is disulfide bond formation and/or protein folding indeed disrupted?
    2. Increased signal of Bip1 in the expanded perinuclear ER is shown and is suggested as consistent with immobilization of BiP upon binding of misfolded proteins. The authors suggest that this increased signal must reflect Bip1 redistribution because "Bip1 levels are constant". Yet, the western image (Figure 4B) looks to show increased level of Bip1 protein up HU treatment. Given the abundance of Bip1 in cells, it seems possible that a two-fold increase in newly synthesized proteins in the perinuclear region may account for the increased signal. These original data cited by the authors uses photobleaching (not just fluorescence intensity) to show a change in crowding / mobility, which the authors should consider to support their conclusion. Alternatively, a detected increased engagement of Bip1 with substrates (e.g. pulldown experiment) would be similarly strengthening.
    3. It is curious that cycloheximide (CHX) has a distinct impact on HU versus DIA treatment. Blocking protein synthesis with CHX exacerbates the phenotype with DIA, but not HU. The authors use the data with CHX to argue that their drug treatments are interfering with folding during synthesis and translation into the ER. If so, what is the rationale as to why CHX treatment decreases expansion upon HU treatment? Relatedly, is protein synthesis and/or ER import impacted upon treatment with HU and/or DIA?
    4. While the authors suggest that there is disulfide stress in the ER / nucleus, the redox environment in these compartments is not tested directly (only cytoplasmic probes).

    Addition suggestions / comments:

    1. What do the authors envision is the role of the cytoplasmic chaperone foci? Do CHX / Pm treatment with HU/DIA reverse the chaperone foci? The authors argue that cytoplasmic foci are "independent" from ER expansion and are "not a direct consequence of thiol stress" based on the observation that DTT does not reverse these foci. This seems like a strong statement based on the limited analysis of these foci.
    2. Based on the transcriptional data, the authors speculate a potential role on role on iron-sulfur cluster protein biogenesis. This would seem to be rather straightforward to test.
    3. The authors suggest that "pre-treatment" with DTT before HU addition suppresses formation of the N-Caps. However, these samples (Figure 2J) contain DTT coincident with the treatment as well. To say it is the effect of pre-treatment, the DTT should be added and then washed out prior to HU or DIA addition. Alternatively, the language used to describe these experiments and their outcomes could be revised.
    4. For a manuscript with 128 references there is rather limited discussion of the data in the context of the wider literature. The discussion primarily focuses on a recap of the results. The authors do cite several prior works focused on redox-dependent nuclear expansion. However, while cited, there is no real discussion of the relationship between this work in the context of that previously published (including several known disulfide bonded proteins that are involved in nuclear/ER architecture).

    Minor points

    1. Figure numbering goes from figure 4 to S6 to 5.
    2. It would be helpful to the reader to explain what some of the reporters are in brief. For example, Guk1-9-GFP and Rho1.C17R-GFP reporters.
    3. Supplementary Figure 3. The main text suggests panel 3A is focused on diamide treatment. The figure legend discusses this in terms of HU treatment. Which is correct?
    4. The authors use ref 110 and 111 to suggest the importance of UPR-independent signaling. However, they do not point out that this UPR-independent signalling referred to in these papers is dependent on the UPR transmembrane kinase IRE1.

    Significance

    An interesting finding that is well-supported as a phenotype. What would raise the impact would be data that connect these observations more directly with a mechanism. In particular, there are suggestions of a disruption in ER folding and/or the ER redox environment that are logical but not directly tested. How one viewed these additional experiments will depend on what journal is considering the manuscript.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Sanchez-Molina describe the impact of hydroxyurea on the remodeling of the nuclear pore complex (clustering) and the expansion of both cortical and perinuclear ER. The study is carried out in S. pombe, and the observations confirmed in S. cerevisiae. Results are clear and analyzed properly, however considering the differences in UPR signaling in both yeast strains the conclusions raised may remain to be fully documented.

    Major issues

    Regarding the conclusions on IRE1 signaling, both yeast species have different IRE1 activities https://elifesciences.org/articles/00048), the total deletion of IRE1 in S pombe appears to indicate that expansion of perinuclear ER is independent of IRE1, however since IRE1 signaling has exclusively a negative impact on mRNA expression, it might be relevant to identify mRNA whose expression is stabilized under those circumstances and evaluate whether those could confer a mechanism which would also yield perinuclear ER expansion (eg differential deregulation of ER stress controlled lipid biosynthesis required for lipid membrane synthesis). In S cerevisiae, do the authors observe HAC1 mRNA splicing?

    The authors indicate that HU and DIA lead to thiol stress, it might be relevant to evaluate the thiol-redox status of major secretory proteins in S pombe (or even cargo reporters if necessary) to fully document the stress impact on global protein redox status. What happens if HU-treated yeast cells are grown in the presence of n-acetyl cysteine?

    The appearance of cytosolic aggregates is intriguing, do the authors have any idea on the nature of the protein aggregates? Are those resulting from proficient retrotranslocation (or reflux of misfolded proteins from the ER? Are those aggregates membrane bound or do they correspond to aggresomes as initially defined?

    The Walter lab has demonstrated a tight balance between ER phagy and ER membrane expansion (https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.0040423), which could also impact on the presence of protein aggregates in the cytosol. Does HU impact on the regulation of autophagy?

    Nucleotide depletion was previously shown to lead to HSP16 expression through activation of the spc1 MAPK pathway (10.1093/nar/29.14.3030), one might think that HU (or diamide) could lead to this through a nucleotide dependent mechanism and not necessary through a thiol-redox protein misfolding stress. This issue has to be sorted out to ensure that the HSP effect is independent of nucleotide depletion.

    Minor issues

    P1 - UPR = Unfolded Protein Response

    P22 - HSP upregulation "might" be indicative of a folding stress

    The abstract does not reflect the findings presented in the manuscript. In addition, I would recommend the authors to revise the story telling in their manuscript to push forward the message on either the specific phenotype associated with perinuclear ER or on the characterization of protein misfolding stress.

    Significance

    This is a nice manuscript describing the likely effects of HU on protein misfolding and several consequences including the remodeling of the nuclear pore complex, the expansion of both cortical and perinuclear ER. The underlying mechanisms remain however unclear (for each parameter evaluated) and the manuscript would definitely benefit from the elucidation of one of those (if not more).

    The work in yeast is novel and might bring light on mechanisms existing in mammalian systems. Since HU is used as a therapeutic, the characterization of the molecular mechanisms associated with its mode(s) of action will definitely be useful for better (targeted) efficiency.

    The audience for this work is more targeted towards people working on yeast cell biology, however, the authors could expand the discussion section to make it of a broader scope.

    I am expert on ER stress signaling

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    Reply to the reviewers

    Reviewer 1

    Major issue #1. Regarding the conclusions on IRE1 signaling, both yeast species have different IRE1 activities (https://elifesciences.org/articles/00048), the total deletion of IRE1 in S pombe appears to indicate that expansion of perinuclear ER is independent of IRE1, however since IRE1 signaling has exclusively a negative impact on mRNA expression, it might be relevant to identify mRNA whose expression is stabilized under those circumstances and evaluate whether those could confer a mechanism which would also yield perinuclear ER expansion (eg differential deregulation of ER stress controlled lipid biosynthesis required for lipid membrane synthesis). In S. cerevisiae, do the authors observe HAC1 mRNA splicing?

    We have not tested whether HAC1 mRNA is processed in S. cerevisiae. To address this question, we will perform RT-PCR to test it.

    In addition, as requested by the reviewers, we will further test the involvement of Ire1 in the HU/DIA-induced phenotype in S. pombe. For that, we will reassess our RNA-seq data and compare it with data from (Kimmig et al., 2012) (UPR activation in S. pombe). We will test the levels and splicing of mRNA of Bip1 upon HU/DIA treatments by RT-PCR and finally we will test the levels of Gas2p which has been described to decrease upon Ire1/UPR activation in S. pombe.

    We are confident in that the results of these experiments and the re-analysis of our RNA-Seq data will help us to infer the mechanisms that modulate the ER response to HU or DIA treatment.

    Major issue #2. The authors indicate that HU and DIA lead to thiol stress, it might be relevant to evaluate the thiol-redox status of major secretory proteins in S. pombe (or even cargo reporters if necessary) to fully document the stress impact on global protein redox status.

    We agree with the reviewer that it is important to determine the redox and the functional state of the secretory pathway in our conditions to fully understand the cellular consequences of these treatments, especially in the case of HU, as it is routinely used in clinics.

    In this context, we have already included new data showing that HU or DIA treatment leads to alterations in the Golgi apparatus and in the distribution of secretory proteins (Figures 3A-B).

    In addition, we plan to perform mass spectrometry experiments to detect protein glutathionylation in our conditions, as it has been previously shown that DIA treatment leads to glutathionylation of key ER proteins such as Bip1, Pdi or Ero1 (Lind et al., 2002; Wang & Sevier, 2016), which might by reproduced upon HU treatment. We will test specifically the redox state of Bip1, Pdi and/or Ero1 by immunoprecipitation and western blot.

    Finally, we plan to test the folding and processing of specific secretory cargoes by western blot in our experimental conditions (See below, Reviewer 2, Major issue #1).

    What happens if HU-treated yeast cells are grown in the presence of n-acetyl cysteine?

    We have tested whether the addition of this antioxidant could prevent and/or revert the N-Cap phenotype. We found that NAC in combination with HU increased N-Cap incidence (Figure 5H). As NAC is a GSH precursor and we find that GSH is required to develop the phenotype of N-Cap (Figure 5A-B, D, G), this result further supports that the HU-induced cellular damage might involve ectopic glutathionylation of proteins.

    Unfortunately, we have not tested NAC in combination with DIA, as NAC seems to reduce DIA as soon as they get in contact, as judged by the change in the characteristic orange color of DIA, the same that happens when we combine GSH and DIA (Supplementary Figure 5A-B).

    In this regard, the following information has been added to the manuscript (page 32-33, highlighted in blue):

    "We also tested GSH addition to the medium in combination with either HU or DIA. When mixed with DIA, we noticed that the color of the culture changed after GSH addition (Figure S5A), which suggests that GSH and DIA can interact extracellularly, thus preventing us from being able to draw conclusions from those experiments. On the other hand, combining GSH with HU increased N-Cap incidence (Figure 5G), as expected based on our previous observations. Additionally, we checked whether the addition of the antioxidant N-acetyl cysteine (NAC), a GSH precursor, impacted upon the N-Cap phenotype. The results were the same as with GSH addition: when combined with HU, NAC increased N-Cap incidence (Figure 5H), whereas in combination, the two compounds interacted extracellularly (Figure S5B). These data align with NAC being a precursor of GSH, as incrementing GSH levels augments the penetrance of the HU-induced phenotype".

    Major issue #3. The appearance of cytosolic aggregates is intriguing, do the authors have any idea on the nature of the protein aggregates?

    DIA is a strong oxidant, and HU treatment results in the production of reactive oxygen species (ROS). Therefore, one hypothesis would be that cytoplasmic chaperone foci represent oxidized and/or misfolded soluble proteins. Indeed, this hypothesis is supported by the appearance of cytoplasmic foci containing the guk1-9-GFP and Rho1.C17R-GFP soluble reporters of misfolding upon HU or DIA treatment (Figure 4I-J). We have already tested if they contain Vgl1, which is one of the main components of heat shock induced stress granules in *S. pombe *(Wen et al., 2010). However, we found that HU or DIA-induced foci lacked this stress granule marker, and indeed Vgl1 did not form any foci in response to these treatments. Therefore, our aggregates differ from the canonical stress-induced granules. We have yet to include this data in the manuscript, but we plan to do that for the final version.

    To further explore the nature of the cytoplasmic aggregates induced by HU and DIA, we will test whether Hsp104-containing foci colocalize with guk1-9-GFP and/or Rho1.C17R-GFP foci.

    Are those resulting from proficient retrotranslocation or reflux of misfolded proteins from the ER?

    To test whether these cytosolic aggregates result from retrotranslocation from the ER, we plan to use the vacuolar Carboxipeptidase Y mutant reporter CPY*, which is misfolded. This misfolded protein is imported into the ER lumen but does not reach the vacuole. Instead, it is retrotranslocated to the cytoplasm, where it is ubiquitinated and degraded by the proteasome (Mukaiyama et al., 2012). We will analyze by fluorescence microscopy the localization of CPY*´-GFP and Hsp104-containing aggregates upon HU or DIA treatment and with or without proteasome inhibitors. We can also test the levels, processing and ubiquitination of CPY*-GFP by western blot, as ubiquitination of retrotranslocated proteins occurs once they are in the cytoplasm.

    Are those aggregates membrane bound or do they correspond to aggresomes as initially defined? The Walter lab has demonstrated a tight balance between ER phagy and ER membrane expansion (https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.0040423), which could also impact on the presence of protein aggregates in the cytosol.

    Our results suggest that these aggregates are not bound to ER membranes, as they do not appear in close proximity to the ER area marked by mCherry-AHDL in fluorescence microscopy images.

    To fully rule out this possibility, we will test whether these Hsp104-aggregates colocalize with ER transmembrane proteins such as Rtn1 or Yop1, with Gma12-GFP that marks the Golgi apparatus and with the dye FM4-64 that stains endosomal-vacuole membranes.

    We have tested whether deletion of key genes involved in autophagy affected the N-Cap phenotype. To this end, we used deletions of ypt1, vac8 and atg8 in strains expressing Cut11-GFP and/or mCherry-AHDL and found that none of them affected N-Cap formation. These data suggest that the core machinery of autophagy is not critical for HU/DIA-induced ER expansion. We plan to include this data in the final version of the manuscript along with the rest of experiments proposed.

    To get deeper insights and to fully rule out a possible contribution of macro-autophagy to the HU- and DIA-induced phenotypes, we plan to analyze by western blot whether GFP-Atg8 is induced and cleaved upon HU or DIA treatments which would be indicative of macroautophagy activation.

    To test whether the cytoplasmic aggregates are the result of an imbalance between ER-expansion and ER-phagy we plan to analyze the localization of GFP-Atg8 and Hsp104-RFP in the atg7Δ mutant, impaired in the core macro-autophagy machinery. In these conditions, the number or size of the cytoplasmic aggregates might be impacted.

    On the other hand, it has been recently shown that an ER-selective microautophagy occurs in yeasts upon ER stress (Schäfer et al., 2020; Schuck et al., 2014). This micro-ER-phagy involves the direct uptake of ER membranes into lysosomes, is independent of the core autophagy machinery and depends on the ESCRT system and is influenced by the Nem1-Spo7 phosphatase. ESCRT directly functions in scission of the lysosomal membrane to complete the uptake of the ER membrane. Interestingly, N-Caps are fragmented in the absence of cmp7 and specially in the absence of vps4 or lem2, the nuclear adaptor of the ESCRT (Figure 3E), We had initially interpreted these results as the need to maintain nuclear membrane identity during the process of ER expansion (Kume et al., 2019); however, the appearance of fragmented ER upon HU treatment in the absence of ESCRT might also be due to an inability to complete microautophagic uptake of ER membranes. To test this hypothesis, we plan to analyze whether the fragmented ER in these conditions co-localize with lysosome/vacuole markers.

    Major issue #4. Nucleotide depletion was previously shown to lead to HSP16 expression through activation of the spc1 MAPK pathway (https://academic.oup.com/nar/article/29/14/3030/2383924), one might think that HU (or diamide) could lead to this through a nucleotide dependent mechanism and not necessary through a thiol-redox protein misfolding stress. This issue has to be sorted out to ensure that the HSP effect is independent of nucleotide depletion.

    As stated in (Taricani et al., 2001), hsp16 expression is strongly induced in a cdc22-M45 mutant background. We performed experiments in this mutant that were included in the original version of the manuscript and remain in the current version (Sup. Fig. 2C) and, under restrictive conditions, we do not see spontaneous N-Cap formation. If Hsp16 overexpression and nucleotide depletion were key to the mechanism triggering N-Cap appearance, we would expect this mutant to eventually form N-Caps when placed at restrictive temperature. Furthermore, Taricani et al. show that Hsp16 expression was abolished in a Δatf1 mutant background in the presence of HU, and we found that this mutant is still able to produce N-Caps in HU; therefore, our results strongly suggest that the phenotype of N-cap is independent on the MAPK pathway and on the expression of hsp16.

    Minor issues

    1. __P1 - UPR = Unfolded Protein Response: __Corrected in the manuscript
    2. 2__. P22 - HSP upregulation "might" be indicative of a folding stress:__ Corrected in the manuscript
    3. __ The abstract does not reflect the findings presented in the manuscript. In addition, I would recommend the authors revise the storytelling in their manuscript to push forward the message on either the specific phenotype associated with perinuclear ER or on the characterization of protein misfolding stress.__ We have modified the abstract to better reflect our findings and will further revise our arguments in the final version of the manuscript once we have the results of the experiments proposed

    Reviewer 2

    Major issue #1. The authors state the cytoplasmic and ER folding are both disrupted. The impact on ER protein biogenesis would be bolstered with some biochemical data focused on the folding of one or more nascent secretory proteins. Is disulfide bond formation and/or protein folding indeed disrupted?

    We have addressed the status of secretion in cells treated with HU or DIA by assessing the morphology of the Golgi apparatus and the localization of several secretory proteins by fluorescence microscopy and found that both HU and DIA treatments impact the secretion system. In addition, we plan on addressing the redox status of ER proteins (Bip1, Pdi or Ero1) by biochemical approaches. Please see the answer to major issue #2 from reviewer 1.

    We will also analyze by western blot the biogenesis and processing of the wildtype vacuolar Carboxypeptidase Y (Cpy1-GFP) and alkaline phosphase (Pho8-GFP), two widely used markers to test the functionality of the ER/endomembrane system.

    Major issue #2. Increased signal of Bip1 in the expanded perinuclear ER is shown and is suggested as consistent with immobilization of BiP upon binding of misfolded proteins. The authors suggest that this increased signal must reflect Bip1 redistribution because "Bip1 levels are constant". Yet, the western image (Figure 4B) looks to show increased level of Bip1 protein up HU treatment. Given the abundance of Bip1 in cells, it seems possible that a two-fold increase in newly synthesized proteins in the perinuclear region may account for the increased signal. These original data cited by the authors uses photobleaching (not just fluorescence intensity) to show a change in crowding / mobility, which the authors should consider to support their conclusion. Alternatively, a detected increased engagement of Bip1 with substrates (e.g. pulldown experiment) would be similarly strengthening.

    This same issue arose with reviewer 3, so we decided to change the image of the western blot showing another one with less exposure and added a quantification showing that Bip1-GFP levels remain mostly constant between control conditions and treatments with HU and DIA.

    We have also performed the suggested photobleaching experiment to analyze potential changes in crowding and mobility in Bip1-GFP upon HU treatment. We found that Bip1-GFP signal recovers after photobleaching the perinuclear ER in HU-treated cells that had not yet expanded the ER, showing that Bip1-GFP is dynamic in these conditions. However, Bip1-GFP signal did not recover after photobleaching the whole N-Cap in cells that had fully developed the expanded perinuclear ER phenotype, whereas it did recover when only half of the N-Cap region was bleached. This suggests that Bip1-GFP is mobile within the expanded perinuclear ER but cannot freely diffuse between the cortical and the perinuclear ER once the N-Cap is formed.

    These data have been included in the revised version of the manuscript, in figure 4B, sup. figures 4A-B, and in page 23.

    Major issue #3. It is curious that cycloheximide (CHX) has a distinct impact on HU versus DIA treatment. Blocking protein synthesis with CHX exacerbates the phenotype with DIA, but not HU. The authors use the data with CHX to argue that their drug treatments are interfering with folding during synthesis and translation into the ER. If so, what is the rationale as to why CHX treatment decreases expansion upon HU treatment? Relatedly, is protein synthesis and/or ER import impacted upon treatment with HU and/or DIA?

    As all three reviewers had comments about the CHX and Pm-related data, we revised those experiments and noticed a phenotype occurring upon HU+CHX treatment that had gone unnoticed previously and that changed our understanding about the effect of these drugs on the ER. Briefly, we noticed that, although CHX treatment decreases the HU-induced expansion of the perinuclear ER, it indeed induced expansion but in this case in the cortical area of the ER. This means that the phenotype of ER expansion in HU is not being suppressed by addition of CHX, but rather taking place in another area of the ER (cortical ER). We do not understand why this happens; however, these results show that ER expansion is exacerbated both in DIA and HU when combined with CHX. We have included this data in Figures 3C-D and in page 22.

    We also examined the trafficking of secretory proteins that go from the ER to the cell tips and noticed that this transit was affected under both drugs (Figures 3A-B). This suggests that, although there is still protein synthesis when cells are exposed to the drugs (as can be seen by the higher levels of chaperones induced by both stresses (Figure 4C-E)), their protein synthesis capacity is possibly impinged on to certain degree. All this information is now included in the manuscript (page 19).

    Major issue #4. While the authors suggest that there is disulfide stress in the ER / nucleus, the redox environment in these compartments is not tested directly (only cytoplasmic probes).

    Although we have only included experiments using one redox sensor in the manuscript, we had tested the oxidation of several biosensors during HU and DIA exposure monitoring cytoplasmic, mitochondrial and glutathione-specific probes. We have tried to use ER directed probes however, we have not been successful due to oversaturation of the probe in the highly oxidative environment of the ER lumen.

    Although so far we have not been able to directly test the redox status of the ER with optical probes, we plan to test the folding and redox status of several ER proteins and secretory markers by biochemical approaches, so hopefully these experiments will give us more information on this question (See answer to Reviewer 1, Main Issue #2 and Reviewer 2, Main issue #1).

    Major Issue #5. What do the authors envision is the role of the cytoplasmic chaperone foci? Do CHX / Pm treatment with HU/DIA reverse the chaperone foci?

    Pm causes premature termination of translation, leading to the release of truncated, misfolded, or incomplete polypeptides into the cytosol and the re-engagement of ribosomes in a new cycle of unproductive translation, as puromycin does not block ribosomes (Aviner, 2020; Azzam & Algranati, 1973). This is likely to decrease the number of peptides entering the ER that can be targeted by either HU or DIA, decreasing in turn ER expansion. Indeed, we have found that Pm treatment alone results in the formation of multiple cytoplasmic protein aggregates marked by Hsp104-GFP (Figure 4K), consistent with a continuous release of incomplete and misfolded nascent peptides to the cytoplasm. This would explain why Pm treatment suppresses N-Cap formation when cells are treated with either HU or DIA.

    To further test this idea, we plan to carefully analyze the number, size and dynamics of Hsp104-containing cytoplasmic aggregates in cells treated with HU or DIA and Pm, where N-Caps are suppressed. We expect to find an increase in the accumulation of proteotoxicity in the cytoplasm in these conditions.

    On the other hand, CHX inhibits translation elongation by stalling ribosomes on mRNAs, preventing further peptide elongation but leaving incomplete polypeptides tethered to the blocked ribosomes. This reduces overall protein load entering the ER by blocking new protein synthesis and stabilizes misfolded proteins bound to ribosomes. Accordingly, it has been shown previously that blocking translation with CHX abolishes protein aggregation (Cabrera et al., 2020; Zhou et al., 2014). Similarly, we have found that Hsp104 foci are not observed when we add CHX alone or in combination with HU or DIA (Figures 4K-L). These results suggest that cytoplasmic foci that we observe upon HU or DIA treatment likely contain misfolded proteins derived from ongoing translation.

    As this question has also been raised by reviewer 1, we have decided to further explore the nature of these cytoplasmic foci (please see answer to Reviewer1, Issue 3). Briefly:

    • We plan to test whether they colocalize with the foci of Guk1-9-GFP and Rho1.C17R-GFP reporters of misfolding that appear upon HU or DIA treatments.
    • We will test whether these foci are membrane bound.
    • We plan to test whether the cytoplasmic foci represent proteins retro-translocated from the ER.
    • We will also test whether autophagy or an imbalance between ER expansion and ER-phagy might contribute to the accumulation of cytoplasmic protein foci. The new data regarding the suppression of cytoplasmic foci by CHX treatment has already been included in the current version of the manuscript in Figure 4K and in the text (page 30).

    The authors argue that cytoplasmic foci are "independent" from ER expansion and are "not a direct consequence of thiol stress" based on the observation that DTT does not reverse these foci. This seems like a strong statement based on the limited analysis of these foci.

    We agree with the reviewer. We have toned down our statements about the relationship between thiol stress, the cytoplasmic chaperone foci and their relationship with ER expansion. We have removed from the text the statement that cytoplasmic foci are independent from ER expansion and thiol stress and have further revised our claims about CHX and Pm in the main text and the discussion to address these and the other reviewers' concerns.

    Major Issue #6. Based on the transcriptional data, the authors speculate a potential role on role on iron-sulfur cluster protein biogenesis. This would seem to be rather straightforward to test.

    To address this issue, we plan to analyze the localization of proteins involved in iron-sulfur cluster assembly and/or containing iron-sulfur clusters by in vivo fluorescence microscopy, such as DNA polymerase Dna2 or Grx5, during HU or DIA treatments.

    Related to this, we have found that a subunit of the ribonucleotide reductase (RNR) aggregated in the cytoplasm upon HU exposure (Figure S2B). It is worth noting that RNR is an iron-containing protein whose maturation needs cytosolic Grxs (Cotruvo & Stubbe, 2011; Mühlenhoff et al., 2020). The catalytic site, the activity site (which governs overall RNR activity through interactions with ATP) and the specificity site (which determines substrate choice) are located in the R1 (Cdc22) subunits, which are the ones that aggregate, while the R2 subunits (Suc22) contain the di-nuclear iron center and a tyrosyl radical that can be transferred to the catalytic site during RNR activity (Aye et al., 2015). The fact that a subunit of RNR aggregates could be related to an impingement on its synthesis and/or maturation due to defects in iron-sulfur cluster formation, as it has been recently published that RNR cofactor biosynthesis shares components with cytosolic iron-sulfur protein biogenesis and that the iron-sulfur cluster assembly machinery is essential for iron loading and cofactor assembly in RNR in yeast (Li et al., 2017). This information has been added to the discussion.

    Major Issue #7. The authors suggest that "pre-treatment" with DTT before HU addition suppresses formation of the N-Caps. However, these samples (Figure 2J) contain DTT coincident with the treatment as well. To say it is the effect of pre-treatment, the DTT should be added and then washed out prior to HU or DIA addition. Alternatively, the language used to describe these experiments and their outcomes could be revised.

    We modified the language used to describe the experiment in the manuscript, as suggested by the reviewer, to clarify that while DTT is kept in the medium, N-Caps never form. In addition, we have also performed a pre-treatment with DTT; adding 1 mM DTT one hour before, washing the reducing agent out and adding HU to the medium then. The result indicates that pre-treating cells with DTT significantly reduces N-Cap formation after a 4-hour incubation with HU, which suggests that triggering reducing stress "protects" cells from the oxidative damage induced by HU and DIA. This information has been also added to the manuscript (Figure 2J).

    Major Issue #8. For a manuscript with 128 references there is rather limited discussion of the data in the context of the wider literature. The discussion primarily focuses on a recap of the results. The authors do cite several prior works focused on redox-dependent nuclear expansion. However, while cited, there is no real discussion of the relationship between this work in the context of that previously published (including several known disulfide bonded proteins that are involved in nuclear/ER architecture).

    We have revised and expanded our discussion. In addition, in the final revision of our work we will increase the discussion in the context of the new results obtained.

    Minor points

    1. __ Figure numbering goes from figure 4 to S6 to 5.__ We have updated the numbering of the figures after merging several supplementary figures, so now this issue is fixed.

    __ It would be helpful to the reader to explain what some of the reporters are in brief. For example, Guk1-9-GFP and Rho1.C17R-GFP reporters__.

    Both the Guk1-9-GFP and Rho1.C17R-GFP are two thermosensitive mutants in guanylate kinase and Rho1 GTPase respectively, that have been previously used in S. pombe as soluble reporters of misfolding in conditions of heat stress. During mild heat shock, both mutants aggregate into reversible protein aggregate centers (Cabrera et al., 2020). This information has now been added to the manuscript.

    __ Supplementary Figure 3. The main text suggests panel 3A is focused on diamide treatment. The figure legend discusses this in terms of HU treatment. Which is correct?__

    We thank the reviewer for pointing out this mistake. The experiment was performed in 75 mM HU, the legend was correct. It has now been corrected in the manuscript.

    __ The authors use ref 110 and 111 to suggest the importance of UPR-independent signaling. However, they do not point out that this UPR-independent signaling referred to in these papers is dependent on the UPR transmembrane kinase IRE1.__

    We have included pertinent clarification in the new discussion.

    Reviewer 3

    Major issue #1. It is hard to see how the claim of ER stress can be supported if BiP levels do not change (Fig. 4B). Also, this figure is overexposed. The RNA-seq data should be able to establish ER stress as well, but no rigorous analysis of ER stress markers is presented.

    Regarding the levels of Bip1, we now show in Figure 4 a less exposed image of the western blot, and a quantification of Bip1-GFP intensity from three independent experiments. We find that, in our experimental conditions, neither HU nor DIA treatments significantly altered Bip1 levels.

    With respect to the RNA-Seq, as we mentioned in the major issue 1 from reviewer 1, we plan to reassess our data to further clarify and add information about ER stress markers induced or repressed by HU and DIA. We also will test the levels of Bip1 and several UPR targets by RT-PCR and by western blot.

    Major issue #2. The interpretation of the CHX and puromycin experiments of Figure 3A-B is hard to follow. My best guess is that the authors argue that CHX decreases misfolded protein load and that puromycin increases misfolded protein load, and that since DIA is a stronger oxidative stress than HU hence CHX is only protective under HU and not DIA. However, while CHX decreases misfolded protein load, puromycin hasn't been show directly to increase it and I don't see how this explains puromycin being protective at all.

    We have found that puromycin treatment alone results in the formation of cytoplasmic foci containing Hsp104, suggesting that puromycin indeed increases folding stress in the cytoplasm. We have now included this data in Figure 4K (please see Main Issue #5 from Reviewer 2). Pm suppresses the formation of N-caps induced by HU or DIA; however, we have not addressed cell survival or fitness in these conditions and therefore we cannot conclude about being protective.

    In addition, upon the reevaluation of our data, we have realized that CHX treatment suppresses HU-induced perinuclear expansion, although it does not suppress but instead enhances ER expansion in the cortical region. This data has been added to the present version of the manuscript in Figure 3C-D (page 22).

    Furthermore, puromycin causes Ca leakage from the ER (which can be recapitulated with thapsigargin and blocked with anisomycin; easy experiments), which could be responsible for the differences from CHX, and the model does not address the effects on downstream stress signaling. The authors should be much more clear regarding their argument, since this data is used to support the argument of disrupted ER proteostasis.

    As the reviewer requested, we plan to test the effect of anisomycin (thapsigargin has been described to not work in yeast, as they lack a (SERCA)‐type Ca2+ pump (Strayle et al., 1999), which this drugs targets.

    Regarding the downstream effects of HU or DIA treatment on ER proteostasis, we plan to further explore the effect of these drugs on the secretory system (please see major issue #2 from Reviewer 1) and to evaluate the redox state and processing of several key ER and secretory proteins. We will further explore the nature of the aggregates that appear in the cytoplasm in our experimental conditions, which will also shed light into the downstream effects of these drugs in cytoplasmic proteostasis (please see answer to issue #5 from Reviewer 2).

    Major issue #3. The claim that a canonical UPR is not induced is weak. First, the transcriptional program of S. cerevisiae from Travers et al is used as the canonical UPR, and compared to HU/DIA induced stress in S. pombe. These organisms may not be similar enough to assume that they have transcriptionally identical UPRs. Second, no consideration is given to the mechanism by which the different transcripts are modulated between "canonical" and HU/DIA induced UPR. Is it solely through RIDD, or does it point to differences in sensing or signaling transduction?

    We plan on readdressing this topic by analyzing the genes that have been described to be differentially expressed during UPR activation in S. pombe and comparing them with our data, first by reevaluating our transcriptomic data and second by choosing Bip1 and some other of the differentially expressed genes in (Kimmig et al., 2012) (for example, Gas2, Pho1 or Yop1) and assessing by RT-PCR their mRNA levels in our experimental conditions. As an alternative approach, we will also analyse the levels of UPR targets by western blot upon HU or DIA treatment.

    We are confident that the results of these experiments and the re-analysis of our RNA-Seq data will allow us to infer the mechanisms that modulate the ER response to HU or DIA treatment.

    Finally, the p-values used are unadjusted (e.g. by Bonferroni's method or by ANOVA or at least controlled by an FDR approach) and unmodulated (extremely important when n = 3 and variance is poorly sampled), which makes them not dependable. It looks like HSF1 targets are induced, which should be addressed.

    We thank the reviewer for pointing this out. We forgot to include this information which now appears in the M&M section as follows:

    "A gene was considered as differentially expressed when it showed an absolute value of log2FC(LFC){greater than or equal to}1 and an adjusted p-valueIn this regard, we plan to perform proteome-wide mass spectrometry experiments to detect protein glutathionylation in our conditions, as it has been previously shown that DIA treatment leads to glutathionylation of key ER proteins such as Bip1, Pdi or Ero1 (Lind et al., 2002; Wang & Sevier, 2016), which might by reproduced upon HU treatment. We will also test specifically the redox state of Bip1, Pdi and/or Ero1 by immunoprecipitation and western blot. We also plan to test the folding and processing of specific secretory cargoes by western blot in our experimental conditions (see below, and Reviewer 2, Major issue #1).

    We have already tested whether mutant strains with deletions of key enzymes in both cytoplasmic and ER redox systems are able to expand the ER upon HU or DIA treatment. We have found that only *pgr1Δ *(glutathione reductase), *gsa1Δ *(glutathione synthetase) and gcs1Δ (glutamate-cysteine ligase) mutants fully suppressed N-Cap formation, which suggests that glutathione has an important role in the phenotype of ER expansion. We have now added the *pgr1Δ *mutant strain to the main text of the manuscript (Figure 5C, page 31).

    Major issue #5. Figure S5 presents weak ER expansion in fribrosarcoma cells in response to HU (at very low concentrations and DIA is not included). The lack of any other phenotypes being presented could suggest that such experiments were done but didn't show any effect. The authors should straightforwardly discuss whether they performed experiments looking for perinuclear ER expansion or NPC clustering, and if not, what challenges precluded such experiments. Given how important this line of experimentation is for establishing generality, much more discussion is needed here.

    We not only investigated the effects of HU on the ER in mammalian cells, but also of DIA. The results from this experiment mimicked the effect of HU (an increase in ER-ID fluorescence intensity in DIA). We merely excluded this information from the manuscript because we were focusing on HU at that point due to its importance as it is used currently in clinics. In this new version of the manuscript, we have included an extra panel in supplementary figure 5 to show the results from DIA in mammalian cells.

    Minor concerns

    1) Figure 1A should show individual data points (i.e. 3 averages of independent experiments) in the bar graph.

    Although we initially changed the graph, we believe the bar plot disposition facilitates its comprehension and went back to the initial one. Also, as the rest of the graphs similar to 1A are all expressed as bar plots, changing one would mean that, to avoid visual noise, we should change all. Therefore, we preferred keeping the figure as it was in the original version. However, we include here the graph with each of the averages of the independent experiments.

    2) It is argued that Figure 1B demonstrates that the SPB is clustered with the NPC cluster. However, a single image is not enough to support this claim, as the association could be coincidental.

    We have changed the image to show a whole population of cells, with several of them having NPC clusters, and we have indicated the position of SPB in each of them (all colocalizing with the N-Cap).

    3) Figures 1B through 1D do not indicate the HU concentration.

    We thank the reviewer for pointing out this mistake. Figures 1B and 1C represent cells exposed to 15 mM HU for 4 hours, while the graph in 1D shows the results from cells exposed to 75 mM HU over a 4-hour period. This information has been now added to the corresponding figure legend.

    4) I was confused by the photobleaching experiments of Figure S1. How do the authors know that there is complete photobleaching of the cytoplasm or nucleus in the absence of a positive control? If photobleaching is incomplete, they could be measuring motility without compartments rather than transport between compartments, and hence the conclusion that trafficking is unaffected could be wrong.

    Our control is the background of each microscopy image; we make sure that after the laser bleaches a cell, the bleached area coincides with the background noise. That way, we make sure that fluorescence from any remaining GFP is completely removed from the bleached area.

    5) On page 8, they say "exposure to DIA" when they intend HU.

    This has been corrected in the manuscript.

    6) In Figure S3A, the colocalization of INM proteins with the ER are presented. It is not clearly explained what conclusions are meant to be drawn from this figure, but it seems it would have been more useful to compare INM and Cut11, to see whether the NPCs are localizing at the INM or ONM.

    We have added an explanation in the main text to clarify the main conclusions derived from this figure. We think that NPCs localize in a section of the nucleus where the two membranes (INM and ONM) are still bound together.

    7) I had to read Figure 2C's description and caption several times to understand the experiment. A schematic would be helpful. 20 mM HU is low compared to most conditions used. Does repositioning eventually take place for 75 mM HU or 3 mM DIA treatment, or do the cells just die before they get a chance?

    20 mM HU was used in this experiment to provide a time frame suitable for analysis after HU addition, as a higher HU concentration increases the repositioning time. We found that both HU (75mM 4h) and DIA (3mM 4h)-induced ER expansions are reversible upon drug washout. If HU is kept in the media, ER expansions are eventually resolved. However, DIA is a strong oxidant and if it is kept in the media ER expansions are not resolved and cells do not survive.

    8) Figure 2D shows little oxidative consequence from 75 mM HU treatment until 40 min., the same time that phenotypes are observed (Figure 1D). Is this relationship consistent with the kinetics of other concentrations of HU, or of DIA? Seems like a pretty important mechanistic consideration that can rationalize the effects of the two oxidants.

    Thanks to this comment, we realized the notation underneath Figure 1D (1E in the new version of the manuscript) could lead to misunderstandings, as the timings there were "random". We have now made a clarification for this panel to be clearer: the timings are normalized to the moment when NPCs cluster. The fact that, before, that moment coincided with "40 minutes" does not mean N-Caps appear at that time point-quite the opposite, as most of them start to appear after >2 hours have passed in HU. We hope this can be better understood now.

    9) Figure S4 is missing the asterisk on the lower left cell.

    Fixed in the corresponding figure.

    10) How is roundness determined in Figure S4B?

    Roundness in Figure S4B (now S2E) is determined the same way as in Figure 1D, and as is described in the Method section (copied below). A clarification has been added to the legend to address that.

    The 'roundness' parameter in the 'Shape Descriptors' plugin of Fiji/ImageJ was used after applying a threshold to the image in order to select only the more intense regions and subtract background noise (Schindelin et al., 2012). Roundness descriptor follows the function:

            Round=4 X [Area]/π X [Major axis]2

    where [Area] constitutes the area of an ellipse fitted to the selected region in the image and [Major axis] is the diameter of the round shape that in this case would fit the perimeter of the nucleus.

    11) What threshold is used to determine whether cells analyzed in Figures S4C have "small ER" or "large ER"?

    Large ER are considered when their area along the projection of a 3-Z section is over 4 μm2 (more than twice the mean area of the ER in cells with N-Caps in milder conditions). This has now been clarified in the legend of the corresponding figure.

    __12) The authors interpret Figure 4K as indicating that ER expansion is not involved in the generation of punctal misfolded protein aggregates. However, the washout occurs only after the proteins have already aggregated. The proper interpretation is that the aggregates are not reversible by resolution of the stress, and hence are not physically reliant on disulfide bonds. __

    We agree with the reviewer and have modified the interpretation of the indicated figure accordingly (page 30).

    The speculation that these proteins are iron dependent is a stretch; there is no reason to believe that losses of iron metabolism are the most important stress in these cells. It seems at least as likely that oxidizing cysteine-containing proteins in the cytosol or messing with the GSH/GSSG ratio in the cytosol would make plenty of proteins misfold; oxidative stress in budding yeast does activate hsf1. However, this point could be addresses by centrifugation and mass spectrometry to identify the aggregated proteome. It is also surprising that the authors did not investigate ER protein aggregation, perhaps by looking at puncta formation of chaperones beyond BiP. By contrast, the fact that gcs1 deletion prevents ER expansion but does not prevent Hsp104 puncta does support the idea that cytoplasmic aggregation is not dependent on ER expansion.

    To address this suggestion, we plan to analyze the localization of other chaperones and components of the protein quality control such as the ER Hsp40 Scj1 or the ribosome-associated Hsp70 Sks2.

    13) Figure 4L is cited on page 28 when Figure 4K is intended.

    This has been corrected in the text, although new panels have been added and now it is 4N.

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    Referee #3

    Evidence, reproducibility and clarity

    This article makes the following claims, using S. pombe as their model system. Hydroxyurea (HU) and diamide (DIA) induce ER stress, an atypical UPR, and cytoplasmic protein aggregation. HU and DIA induce IRE1-independent and GSH-dependent reversible ER perinuclear expansion which causes nuclear pore clustering with no effect on protein trafficking, and can be reversed by DTT.

    Major concerns:

    1. It is hard to see how the claim of ER stress can be supported if BiP levels do not change (Fig. 4B). Also, this figure is overexposed. The RNA-seq data should be able to establish ER stress as well, but no rigorous analysis of ER stress markers is presented.
    2. The interpretation of the CHX and puromycin experiments of Figure 3A-B is hard to follow. My best guess is that the authors argue that CHX decreases misfolded protein load and that puromycin increases misfolded protein load, and that since DIA is a stronger oxidative stress than HU hence CHX is only protective under HU and not DIA. However, while CHX decreases misfolded protein load, puromycin hasn't been show directly to increase it and I don't see how this explains puromycin being protective at all. Furthermore, puromycin causes Ca leakage from the ER (which can be recapitulated with thapsigargin and blocked with anisomycin; easy experiments), which could be responsible for the differences from CHX, and the model does not address the effects on downstream stress signaling. The authors should be much more clear regarding their argument, since this data is used to support the argument of disrupted ER proteostasis.
    3. The claim that a canonical UPR is not induced is weak. First, the transcriptional program of S. cerevisiae from Travers et al is used as the canonical UPR, and compared to HU/DIA induced stress in S. pombe. These organisms may not be similar enough to assume that they have transcriptionally identical UPRs. Second, no consideration is given to the mechanism by which the different transcripts are modulated between "canonical" and HU/DIA induced UPR. Is it solely through RIDD, or does it point to differences in sensing or signaling transduction? Finally, the p-values used are unadjusted (e.g. by Bonferroni's method or by ANOVA or at least controlled by an FDR approach) and unmodulated (extremely important when n = 3 and variance is poorly sampled), which makes them not dependable. It looks like HSF1 targets are induced, which should be addressed.
    4. Mechanistically, one would expect effects to be mediated by PDIs and oxidoreductases. No effort is made to characterize the redox state of these molecules, nor how that relates to the kinetics of ER expansion and resolution under HU/DIA treatment. No discussion is made of the existing literature on oxidants and ER stress. A few papers: PMID: 29504610, PMID: 31595201.
    5. Figure S5 presents weak ER expansion in fribrosarcoma cells in response to HU (at very low concentrations and DIA is not included). The lack of any other phenotypes being presented could suggest that such experiments were done but didn't show any effect. The authors should straightforwardly discuss whether they performed experiments looking for perinuclear ER expansion or NPC clustering, and if not, what challenges precluded such experiments. Given how important this line of experimentation is for establishing generality, much more discussion is needed here.

    Minor concerns:

    1. Figure 1A should show individual data points (i.e. 3 averages of independent experiments) in the bar graph.
    2. It is argued that Figure 1B demonstrates that the SPB is clustered with the NPC cluster. However, a single image is not enough to support this claim, as the association could be coincidental.
    3. Figures 1B through 1D do not indicate the HU concentration.
    4. I was confused by the photobleaching experiments of Figure S1. How do the authors know that there is complete photobleaching of the cytoplasm or nucleus in the absence of a positive control? If photobleaching is incomplete, they could be measuring motility without compartments rather than transport between compartments, and hence the conclusion that trafficking is unaffected could be wrong.
    5. On page 8, they say "exposure to DIA" when they intend HU.
    6. In Figure S3A, the colocalization of INM proteins with the ER are presented. It is not clearly explained what conclusions are meant to be drawn from this figure, but it seems it would have been more useful to compare INM and Cut11, to see whether the NPCs are localizing at the INM or ONM.
    7. I had to read Figure 2C's description and caption several times to understand the experiment. A schematic would be helpful. 20 mM HU is low compared to most conditions used. Does repositioning eventually take place for 75 mM HU or 3 mM DIA treatment, or do the cells just die before they get a chance?
    8. Figure 2D shows little oxidative consequence from 75 mM HU treatment until 40 min., the same time that phenotypes are observed (Figure 1D). Is this relationship consistent with the kinetics of other concentrations of HU, or of DIA? Seems like a pretty important mechanistic consideration that can rationalize the effects of the two oxidants.
    9. Figure S4 is missing the asterisk on the lower left cell.
    10. How is roundness determine in Figure S4B?
    11. What threshold is used to determine whether cells analyzed in Figures S4C have "small ER" or "large ER"?
    12. The authors interpret Figure 4K as indicating that ER expansion is not involved in the generation of punctal misfolded protein aggregates. However, the washout occurs only after the proteins have already aggregated. The proper interpretation is that the aggregates are not reversible by resolution of the stress, and hence are not physically reliant on disulfide bonds. The speculation that these proteins are iron dependent is a stretch; there is no reason to believe that losses of iron metabolism are the most important stress in these cells. It seems at least as likely that oxidizing cysteine-containing proteins in the cytosol or messing with the GSH/GSSG ratio in the cytosol would make plenty of proteins misfold; oxidative stress in budding yeast does activate hsf1. However, this point could be addresses by centrifugation and mass spectrometry to identify the aggregated proteome. It is also surprising that the authors did not investigate ER protein aggregation, perhaps by looking at puncta formation of chaperones beyond BiP. By contrast, the fact that gcs1 deletion prevents ER expansion but does not prevent Hsp104 puncta does support the idea that cytoplasmic aggregation is not dependent on ER expansion.
    13. Figure 4L is cited on page 28 when Figure 4K is intended.

    Significance

    This paper is for the most part well-written, presenting a logical chain of experiments that fully support the most important claims that have been made. Specifically, they show that HU and DIA induce reversible perinuclear expansion and nuclear pore clustering in an IRE1-independent and GSH-dependent manner, and that DTT can prevent and accelerate recovery of this phenotype. Both oxidants clearly induce protein aggregation in the cytosol. The evidence that perinuclear expansion is responsible for nuclear pore clustering is compelling, with strong support from the kinetics and the nup120 deletion experiments. Some conclusions are not supported, including the claim of an atypical UPR and of ER stress, but the validity of these claims does not substantively affect the overall importance of the paper and could be handled by withdrawal or tempering of the claims. The lack of a molecular mechanism connecting oxidation with ER expansion moderately detracts from the potential impact. Adequate experimental detail is provided unless otherwise noted

    This paper is likely to be important for cell biologists interested in interorganelle communication and how the cell responds to oxidative stress. Modulating ER oxidoreductase activity has been shown to be a powerful way to regulate ER stress and proteostasis, and this paper shows how specific oxidative stresses that have not widely been investigated in this context, as opposed to the more commonly studied reductive and electrophilic stresses, can remodel the ER with cell-wide consequences. More specifically, the nuclear pore and nuclear morphology phenotypes, while not yet functionally significant in yeast, could be significant in other unexplored ways identified in the future. Towards that end, it would be valuable to see if these gross phenotypes reproduce in any metazoan cell or tissue, rather than just looking at ER expansion as in the current manuscript. My expertise is centered around ER proteostasis and chaperones, and as such I consider this paper important to my field.

  7. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript by Sánchez-Molina et al describes a striking time and dose-dependent clustering of nuclear pores and perinuclear ER expansion in response to hydroxyurea (HU) or diamide (DIA) treatment in S. pombe. Using microscopy, the authors establish clustering is reversible upon drug washout or extended drug treatment. Pretreatment or post-treatment with the reductant DTT prevents or reverses the clustering and expansion effects, as does the release of translating polypeptides from ribosomes (with puromycin). The phenotypes were established to occur independent of the established impact of HU on RNR activity and the cell cycle. The authors suggest instead that the phenotypes (referred to as nuclear-cap (N-Cap) formation) are associated with disulfide-based folding stress. Overlapping transcriptional responses for HU and DIA treatment suggest that cells are experiencing folding stress (based on chaperone induction) and/or a disruption in iron homeostasis (induction of genes involved in iron homeostasis). The observed clustering, ER expansion, and transcriptional profiles are independent of the well-established ER stress response pathway: the UPR.

    The manuscript outlines several interesting phenotypic observations, and they establish the potential for conserved of this ER expansion and nuclear pore clustering from yeast (S. cerevisiae) and mammals (HT1080 fibrosarcoma cells). Data clearly establish the time and dose-dependent formation of these interesting structures. Additional experiments with combined drug treatments points towards a role for changes in the redox environment in cells, an impact on cytoplasmic protein aggregation, and a potential impact on the ER folding environment / ER redox environment.

    Data obtained with thiol oxidants and reductants, alongside translation inhibitors, suggest a potential connection between the N-Cap phenotype and oxidative folding within the ER. Yet, this latter observation remains a suggestive link with less clear mechanistic connections. Some experiments that would more directly assess the suggested changes within the nuclear/ER region are outlined below.

    1. The authors state the cytoplasmic and ER folding are both disrupted. The impact on ER protein biogenesis would be bolstered with some biochemical data focused on the folding of one or more nascent secretory proteins. Is disulfide bond formation and/or protein folding indeed disrupted?
    2. Increased signal of Bip1 in the expanded perinuclear ER is shown and is suggested as consistent with immobilization of BiP upon binding of misfolded proteins. The authors suggest that this increased signal must reflect Bip1 redistribution because "Bip1 levels are constant". Yet, the western image (Figure 4B) looks to show increased level of Bip1 protein up HU treatment. Given the abundance of Bip1 in cells, it seems possible that a two-fold increase in newly synthesized proteins in the perinuclear region may account for the increased signal. These original data cited by the authors uses photobleaching (not just fluorescence intensity) to show a change in crowding / mobility, which the authors should consider to support their conclusion. Alternatively, a detected increased engagement of Bip1 with substrates (e.g. pulldown experiment) would be similarly strengthening.
    3. It is curious that cycloheximide (CHX) has a distinct impact on HU versus DIA treatment. Blocking protein synthesis with CHX exacerbates the phenotype with DIA, but not HU. The authors use the data with CHX to argue that their drug treatments are interfering with folding during synthesis and translation into the ER. If so, what is the rationale as to why CHX treatment decreases expansion upon HU treatment? Relatedly, is protein synthesis and/or ER import impacted upon treatment with HU and/or DIA?
    4. While the authors suggest that there is disulfide stress in the ER / nucleus, the redox environment in these compartments is not tested directly (only cytoplasmic probes).

    Addition suggestions / comments:

    1. What do the authors envision is the role of the cytoplasmic chaperone foci? Do CHX / Pm treatment with HU/DIA reverse the chaperone foci? The authors argue that cytoplasmic foci are "independent" from ER expansion and are "not a direct consequence of thiol stress" based on the observation that DTT does not reverse these foci. This seems like a strong statement based on the limited analysis of these foci.
    2. Based on the transcriptional data, the authors speculate a potential role on role on iron-sulfur cluster protein biogenesis. This would seem to be rather straightforward to test.
    3. The authors suggest that "pre-treatment" with DTT before HU addition suppresses formation of the N-Caps. However, these samples (Figure 2J) contain DTT coincident with the treatment as well. To say it is the effect of pre-treatment, the DTT should be added and then washed out prior to HU or DIA addition. Alternatively, the language used to describe these experiments and their outcomes could be revised.
    4. For a manuscript with 128 references there is rather limited discussion of the data in the context of the wider literature. The discussion primarily focuses on a recap of the results. The authors do cite several prior works focused on redox-dependent nuclear expansion. However, while cited, there is no real discussion of the relationship between this work in the context of that previously published (including several known disulfide bonded proteins that are involved in nuclear/ER architecture).

    Minor points

    1. Figure numbering goes from figure 4 to S6 to 5.
    2. It would be helpful to the reader to explain what some of the reporters are in brief. For example, Guk1-9-GFP and Rho1.C17R-GFP reporters.
    3. Supplementary Figure 3. The main text suggests panel 3A is focused on diamide treatment. The figure legend discusses this in terms of HU treatment. Which is correct?
    4. The authors use ref 110 and 111 to suggest the importance of UPR-independent signaling. However, they do not point out that this UPR-independent signalling referred to in these papers is dependent on the UPR transmembrane kinase IRE1.

    Significance

    An interesting finding that is well-supported as a phenotype. What would raise the impact would be data that connect these observations more directly with a mechanism. In particular, there are suggestions of a disruption in ER folding and/or the ER redox environment that are logical but not directly tested. How one viewed these additional experiments will depend on what journal is considering the manuscript.

  8. Review Commons

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

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Sanchez-Molina describe the impact of hydroxyurea on the remodeling of the nuclear pore complex (clustering) and the expansion of both cortical and perinuclear ER. The study is carried out in S. pombe, and the observations confirmed in S. cerevisiae. Results are clear and analyzed properly, however considering the differences in UPR signaling in both yeast strains the conclusions raised may remain to be fully documented.

    Major issues

    Regarding the conclusions on IRE1 signaling, both yeast species have different IRE1 activities https://elifesciences.org/articles/00048), the total deletion of IRE1 in S pombe appears to indicate that expansion of perinuclear ER is independent of IRE1, however since IRE1 signaling has exclusively a negative impact on mRNA expression, it might be relevant to identify mRNA whose expression is stabilized under those circumstances and evaluate whether those could confer a mechanism which would also yield perinuclear ER expansion (eg differential deregulation of ER stress controlled lipid biosynthesis required for lipid membrane synthesis). In S cerevisiae, do the authors observe HAC1 mRNA splicing?

    The authors indicate that HU and DIA lead to thiol stress, it might be relevant to evaluate the thiol-redox status of major secretory proteins in S pombe (or even cargo reporters if necessary) to fully document the stress impact on global protein redox status. What happens if HU-treated yeast cells are grown in the presence of n-acetyl cysteine?

    The appearance of cytosolic aggregates is intriguing, do the authors have any idea on the nature of the protein aggregates? Are those resulting from proficient retrotranslocation (or reflux of misfolded proteins from the ER? Are those aggregates membrane bound or do they correspond to aggresomes as initially defined?

    The Walter lab has demonstrated a tight balance between ER phagy and ER membrane expansion (https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.0040423), which could also impact on the presence of protein aggregates in the cytosol. Does HU impact on the regulation of autophagy?

    Nucleotide depletion was previously shown to lead to HSP16 expression through activation of the spc1 MAPK pathway (10.1093/nar/29.14.3030), one might think that HU (or diamide) could lead to this through a nucleotide dependent mechanism and not necessary through a thiol-redox protein misfolding stress. This issue has to be sorted out to ensure that the HSP effect is independent of nucleotide depletion.

    Minor issues

    P1 - UPR = Unfolded Protein Response

    P22 - HSP upregulation "might" be indicative of a folding stress

    The abstract does not reflect the findings presented in the manuscript. In addition, I would recommend the authors to revise the story telling in their manuscript to push forward the message on either the specific phenotype associated with perinuclear ER or on the characterization of protein misfolding stress.

    Significance

    This is a nice manuscript describing the likely effects of HU on protein misfolding and several consequences including the remodeling of the nuclear pore complex, the expansion of both cortical and perinuclear ER. The underlying mechanisms remain however unclear (for each parameter evaluated) and the manuscript would definitely benefit from the elucidation of one of those (if not more).

    The work in yeast is novel and might bring light on mechanisms existing in mammalian systems. Since HU is used as a therapeutic, the characterization of the molecular mechanisms associated with its mode(s) of action will definitely be useful for better (targeted) efficiency.

    The audience for this work is more targeted towards people working on yeast cell biology, however, the authors could expand the discussion section to make it of a broader scope.

    I am expert on ER stress signaling

  9. Version published to 10.1101/2024.10.17.618805 on bioRxiv